S. cerevisiae

J Biol Chem, 2003 Jun 27, 278(26), 24225 - 32 Epub 2003 Apr 17.
Nab2p and the Thp1p-Sac3p complex functionally interact at the interface between transcription and mRNA metabolism; Gallardo M et al.; THP1 is a conserved eukaryotic gene whose null mutations confer, in yeast, transcription and genetic instability phenotypes and RNA export defects similar to those of the THO/TREX complex null mutations . In a search for multicopy suppressors of the transcription defect of thp1Delta cells, we identified the poly(A)+ RNA-binding heterogeneous nuclear ribonucleoprotein Nab2p . Multicopy NAB2 also suppressed the RNA export defect of thp1Delta cells . This result suggests a functional relationship between Thp1p and Nab2p . Consistently, the leaky mutation nab2-1 conferred a transcription defect and hyper-recombination phenotype similar to those of thp1Delta, although to a minor degree . Reciprocally, a purified His6-tagged Thp1p fusion bound RNA in vitro . In a different approach, we show by Western analyses that a highly purified Thp1p-Sac3p complex does not contain components of THO/TREX and that sac3Delta confers a transcription defect and hyper-recombination phenotype identical to those of thp1Delta . mRNA degradation was not affected in thp1Delta mutants, implying that their expression defects are not due to mRNA decay . This indicates that Thp1p-Sac3p is a structural and functional unit . Altogether, our results suggest that Thp1p-Sac3p and Nab2p are functionally related heterogeneous nuclear ribonucleoproteins that define a further link between mRNA metabolism and transcription.

FEMS Yeast Res, 2003 Mar, 3(1), 27 - 34
The Hansenula polymorpha PDD7 gene is essential for macropexophagy and microautophagy; Komduur JA et al.; Hansenula polymorpha PDD genes are involved in the selective degradation of peroxisomes via macropexophagy . We have isolated various novel pdd mutants by a gene-tagging method . Here we describe the isolation and characterisation of PDD7, which encodes a protein with high sequence similarity (40% identity) to Saccharomyces cerevisiae Apg1p/Aut3p, previously described to be involved in random autophagy and the cytoplasm-to-vacuole targeting pathway . Our data indicate that HpPdd7p is essential for two processes that degrade peroxisomes, namely the highly selective process of macropexophagy and microautophagy, which occurs in H . polymorpha upon nitrogen starvation.

Mol Cell Proteomics, 2003 Mar, 2(3), 173 - 81 Epub 2003 Apr 07.
Genome-wide Analyses of Carboxyl-terminal Sequences; Chung JJ et al.; Sequence motifs at the protein carboxyl termini in linear polypeptides are uniquely positioned and functionally capable of serving as recognition signatures for a variety of cellular and biochemical processes . At the proteome level, it is unknown whether and what carboxyl-terminal sequences might be particularly conserved, which may be directly related to specific biological functions shared among certain groups of proteins . To investigate this question, we analyzed the terminal sequences of reported yeast open reading frames, which presumably constitute the predicted, entire proteome of Saccharomyces cerevisiae . The results show that there are both known and novel terminal sequences . They are conserved at a frequency similar to that of functionally important, experimentally confirmed signals such as the HDEL sequence that mediates the endoplasmic reticulum retention and/or retrieval . The findings support the notion that there may be additional carboxyl-terminal signals, and the conserved motifs could be experimentally tested for currently unknown biological functions . Similar analyses were also applied to the limited proteome databases of other organisms with overall consistent findings . Therefore, indexing a proteome according to its carboxyl-terminal sequences may provide a means for functional classification and determination of proteins.

EMBO J, 2003 Apr 15, 22(8), 1846 - 56
Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes; Simic R et al.; Transcription in eukaryotes is influenced by the chromatin state of the template, and chromatin remodeling factors have well-documented roles in regulating transcription initiation by RNA polymerase (pol) II . Chromatin also influences transcription elongation; however, little is known about the role of chromatin remodeling factors in this process . Here, we present evidence that the Saccharomyces cerevisiae chromatin remodeling factor Chd1 functions during transcription elongation . First, we identified Chd1 in a two-hybrid screen for proteins that interact with Rtf1, a member of the Paf1 complex that associates with RNA pol II and regulates transcription elongation . Secondly, we show through co-immunoprecipitation studies that Chd1 also interacts with components of two essential elongation factors, Spt4-Spt5 and Spt16-Pob3 . Thirdly, we demonstrate that deletion of CHD1 suppresses a cold-sensitive spt5 mutation that is also suppressed by defects in the Paf1 complex and RNA pol II . Finally, we demonstrate that Chd1, Rtf1 and Spt5 associate with actively transcribed regions of chromatin . Collectively, these findings suggest an important role for Chd1 and chromatin remodeling in the control of transcription elongation.

Curr Biol, 2003 Apr 15, 13(8), R326 - 37
Mechanisms of protein import into mitochondria; Truscott KN et al.; Apart from a handful of proteins encoded by the mitochondrial genome, most proteins residing in this organelle are nuclear-encoded and synthesised in the cytosol . Thus, delivery of proteins to their final destination depends on a network of specialised import components that form at least four main translocation complexes . The import machinery ensures that proteins earmarked for the mitochondrion are recognised and delivered to the organelle, transported across membranes, sorted to the correct compartment and assisted in overcoming energetic barriers.

Drugs, 2003, 63(10), 1021 - 51
Recombinant hepatitis B vaccine (Engerix-B): a review of its immunogenicity and protective efficacy against hepatitis B; Keating GM et al.; Engerix-B (Hep-B{Eng}) is a noninfectious recombinant DNA vaccine containing hepatitis B surface antigen (HBsAg) . It is produced from genetically engineered yeast (Saccharomyces cerevisiae) . Intramuscular Hep-B(Eng) {0-, 1-, 6-month schedule} has excellent immunogenicity in healthy neonates and infants, children, adolescents and adults, with seroprotection rates of 85-100% seen approximate, equals 1 month after the final dose of vaccine; seroprotection was defined as an antibody against HBsAg (anti-HBs) titre of > or =10 IU/L . The use of alternative Hep-B(Eng) immunisation schedules (e.g . a 0-, 1-, 2-, 12-month schedule in neonates and infants, 0-, 12-, 24-month or two-dose schedules in children and adolescents, and accelerated schedules in adults) have also been associated with high rates of seroprotection . Seroprotection rates were generally similar with Hep-B(Eng) and the recombinant vaccine Recombivax HB (Hep-B{Rax}) or plasma-derived vaccines (PDVs) approximate, equals 1 month after the final dose (although anti-HBs geometric mean titres were significantly higher with Hep-B{Eng} than with Hep-B{Rax}) . One month after the final dose, adults had significantly higher seroprotection rates with the recombinant triple-antigen vaccine Bio-Hep-B (Hep-B{Bio}) than with Hep-B(Eng), although seroprotection rates in healthy infants were similar with Hep-B(Eng) and Hep-B(Bio) . Hep-B(Eng) had excellent immunogenicity in several groups considered at high risk of acquiring hepatitis B (e.g . neonates born to hepatitis B carrier mothers and healthcare workers) . The immunogenicity of Hep-B(Eng) was reduced in patients with conditions associated with impaired immune function (e.g . patients undergoing haemodialysis or being treated for malignancy), although it had good immunogenicity in patients with diabetes mellitus.Hep-B(Eng) had excellent protective efficacy against HBsAg carriage in healthy infants and children, and in neonates born to hepatitis B carrier mothers (protective efficacy of 95-99%) . Hep-B(Eng) also demonstrated good protective efficacy in a number of other high-risk groups . Hep-B(Eng) is generally well tolerated with a tolerability profile similar to that of Hep-B(Rax), Hep-B(Bio) and PDVs . In conclusion, Hep-B(Eng) is a well established, highly immunogenic hepatitis B vaccine with good tolerability and excellent protective efficacy; it offers flexibility through a variety of immunisation schedules . In addition, it appears that Hep-B(Eng) confers immunity for at least 10 years . Hep-B(Eng) has an important role in mass vaccination campaigns against hepatitis B, as well as in groups considered at high risk of acquiring hepatitis B.

Mol Cell Biol, 2003 May, 23(9), 3339 - 51
Regulation of the bone-specific osteocalcin gene by p300 requires Runx2/Cbfa1 and the vitamin D3 receptor but not p300 intrinsic histone acetyltransferase activity; Sierra J et al.; p300 is a multifunctional transcriptional coactivator that serves as an adapter for several transcription factors including nuclear steroid hormone receptors . p300 possesses an intrinsic histone acetyltransferase (HAT) activity that may be critical for promoting steroid-dependent transcriptional activation . In osteoblastic cells, transcription of the bone-specific osteocalcin (OC) gene is principally regulated by the Runx2/Cbfa1 transcription factor and is stimulated in response to vitamin D(3) via the vitamin D(3) receptor complex . Therefore, we addressed p300 control of basal and vitamin D(3)-enhanced activity of the OC promoter . We find that transient overexpression of p300 results in a significant dose-dependent increase of both basal and vitamin D(3)-stimulated OC gene activity . This stimulatory effect requires intact Runx2/Cbfa1 binding sites and the vitamin D-responsive element . In addition, by coimmunoprecipitation, we show that the endogenous Runx2/Cbfa1 and p300 proteins are components of the same complexes within osteoblastic cells under physiological concentrations . We also demonstrate by chromatin immunoprecipitation assays that p300, Runx2/Cbfa1, and 1alpha,25-dihydroxyvitamin D(3) receptor interact with the OC promoter in intact osteoblastic cells expressing this gene . The effect of p300 on the OC promoter is independent of its intrinsic HAT activity, as a HAT-deficient p300 mutant protein up-regulates expression and cooperates with P/CAF to the same extent as the wild-type p300 . On the basis of these results, we propose that p300 interacts with key transcriptional regulators of the OC gene and bridges distal and proximal OC promoter sequences to facilitate responsiveness to vitamin D(3).

Mol Cell Biol, 2003 May, 23(9), 3329 - 38
Loss of the Rpb4/Rpb7 subcomplex in a mutant form of the Rpb6 subunit shared by RNA polymerases I, II, and III; Tan Q et al.; We have identified a conditional mutation in the shared Rpb6 subunit, assembled in RNA polymerases I, II, and III, that illuminated a new role that is independent of its assembly function . RNA polymerase II and III activities were significantly reduced in mutant cells before and after the shift to nonpermissive temperature . In contrast, RNA polymerase I was marginally affected . Although the Rpb6 mutant strain contained two mutations (P75S and Q100R), the majority of growth and transcription defects originated from substitution of an amino acid nearly identical in all eukaryotic counterparts as well as bacterial omega subunits (Q100R) . Purification of mutant RNA polymerase II revealed that two subunits, Rpb4 and Rpb7, are selectively lost in mutant cells . Rpb4 and Rpb7 are present at substoichiometric levels, form a dissociable subcomplex, are required for RNA polymerase II activity at high temperatures, and have been implicated in the regulation of enzyme activity . Interaction experiments support a direct association between the Rpb6 and Rpb4 subunits, indicating that Rpb6 is one point of contact between the Rpb4/Rpb7 subcomplex and RNA polymerase II . The association of Rpb4/Rpb7 with Rpb6 suggests that analogous subunits of each RNA polymerase impart class-specific functions through a conserved core subunit.

Mol Cell Biol, 2003 May, 23(9), 3141 - 51
The Hsp70 and TRiC/CCT chaperone systems cooperate in vivo to assemble the von Hippel-Lindau tumor suppressor complex; Melville MW et al.; The degree of cooperation and redundancy between different chaperones is an important problem in understanding how proteins fold in the cell . Here we use the yeast Saccharomyces cerevisiae as a model system to examine in vivo the chaperone requirements for assembly of the von Hippel-Lindau protein (VHL)-elongin BC (VBC) tumor suppressor complex . VHL and elongin BC expressed in yeast assembled into a correctly folded VBC complex that resembles the complex from mammalian cells . Unassembled VHL did not fold and remained associated with the cytosolic chaperones Hsp70 and TRiC/CCT, in agreement with results from mammalian cells . Analysis of the folding reaction in yeast strains carrying conditional chaperone mutants indicates that incorporation of VHL into VBC requires both functional TRiC and Hsp70 . VBC assembly was defective in cells carrying either a temperature-sensitive ssa1 gene as their sole source of cytosolic Hsp70/SSA function or a temperature-sensitive mutation in CCT4, a subunit of the TRiC/CCT complex . Analysis of the VHL-chaperone interactions in these strains revealed that the cct4ts mutation decreased binding to TRiC but did not affect the interaction with Hsp70 . In contrast, loss of Hsp70 function disrupted the interaction of VHL with both Hsp70 and TRiC . We conclude that, in vivo, folding of some polypeptides requires the cooperation of Hsp70 and TRiC and that Hsp70 acts to promote substrate binding to TRiC.

Mol Cell Biol, 2003 May, 23(9), 3126 - 40
Cell cycle activation of the Swi6p transcription factor is linked to nucleocytoplasmic shuttling; Queralt E et al.; The control of the subcellular localization of cell cycle regulators has emerged as a crucial mechanism in the regulation of cell division . In the present work, we have characterized the function of the karyopherin Msn5p in the control of the cell cycle of Saccharomyces cerevisiae . Phenotypic analysis of the msn5 mutant revealed an increase in cell size and a functional interaction between Msn5p and the cell cycle transcription factor SBF (composed of the Swi4p and Swi6p proteins), indicating that Msn5p is involved in Start control . In fact, we have shown that the level of Cln2p protein is drastically reduced in an msn5 mutant . The effect on CLN2 expression is mediated at a transcriptional level, Msn5p being necessary for proper SBF-dependent transcription . On the contrary, loss of MSN5 has no effect on the closely related transcription factor MBF (composed of the Mbp1p and Swi6p proteins) . Regulation of SBF by Msn5p is exerted by control of the localization of the regulatory subunit Swi6p . Swi6p shuttles between the nucleus and the cytoplasm during the cell cycle, and we have found that Msn5p is required for Swi6p export from the nucleus during the G(2)-M phase . What is more important, we have demonstrated that export of Swi6p to the cytoplasm is required for SBF activity, providing evidence for a functional switch of Swi6p linked to its nucleocytoplasmic shuttling during the cell cycle.

J Biol Chem, 2003 Jun 20, 278(25), 22574 - 7 Epub 2003 Apr 14.
REX1, a novel gene required for DNA repair; Cenkci B et al.; Nucleotide excision repair is a major pathway for repairing UV light-induced DNA damage in most organisms . Using insertional mutagenesis, we isolated a UV-sensitive mutant of Chlamydomonas reinhardtii that is blocked in the excision of cyclobutane pyrimidine dimers . The mutant is also sensitive to the alkylating agent, methyl methanesulphonate . We have cloned REX1, a novel gene that rescues the mutant . The gene is unusual in a eukaryotic organism in that it is predicted to encode two different proteins, a small protein (8.9 kDa) and a larger protein (31.8 kDa) . Neither protein is homologous to known DNA repair proteins . Partial complementation is achieved with subclones of the gene encoding only the 8.9-kDa protein . The 8.9-kDa protein has homologues in many organisms including Saccharomyces cerevisiae, Arabidopsis, and humans . The 31.8-kDa protein appears to be less conserved . These findings may be of general importance for DNA repair in other organisms.

J Biol Chem, 2003 Jun 27, 278(26), 23410 - 7 Epub 2003 Apr 14.
The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 protein; Kantake N et al.; Replication protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination . Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication . We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)) . This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11).ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPA.ssDNA complex . RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 protein.ssDNA nucleoprotein complex is assembled prior to introduction of the mutant RPA . However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 protein more slowly than wild-type RPA and, as a consequence, Rad51 protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11) . Rad52 protein can stimulate displacement of RPA(rfa1-t11) from ssDNA by Rad51 protein, but the rate of displacement remains slow compared with wild-type RPA . These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 protein and that RPA displacement by Rad51 protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation.

BMC Dev Biol . 2003 Apr 09;3(1):3.
IfkA, a presumptive eIF2 alpha kinase of Dictyostelium, is required for proper timing of aggregation and regulation of mound size; Fang R et al.; BACKGROUND: The transition from growth to development in Dictyostelium is initiated by amino acid starvation of growing amobae . In other eukaryotes, a key sensor of amino acid starvation and mediator of the resulting physiological responses is the GCN2 protein, an eIF2alpha kinase . GCN2 downregulates the initiation of translation of bulk mRNA and enhances translation of specific mRNAs by phosphorylating the translation initiation factor eIF2alpha . Two eIF2alpha kinases were identified in Dictyostelium and studied herein . RESULTS: Neither of the eIF2alpha kinases appeared to be involved in sensing amino acid starvation to initiate development . However, one of the kinases, IfkA, was shown to phosphorylate eIF2alpha from 1 to 7 hours after the onset of development, resulting in a shift from polysomes to free ribosomes for bulk mRNA . In the absence of the eIF2alpha phosphorylation, ifkA null cells aggregated earlier than normal and formed mounds and ultimately fruiting bodies that were larger than normal . The early aggregation phenotype in ifkA null cells reflected an apparent, earlier than normal establishment of the cAMP pulsing system . The large mound phenotype resulted from a reduced extracellular level of Countin, a component of the counting factor that regulates mound size . In wild type cells, phosphorylation of eIF2alpha by IfkA resulted in a specific stabilization and enhanced translational efficiency of countin mRNA even though reduced translation resulted for bulk mRNA . CONCLUSIONS: IfkA is an eIF2alpha kinase of Dictyostelium that normally phosphorylates eIF2alpha from 1 to 7 hours after the onset of development, or during the preaggregation phase . This results in an overall reduction in the initiation of protein synthesis during this time frame and a concomitant reduction in the number of ribosomes associated with most mRNAs . For some mRNAs, however, initiation of protein synthesis is enhanced or stabilized under the conditions of increased eIF2alpha phosphorylation . This includes countin mRNA.

Cell Cycle, 2003 Mar-Apr, 2(2), 105 - 11
Vascular smooth muscle polyploidization--from mitotic checkpoints to hypertension; Hixon ML et al.; Aging and hypertension are accompanied by an increase in mass and rigidity of arterial walls . At capacitance arteries, the enlargement and stiffness of the medial smooth muscle layer promote systolic hypertension and contribute to left ventricular hypertrophy and cardiovascular morbidity . Morphological studies have demonstrated that vascular smooth muscle cell (VSMC) hypertrophy, with minimal hyperplasia, causes the enlargement of vascular smooth muscle at capacitance arteries, and that VSMC hypertrophy is strongly associated with VSMC polyploidization . Recent studies demonstrate that hypertrophic signals, such as those elicited by Angiotensin II, abrogate the mechanisms of control of M phase in VSMC and induce cell cycle re-entry and polyploidization . These polyploid VSMC have a lower replicative rate, but a higher mass, protein content and matrix production than their diploid counterparts . Both, the protein kinase Aktl and the cyclin kinase-associated protein CKsl, have been implicated in the mechanism of VSMC polyploidization during hypertension . Here, we review the function of these proteins at the mitotic spindle cell cycle checkpoint and their role in the process of VSMC polyploidization.

J Biol Chem, 2003 Jun 20, 278(25), 22453 - 9 Epub 2003 Apr 14.
A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants; Venema K et al.; In this study we have identified the first plant K+/H+ exchanger, LeNHX2 from tomato (Lycopersicon esculentum Mill . cv . Moneymaker), which is a member of the intracellular NHX exchanger protein family . The LeNHX2 protein, belonging to a subfamily of plant NHX proteins closely related to the yeast NHX1 protein, is abundant in roots and stems and is induced in leaves by short term salt or abscisic acid treatment . LeNHX2 complements the salt- and hygromycin-sensitive phenotype caused by NHX1 gene disruption in yeast, but affects accumulation of K+ and not Na+ in intracellular compartments . The LeNHX2 protein co-localizes with Prevacuolar and Golgi markers in a linear sucrose gradient in both yeast and plants . A histidine-tagged version of this protein could be purified and was shown to catalyze K+/H+ exchange but only minor Na+/H+ exchange in vitro . These data indicate that proper functioning of the endomembrane system relies on the regulation of K+ and H+ homeostasis by K+/H+ exchangers.

Genome Res, 2003 May, 13(5), 896 - 904 Epub 2003 Apr 14.
Predicting gene function from patterns of annotation; King OD et al.; The Gene Ontology (GO) Consortium has produced a controlled vocabulary for annotation of gene function that is used in many organism-specific gene annotation databases . This allows the prediction of gene function based on patterns of annotation . For example, if annotations for two attributes tend to occur together in a database, then a gene holding one attribute is likely to hold the other as well . We modeled the relationships among GO attributes with decision trees and Bayesian networks, using the annotations in the Saccharomyces Genome Database (SGD) and in FlyBase as training data . We tested the models using cross-validation, and we manually assessed 100 gene-attribute associations that were predicted by the models but that were not present in the SGD or FlyBase databases . Of the 100 manually assessed associations, 41 were judged to be true, and another 42 were judged to be plausible.

Eur J Cardiothorac Surg, 2003 Apr, 23(4), 497 - 502
Improvements in lung compliance after pulmonary transplantation: correlation with interleukin 8 expression; Rao JN et al.; OBJECTIVE: Previous studies have suggested reductions in lung reperfusion injury when initial reperfusion is undertaken with the addition of pharmacological modulators . We investigated three pharmacological agents in a porcine model of left single lung transplantation to determine the effect on lung compliance and its relationship with the expression of the cytokine, interleukin-8 (IL-8) . METHODS: Donor lungs were preserved with modified Euro-Collins for a mean ischaemic time of 18.6 h . Pulmonary venous oxygenation, lung compliance and IL-8 expression were assessed over a 12-h period . Group A (n=5) was a control group with no interventions added, Group B was reperfused with the addition of intravenous inositol hexakisphosphate (InSP6) (0.02 mg/kg per min), Group C received the nitric oxide donor, 3-morpholinosydnonimine (SIN-1) (0.02 mg/kg per min) and Group D received intravenous Pentoxifylline (2 mg/kg per h) . All interventions were administered at a pulmonary artery pressure of 20 mmHg . RESULTS: Group D yielded the best oxygenation (P=0.0041) while Groups B and C were similar . All were superior to Group A (P<0.001) . Lung compliance was significantly improved in Groups B, C and D compared to group A . In Group D, the greatest improvements in lung compliance were observed (P<0.0001) . Similar observations were seen with regard to pulmonary vascular resistance . IL-8 expression was delayed until after 30 min of reperfusion in Group D, but was evident after 10 min in all the other groups . This correlates with the compliance and oxygenation data . CONCLUSIONS: The addition of InSP6 or SIN-1 at reperfusion significantly attenuates reperfusion injury compared with controls and improves lung compliance . The unique comparison with Pentoxifylline afforded by this study indicates that at the doses studied Pentoxifylline appears to be superior, correlating with a greater inhibition of IL-8 expression.

Mol Microbiol, 2003 May, 48(3), 823 - 32
Opi1p, Ume6p and Sin3p control expression from the promoter of the INO2 regulatory gene via a novel regulatory cascade; Kaadige MR et al.; The INO2 gene of Saccharomyces cerevisiae is required for expression of most of the phospholipid biosynthetic genes . INO2 expression is regulated by a complex cascade that includes autoregulation, Opi1p-mediated repression and Ume6p-mediated activation . To screen for mutants with altered INO2 expression directly, we constructed an INO2-HIS3 reporter that provides a plate assay for INO2 promoter activity . This reporter was used to isolate mutants (dim1) that fail to repress expression of the INO2 gene in an otherwise wild-type strain . The dim1 mutants contain mutations in the OPI1 gene . To define further the mechanism for Ume6p regulation of INO2 expression, we isolated suppressors (rum1, 2, 3) of the ume6Delta mutation that overexpress the INO2-HIS3 gene . Two of the rum mutant groups contain mutations in the OPI1 and SIN3 genes showing that opi1 and sin3 mutations are epistatic to the ume6Delta mutation . These results are surprising given that Ume6p, Sin3p and Rpd3p are known to form a complex that represses the expression of a diverse set of yeast genes . This prompted us to examine the effect of sin3Delta and rpd3Delta mutants on INO2-cat expression . Surprisingly, the sin3Delta allele overexpressed INO2-cat, whereas the rpd3Delta mutant had no effect . We also show that the UME6 gene does not affect the expression of an OPI1-cat reporter . This suggests that Ume6p does not regulate INO2 expression indirectly by regulating OPI1 expression.

Genes Cells, 2003 May, 8(5), 493 - 500
AKRL1 and AKRL2 activate the JNK pathway; Harada T et al.; BACKGROUND: c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, is activated by specific cytokines and various environmental stresses . MKK4 and MKK7 are shown to be direct activators of JNK . Although several upstream components of the JNK pathway, including members of the MAPKKK family have been described, the components lying between the receptors or sensors and JNK have not been fully characterized . RESULTS: We have identified AKRL1 and AKRL2 (Akr1p-like 1 and 2) as novel activators of the JNK pathway . AKRL1 and AKRL2 proteins have a considerable sequence similarity to Akr1p, a protein essential for endocytosis in Saccharomyces cerevisiae . Expression of AKRL1 or AKRL2 activates JNK and its activators MKK4 and MKK7 . This AKRL1/2-induced JNK activation is significantly suppressed by the expression of a kinase-negative mutant of TAK1, a member of the MAPKKK family . AKRL1 and AKRL2 localize to the Golgi . Both the N-terminal half and the C-terminal transmembrane domain of AKRL1/2 are required for the JNK activation . The C-terminal transmembrane domain of AKRL1/2 is required for localization to the Golgi . CONCLUSION: AKRL1 and AKRL2 are localized to Golgi and the novel activators of the JNK pathway.

Genes Cells, 2003 May, 8(5), 413 - 21
Mammalian Mcm2/4/6/7 complex forms a toroidal structure; Yabuta N et al.; BACKGROUND: The Mcm proteins are a family of six homologous proteins (Mcm2-7) that play an important role in DNA replication . They form Mcm4/6/7 and Mcm2/4/6/7 complexes, but their structures are not known . RESULTS: We found that the human Mcm2/4/6/7 tetramer forms a toroidal structure, with a central cavity about 3-4 nm in diameter . Observations were made using electron microscopy, employing the image analysis of single particles . The most predominant averaged image displayed a toroid harbouring four bulges forming corners, one of which was larger than the others . This structure was very similar to the mouse Mcm2/4/6/7 tetramer that was independently prepared and analysed by electron microscopy . These toroidal structures are distinct from that of the Mcm4/6/7 hexamer, which was also examined by electron microscopy . GST(glutathione S-transferase)-pull down and two hybrid experiments suggest that a putative Mcm6-Mcm6 hinge contributes to the formation of the Mcm7/4/6/6/4/7 heterohexamer . CONCLUSIONS: The Mcm2/4/6/7 tetramer forms a toroidal structure that is distinct from that of the Mcm4/6/7 hexamer in size and shape.

Biochemistry (Mosc), 2003 Feb, 68(2), 247 - 51
Induced absorption band of holotransketolase and its interpretation; Kovina MV et al.; It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and thiamine diphosphate (ThDP) is accompanied by appearance, in both the absorption and CD spectra, of a new band . Binding and subsequent conversion of transketolase substrates bring about changes in the intensity of this band . The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of the substrates during the transketolase reaction and thus to kinetically characterize its individual steps . As regards the new absorption band induced by ThDP binding, its nature, until recently, remained unknown . The reason for its appearance was considered to be either the formation of a charge transfer complex between ThDP and tryptophan (phenylalanine) residue or stacking interaction between the residues of aromatic amino acids . They are thought to be brought together as a result of conformational changes of the apoenzyme during its interaction with the coenzyme . However none of these hypotheses had been substantiated experimentally . According to our hypothesis, the induced absorption band is that of the imino form of ThDP resulting from three contributing features of the ThDP binding site of transketolase: the relative hydrophobicity of this site, hydrogen bonding of the N1;-atom of the ThDP aminopyrimidine ring to Glu418, and base stacking interactions between the aminopyrimidine ring of ThDP and Phe445.

Biochemistry, 2003 Apr 22, 42(15), 4311 - 20
New insights into the mechanisms of protein palmitoylation; Linder ME et al.; Since its discovery more than 30 years ago, protein palmitoylation has been shown to have a role in protein-membrane interactions, protein trafficking, and enzyme activity . Until recently, however, the molecular machinery that carries out reversible palmitoylation of proteins has been elusive . In fact, both enzymatic and nonenzymatic S-acylation reaction mechanisms have been proposed . Recent reports of protein palmitoyltransferases in Saccharomyces cerevisiae and Drosophila provide the first glimpse of enzymes that carry out protein palmitoylation . Equally important is the mechanism of depalmitoylation . Two major classes of protein palmitoylthioesterases have been described . One family is lysosomal and is involved in protein degradation . The second is cytosolic and removes palmitoyl moieties preferentially from proteins associated with membranes . This review discusses recent advances in the understanding of mechanisms of addition of palmitate to proteins and removal of palmitate from proteins.

Plant Physiol, 2003 Apr, 131(4), 1737 - 47
Identification, expression, and import of components 17 and 23 of the inner mitochondrial membrane translocase from Arabidopsis; Murcha MW et al.; Characterization of components 17 and 23 of the inner mitochondrial membrane translocase (TIM17:23) from Arabidopsis indicated that there were three genes present for TIM17 and TIM23 and two for TIM44 . AtTIM17 differed from the yeast (Saccharomyces cerevisiae) and mammalian homologs in that two genes encoded proteins that were longer and one gene encoded a shorter protein . All Arabidopsis TIM23 predicted proteins appeared to lack the first 34 amino acids compared with yeast TIM23 . All AtTIM17 and AtTIM23 genes were expressed but displayed different tissue and developmental profiles . Complementation of deletion mutants in yeast indicated that for AtTIM17, the extension at the C terminus not present in yeast had to be removed to achieve complementation, whereas for TIM23, a preprotein and amino acid transporter domain had to be present for complementation . Import assays with AtTIM17 and AtTIM23 indicated that they both contained internal signals for integration into the inner mitochondrial membrane in a membrane potential-dependent manner . The C terminus of imported AtTIM17-2 was susceptible to degradation by externally added protease with intact mitochondria . Removal of the 85 C-terminal amino acids resulted in import and full protection of the truncated protein . This suggests that the novel extension at the C terminus of AtTIM17-2 links the outer and inner membrane in a manner analogous to yeast TIM23.

Plant Physiol, 2003 Apr, 131(4), 1648 - 60
New insight into Phaeodactylum tricornutum fatty acid metabolism . Cloning and functional characterization of plastidial and microsomal delta12-fatty acid desaturases; Domergue F et al.; In contrast to 16:3 plants like rapeseed (Brassica napus), which contain alpha-linolenic acid (18:3(Delta9,12,15)) and hexadecatrienoic acid (16:3(Delta7,10,13)) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5(Delta5,8,11,14,17)) and a different isomer of hexadecatrienoic acid (16:3(Delta6,9,12)) . In this report, we describe the characterization of two cDNAs having sequence homology to Delta12-fatty acid desaturases from higher plants . These cDNAs were shown to code for a microsomal and a plastidial Delta12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively . Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6) . The microsomal desaturases were similarly specific for oleic acid (18:1(Delta9)), suggesting that PtFAD2 is involved in the biosynthesis of EPA . In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed . Although the rapeseed plastidial desaturase showed high activity toward the omega9-fatty acids 18:1(Delta9) and 16:1(Delta7), in line with the fatty acid composition of rapeseed leaves, the enzyme of P . tricornutum was highly specific for 16:1(Delta9) . Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P . tricornutum (16:3(Delta6,9,12)) is of plastidial origin.

Plant Physiol, 2003 Apr, 131(4), 1566 - 75
Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues; Gao Z et al.; The acyclic polyol sorbitol is a primary photosynthetic product and the principal photosynthetic transport substance in many economically important members of the family Rosaceace (e.g . almond {Prunus dulcis (P . Mill.) D.A . Webber}, apple {Malus pumila P . Mill.}, cherry {Prunus spp.}, peach {Prunus persica L . Batsch}, and pear {Pyrus communis}) . To understand key steps in long-distance transport and particularly partitioning and accumulation of sorbitol in sink tissues, we have cloned two sorbitol transporter genes (PcSOT1 and PcSOT2) from sour cherry (Prunus cerasus) fruit tissues that accumulate large quantities of sorbitol . Sorbitol uptake activities and other characteristics were measured by heterologous expression of PcSOT1 and PcSOT2 in yeast (Saccharomyces cerevisiae) . Both genes encode proton-dependent, sorbitol-specific transporters with similar affinities (K(m) sorbitol of 0.81 mM for PcSOT1 and 0.64 mM for PcSOT2) . Analyses of gene expression of these transporters, however, suggest different roles during leaf and fruit development . PcSOT1 is expressed throughout fruit development, but especially when growth and sorbitol accumulation rates are highest . In leaves, PcSOT1 expression is highest in young, expanding tissues, but substantially less in mature leaves . In contrast, PcSOT2 is mainly expressed only early in fruit development and not in leaves . Compositional analyses suggest that transport mediated by PcSOT1 and PcSOT2 plays a major role in sorbitol and dry matter accumulation in sour cherry fruits . Presence of these transporters and the high fruit sorbitol concentrations suggest that there is an apoplastic step during phloem unloading and accumulation in these sink tissues . Expression of PcSOT1 in young leaves before completion of the transition from sink to source is further evidence for a role in determining sink activity.

J Biol Chem, 2003 Jun 27, 278(26), 24125 - 31 Epub 2003 Apr 13.
Importance of the conserved aromatic residues in the scorpion alpha-like toxin BmK M1: the hydrophobic surface region revisited; Sun YM et al.; About one-third of the amino acid residues conserved in all scorpion long chain Na+ channel toxins are aromatic residues, some of which constitute the so-called "conserved hydrophobic surface." At present, in-depth structure-function studies of these aromatic residues using site-directed mutagenesis are still rare . In this study, an effective yeast expression system was used to study the role of seven conserved aromatic residues (Tyr5, Tyr14, Tyr21, Tyr35, Trp38, Tyr42, and Trp47) from the scorpion toxin BmK M1 . Using site-directed mutagenesis, all of these aromatic residues were individually substituted with Gly in association with a more conservative substitution of Phe for Tyr5, Tyr14, Tyr35, or Trp47 . The mutants, which were expressed in Saccharomyces cerevisiae S-78 cells, were then subjected to a bioassay in mice, electrophysiological characterization on cloned Na+ channels (Nav1.5), and CD analysis . Our results show an eye-catching correlation between the LD50 values in mice and the EC50 values on Nav1.5 channels in oocytes, indicating large mutant-dependent differences that emphasize important specific roles for the conserved aromatic residues in BmK M1 . The aromatic side chains of the Tyr5, Tyr35, and Trp47 cluster protruding from the three-stranded beta-sheet seem to be essential for the structure and function of the toxin . Trp38 and Tyr42 (located in the beta2-sheet and in the loop between the beta2- and beta3-sheets, respectively) are most likely involved in the pharmacological function of the toxin.

Receptors Channels, 2002, 8(5-6), 297 - 308
Functional assays for identifying ligands at orphan G protein-coupled receptors; Szekeres PG; The superfamily of G protein-coupled receptors (GPCRs; 7TMs) is one of the largest families of genes identified in humans, and has a proven history of being an excellent source of drug targets . The near completion of the human genome sequencing project has allowed the identification of a plethora of sequences encoding "orphan" GPCRs--putative receptors whose natural ligand(s) remain to be discovered . In many cases, the level of sequence homology with known receptors is insufficient to be able to predict the natural ligand for these orphan receptors, although it is usually possible to determine the likely nature of the cognate ligand e.g . peptide, lipid, nucleotide etc . Deorphanizing these novel GPCRs and evaluating their biological function has become a major target of many of the major pharmaceutical companies as well as several academic groups . Since 1995 more than 50 ligands for orphan GPCRs have been discovered by using the orphan receptor as a biosensor and screening candidate compounds looking for a biological response (the so-called "reverse pharmacology" approach) . Identification of the natural ligands for these receptors marks the beginning of the process of understanding the biology of these newly discovered signalling systems and the development of novel therapies targeted at them . This article will focus on the functional assays which have been used to discover ligands for orphan GPCRs.

BMC Biochem . 2003 Mar 18;4(1):3.
Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system; Schulze WX et al.; BACKGROUND: The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4 . The carriers differ in their kinetic properties . Many transport proteins are known to exist as oligomers . The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact . RESULTS: Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants . All three fusion genes are co-expressed in companion cells . Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system . Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers . The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters . Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters . CONCLUSIONS: The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits . Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers . The biochemical approaches are required to confirm the in planta interaction.

BMC Bioinformatics . 2003 Mar 27;4(1):11.
PreBIND and Textomy--mining the biomedical literature for protein-protein interactions using a support vector machine; Donaldson I et al.; BACKGROUND: The majority of experimentally verified molecular interaction and biological pathway data are present in the unstructured text of biomedical journal articles where they are inaccessible to computational methods . The Biomolecular interaction network database (BIND) seeks to capture these data in a machine-readable format . We hypothesized that the formidable task-size of backfilling the database could be reduced by using Support Vector Machine technology to first locate interaction information in the literature . We present an information extraction system that was designed to locate protein-protein interaction data in the literature and present these data to curators and the public for review and entry into BIND . RESULTS: Cross-validation estimated the support vector machine's test-set precision, accuracy and recall for classifying abstracts describing interaction information was 92%, 90% and 92% respectively . We estimated that the system would be able to recall up to 60% of all non-high throughput interactions present in another yeast-protein interaction database . Finally, this system was applied to a real-world curation problem and its use was found to reduce the task duration by 70% thus saving 176 days . CONCLUSIONS: Machine learning methods are useful as tools to direct interaction and pathway database back-filling; however, this potential can only be realized if these techniques are coupled with human review and entry into a factual database such as BIND . The PreBIND system described here is available to the public at Current capabilities allow searching for human, mouse and yeast protein-interaction information.

Phys Rev E Stat Nonlin Soft Matter Phys . 2003 Mar;67(3 Pt 1):031902 . Epub 2003 Mar 11.
Iterative signature algorithm for the analysis of large-scale gene expression data; Bergmann S et al.; We present an approach for the analysis of genome-wide expression data . Our method is designed to overcome the limitations of traditional techniques, when applied to large-scale data . Rather than alloting each gene to a single cluster, we assign both genes and conditions to context-dependent and potentially overlapping transcription modules . We provide a rigorous definition of a transcription module as the object to be retrieved from the expression data . An efficient algorithm, which searches for the modules encoded in the data by iteratively refining sets of genes and conditions until they match this definition, is established . Each iteration involves a linear map, induced by the normalized expression matrix, followed by the application of a threshold function . We argue that our method is in fact a generalization of singular value decomposition, which corresponds to the special case where no threshold is applied . We show analytically that for noisy expression data our approach leads to better classification due to the implementation of the threshold . This result is confirmed by numerical analyses based on in silico expression data . We discuss briefly results obtained by applying our algorithm to expression data from the yeast Saccharomyces cerevisiae.

J Immunol, 2003 Apr 15, 170(8), 4139 - 47
Communication between NF-kappa B and Sp1 controls histone acetylation within the proximal promoter of the monocyte chemoattractant protein 1 gene; Boekhoudt GH et al.; The induction of the monocyte chemoattractant protein 1 gene (MCP-1) by TNF occurs through an NF-kappaB-dependent distal regulatory region and an Sp1-dependent proximal regulatory region that are separated by 2.2 kb of sequence . To investigate how these regions coordinate activation of MCP-1 in response to TNF, experiments were performed to examine the role of coactivators, changes in local chromatin structure, and the acetylation of histones at the MCP-1 regulatory regions . An E1a-sensitive coactivator was found to be required for expression . In vivo nuclease sensitivity assays identified changes in response to TNF at both the proximal and distal regions that were dependent on the p65 subunit of NF-kappaB and Sp1 . Chromatin immunoprecipitations used to analyze factor assembly and histone acetylation at the distal and proximal regions showed that Sp1 binding to and histone acetylation of the proximal region was dependent on NF-kappaB p65 . Conversely, Sp1 assembly at the proximal region was required for p65 binding to and acetylation of the distal region, suggesting communication between the two regions during gene activation . These data and the NF-kappaB p65-dependent histone acetylation of a middle region sequence suggest a potential order for the assembly, acetylation and accessibility of the MCP-1 regulatory regions in response to TNF.

FEBS Lett, 2003 Apr 10, 540(1-3), 86 - 90
The apoptosis-associated protein BNIPL interacts with two cell proliferation-related proteins, MIF and GFER; Shen L et al.; Bcl-2/adenovirus E1B 19 kDa interacting protein 2-like, BNIP-2-like (BNIPL) is a recently cloned and characterized apoptosis-associated protein that shares 72% homology with BNIP-2 . It is highly expressed in human placenta and lung . A yeast two-hybrid system was used to obtain two BNIPL-interacting proteins, MIF (macrophage migration inhibitory factor) and GFER (growth factor erv1 (Saccharomyces cerevisiae)-like) . The interactions were confirmed by glutathione S-transferase pull-down assay in vitro and co-immunoprecipitation assay in vivo . Colony formation assay and cell proliferation test suggest that overexpression of BNIPL could inhibit the growth of BEL-7402 cells . These findings suggest that BNIPL may physically bind to cell proliferation-related proteins, MIF and GFER .

Mol Biol Cell, 2003 Apr, 14(4), 1610 - 23
Vps51p mediates the association of the GARP (Vps52/53/54) complex with the late Golgi t-SNARE Tlg1p; Conibear E et al.; Multisubunit tethering complexes may contribute to the specificity of membrane fusion events by linking transport vesicles to their target membrane in an initial recognition event that promotes SNARE assembly . However, the interactions that link tethering factors to the other components of the vesicle fusion machinery are still largely unknown . We have previously identified three subunits of a Golgi-localized complex (the Vps52/53/54 complex) that is required for retrograde transport to the late Golgi . This complex interacts with a Rab and a SNARE protein found at the late Golgi and is related to two other multisubunit tethering complexes: the COG complex and the exocyst . Here we show that the Vps52/53/54 complex has an additional subunit, Vps51p . All four members of this tetrameric GARP (Golgi-associated retrograde protein) complex are required for two distinct retrograde transport pathways, from both early and late endosomes, back to the TGN . vps51 mutants exhibit a distinct phenotype suggestive of a regulatory role . Indeed, we find that Vps51p mediates the interaction between Vps52/53/54 and the t-SNARE Tlg1p . The binding of this small, coiled-coil protein to the conserved N-terminal domain of the t-SNARE therefore provides a crucial link between components of the tethering and the fusion machinery.

Mol Biol Cell, 2003 Apr, 14(4), 1583 - 96
Composition and dynamics of human mitochondrial nucleoids; Garrido N et al.; The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied in Saccharomyces cerevisiae . Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA . However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm . In this article we have used immunocytochemistry and biochemical isolation procedures to characterize the composition of human mitochondrial nucleoids . The results show that both the mitochondrial transcription factor TFAM and mitochondrial single-stranded DNA-binding protein colocalize with Twinkle in intramitochondrial foci defined as nucleoids by the specific incorporation of bromodeoxyuridine . Furthermore, mtDNA polymerase POLG and various other as yet unidentified proteins copurify with mtDNA nucleoids using a biochemical isolation procedure, as does TFAM . The results demonstrated that mtDNA in mammalian cells is organized in discrete protein-rich structures within the mitochondrial network . In vivo time-lapse imaging of nucleoids show they are dynamic structures able to divide and redistribute in the mitochondrial network and suggest that nucleoids are the mitochondrial units of inheritance . Nucleoids did not colocalize with dynamin-related protein 1, Drp1, a protein of the mitochondrial fission machinery.

Mol Biol Cell, 2003 Apr, 14(4), 1501 - 16
Synthetic lethal analysis implicates Ste20p, a p21-activated potein kinase, in polarisome activation; Goehring AS et al.; The p21-activated kinases Ste20p and Cla4p carry out undefined functions that are essential for viability during budding in Saccharomyces cerevisiae . To gain insight into the roles of Ste20p, we have used a synthetic lethal mutant screen to identify additional genes that are required in the absence of Cla4p . Altogether, we identified 65 genes, including genes with roles in cell polarity, mitosis, and cell wall maintenance . Herein, we focus on a set that defines a function carried out by Bni1p and several of its interacting proteins . We found that Bni1p and a group of proteins that complex with Bni1p (Bud6p, Spa2p, and Pea2p) are essential in a cla4delta mutant background . Bni1p, Bud6p, Spa2, and Pea2p are members of a group of polarity determining proteins referred to as the polarisome . Loss of polarisome proteins from a cla4delta strain causes cells to form elongated buds that have mislocalized septin rings . In contrast, other proteins that interact with or functionally associate with Bni1p and have roles in nuclear migration and cytokinesis, including Num1p and Hof1p, are not essential in the absence of Cla4p . Finally, we have found that Bni1p is phosphorylated in vivo, and a substantial portion of this phosphorylation is dependent on STE20 . Together, these results suggest that one function of Ste20p may be to activate the polarisome complex by phosphorylation of Bni1p.

Eur J Clin Pharmacol, 2003 Jun, 59(2), 103 - 9 Epub 2003 Apr 09.
Bioactivation of cyclophosphamide: the role of polymorphic CYP2C enzymes; Griskevicius L et al.; Several-fold differences have been observed among patients in the biotransformation of cyclophosphamide . The aim of this study was to investigate the contribution of CYP2C9 and CYP2C19 and their polymorphisms to the variability of cyclophosphamide activation . The formation of 4-hydroxycyclophosphamide was studied in microsomes from a total of 32 different genotyped human livers, as well as in yeast microsomes expressing different genetic variants of CYP2C9 and CYP2C19 . The kinetic data obtained in the yeast system revealed that the intrinsic clearance (V(max)/K(m)) of cyclophosphamide by CYP2C9.2 and CYP2C9.3 samples was approximately threefold lower than that by CYP2C9.1 . However, in liver microsomes, there were no statistically significant differences in the intrinsic clearance of 4-hydroxycyclophosphamide formation between the group of seven CYP2C9*1/*1 livers and the remaining nine with one or two variant CYP2C9 alleles ( P>0.7) . We found a statistically significant correlation ( r(s)=0.65, P=0.003) between 4-hydroxylation of cyclophosphamide and 5'-hydroxylation of R-omeprazole, a measure of CYP2C19 activity in human liver microsomes ( n=19) . No correlation was found between 4-hydroxylation of cyclophosphamide and the formation rate of hydroxycelecoxib, mainly catalysed by CYP2C9 ( r(s)=0.17, P=0.55, n=32) . In conclusion, based on the correlation with the formation of R-5'-hydroxyomeprazole, CYP2C19 may partly contribute to the bioactivation of cyclophosphamide in human liver microsomes, while the role of CYP2C9 appears minor.

J Biol Chem, 2003 Jun 20, 278(25), 22498 - 505 Epub 2003 Apr 08.
A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis; Thiel C et al.; Deficiency of GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii . The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities . An accumulation of Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol was observed in skin fibroblasts of the patient . Incubation of patient fibroblast extracts with Man1GlcNAc2-PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man1GlcNAc2-PP dolichol . Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog . Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2 . Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells . hALG2 was shown to act as an alpha1,3-mannosyltransferase . The resulting Manalpha1,3-ManGlcNAc2-PP dolichol is further elongated by a yet unknown alpha1,6-mannosyltransferase.

Eukaryot Cell, 2003 Apr, 2(2), 265 - 73
Isolation of mutations in the catalytic domain of the snf1 kinase that render its activity independent of the snf4 subunit; Leech A et al.; Activation of the Snf1 kinase requires at least two events, phosphorylation of the activation loop on threonine 210 and an Snf4-dependent process that is not completely defined . Snf4 directly interacts with a region of the regulatory domain of Snf1 that may otherwise act as an autoinhibitory domain . In order to gain insight into the regulation of Snf1 kinase by Snf4, deletions in the regulatory domain of the catalytic subunit were engineered and tested for their effect on Snf1 function in the absence of Snf4 . Deletion of residues 381 to 488 from the Snf1 protein resulted in a kinase that was activated by glucose limitation even in the absence of the Snf4 protein . A larger deletion (amino acids 381 to 608) encompassing virtually the entire regulatory domain resulted in complete inactivation of the Snf1 kinase even in the presence of Snf4 . A genetic screen for amino acid substitutions that conferred an Snf4-independent phenotype identified four point mutations in the Snf1 catalytic domain . One very conservative mutation, leucine 183 to isoleucine, conferred nearly wild-type levels of Snf1 kinase function in the absence of the Snf4 protein . Purified Snf1 kinase was inactive when isolated from snf4Delta cells, whereas the Snf1-L183I kinase exhibited significant activity in the absence of Snf4 . Our data support the idea that Snf1 kinase activity is constrained in cis by an autoinhibitory domain and that the Snf4-mediated activation of Snf1 can be bypassed by subtle conformational changes in the catalytic domain of the Snf1 kinase.

J Mol Biol, 2003 Apr 18, 328(1), 157 - 66
A novel heme and peroxide-dependent tryptophan-tyrosine cross-link in a mutant of cytochrome c peroxidase; Bhaskar B et al.; The crystal structure of a cytochrome c peroxidase mutant where the distal catalytic His52 is converted to Tyr reveals that the tyrosine side-chain forms a covalent bond with the indole ring nitrogen atom of Trp51 . We hypothesize that this novel bond results from peroxide activation by the heme iron followed by oxidation of Trp51 and Tyr52 . This hypothesis has been tested by incorporation of a redox-inactive Zn-protoporphyrin into the protein, and the resulting crystal structure shows the absence of a Trp51-Tyr52 cross-link . Instead, the Tyr52 side-chain orients away from the heme active-site pocket, which requires a substantial rearrangement of residues 72-80 and 134-144 . Additional experiments where heme-containing crystals of the mutant were treated with peroxide support our hypothesis that this novel Trp-Tyr cross-link is a peroxide-dependent process mediated by the heme iron .

Plant Mol Biol, 2003 Mar, 51(5), 731 - 43
Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus; Iturbe-Ormaetxe I et al.; Cloning of OSCs required for triterpene synthesis from legume species that are amenable to molecular genetics will provide tools to address the importance of triterpenes and their derivatives during normal plant growth and development and also in interactions with symbionts and pathogens . Here we report the cloning and characterization of a total of three triterpene synthases from the legume species Medicago truncatula and Lotus japonicus . These include a beta-amyrin synthase from M . truncatula (MtAMY1) and a mixed function triterpene synthase from Lotus japonicus (LjAMY2) . A partial cDNA predicted to encode a beta-amyrin synthase (LjAMY1) was also isolated from L . japonicus . The expression patterns of MtAMY1, LjAMY1 and LjAMY2 and of additional triterpene synthases previously characterised from M . truncatula and pea differ in different plant tissues and during nodulation, suggesting that these enzymes may have distinct roles in plant physiology and development.

Nat Cell Biol, 2003 May, 5(5), 486 - 8
Reconstitution of gamma-secretase activity; Edbauer D et al.; gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD) . Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity . Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro . Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast.

Theor Appl Genet, 2003 Jun, 107(1), 155 - 61 Epub 2003 Apr 01.
Characterization of a DRE-binding transcription factor from a halophyte Atriplex hortensis; Shen YG et al.; Environmental stresses, such as salinity, drought and cold, can induce the expression of a large amount of genes . Among these are many transcription factors that regulate the expression of downstream genes by specifically binding to cis-elements or forming transcriptional complexes with other proteins . In the present study, a DREB-like transcription factor gene, named AhDREB1, was isolated from a halophyte Atriplex hortensis . AhDREB1 encoded a protein containing a conserved EREBP/AP2 domain featuring the DREB family . In yeast one-hybrid analysis AhDREB1 protein was specifically bound to DRE elements and activated the expression of the reporter genes of HIS3 and LacZ . The AhDREB1 gene was expressed in roots, stems and leaves of A . hortensis . Salinity induced its expression in roots, but not in other organs . Overexpression of AhDREB1 in transgenic tobacco led to the accumulation of its putative downstream genes . The performance of the transgenic lines was also tested under stressed conditions and two lines were found to be stress-tolerant . These results suggest that the AhDREB1 protein functions as a DRE-binding transcription factor and play roles in the stress-tolerant response of A . hortensis.

Science, 2003 Apr 4, 300(5616), 139 - 41
Regulated cycling of mitochondrial Hsp70 at the protein import channel; Liu Q et al.; Hsp70 of the mitochondrial matrix (mtHsp70) provides a critical driving force for the import of proteins into mitochondria . Tim44, a peripheral inner-membrane protein, tethers it to the import channel . Here, regulated interactions were found to maximize occupancy of the active, adenosine 5'-triphosphate (ATP)-bound mtHsp70 at the channel through its intrinsic high affinity for Tim44, as well as through release of adenosine diphosphate (ADP)-bound mtHsp70 from Tim44 by the cofactor Mge1 . A model peptide substrate rapidly released mtHsp70 from Tim44, even in the absence of ATP hydrolysis . In vivo, the analogous interaction of translocating polypeptide would release mtHsp70 from the channel . Consistent with the ratchet model of translocation, subsequent hydrolysis of ATP would trap the polypeptide, driving import by preventing its movement back toward the cytosol.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4372 - 6 Epub 2003 Apr 03.
Assessing experimentally derived interactions in a small world; Goldberg DS et al.; Experimentally determined networks are susceptible to errors, yet important inferences can still be drawn from them . Many real networks have also been shown to have the small-world network properties of cohesive neighborhoods and short average distances between vertices . Although much analysis has been done on small-world networks, small-world properties have not previously been used to improve our understanding of individual edges in experimentally derived graphs . Here we focus on a small-world network derived from high-throughput (and error-prone) protein-protein interaction experiments . We exploit the neighborhood cohesiveness property of small-world networks to assess confidence for individual protein-protein interactions . By ascertaining how well each protein-protein interaction (edge) fits the pattern of a small-world network, we stratify even those edges with identical experimental evidence . This result promises to improve the quality of inference from protein-protein interaction networks in particular and small-world networks in general.

J Biol Chem, 2003 Jun 6, 278(23), 20457 - 60 Epub 2003 Apr 02.
Rapamycin-induced translational derepression of GCN4 mRNA involves a novel mechanism for activation of the eIF2 alpha kinase GCN2; Kubota H et al.; When starved for amino acids, Saccharomyces cerevisiae accumulates uncharged tRNAs to activate its sole eukaryotic initiation factor (eIF) 2alpha kinase GCN2 . Subsequent phosphorylation of eIF2alpha impedes general translation, but translationally derepresses the transcription factor GCN4, which induces expression of various biosynthetic genes to elicit general amino acid control response . By contrast, when supplied with enough nutrients, the yeast activates the target of rapamycin signaling pathway to stimulate translation initiation by facilitating the assembly of eIF4F . A cross-talk was suggested between the two pathways by rapamycin-induced translation of GCN4 mRNA . Here we show that rapamycin causes an increase in phosphorylated eIF2alpha to translationally derepress GCN4 . This increment is not observed in the cells expressing mammalian non-GCN2 eIF2alpha kinases in place of GCN2 . It is thus suggested that rapamycin does not inhibit dephosphorylation of eIF2alpha but rather activates the kinase GCN2 . This activation seems to require an interaction between the kinase and uncharged tRNAs, because rapamycin, similar to amino acid starvation, fails to induce eIF2alpha phosphorylation in the cells with GCN2 defective in tRNA binding . However, in contrast with amino acid starvation, rapamycin activates GCN2 without increasing the amount of uncharged tRNAs, but presumably by modifying the tRNA binding affinity of GCN2.

Pharmacol Res, 2003 May, 47(5), 401 - 7
Signaling and biological actions of sphingosine 1-phosphate; Hla T; Sphingosine 1-phosphate (S1P) is a product of sphingomyelin (SM) metabolism . It is present in most eukaryotic organisms; however, in higher vertebrates (bony fishes to mammals), it is utilized as a regulator of cell function by binding to extracellular receptors . Its role as a second messenger function was originally proposed; however, the unequivocal role of S1P as a second messenger in higher eukaryotes is lacking at present . As an extracellular mediator, S1P binds to a family of G-protein-coupled receptors named S1P(1-5), originally referred to as (EDG-1, 3, 5, 6 and 8) . These receptors couple to multiple G-proteins and regulate intracellular signaling pathways . Most tissues express one or more S1P receptor subtypes; therefore, S1P is known as a multifunctional physiologic mediator . Its precise physiologic roles in multiple organ systems are just beginning to be elucidated; however, at present, regulation of cardiogenesis, vascular system formation, oocyte survival and immune cell trafficking has been recently reported . Currently, a S1P receptor agonist, is undergoing clinical trials as an agent to fight organ transplant rejection . Since S1P is a fundamental multifunctional mediator, better understanding of the biology of S1P holds great promise to develop novel tools to control various diseases.

Biol Chem, 2003 Jan, 384(1), 105 - 16
Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster; Grebe M et al.; Mutants created by site-directed mutagenesis were used to elucidate the function of amino acids involved in ligand binding to ecdysteroid receptor (EcR) and heterodimer formation with ultraspiracle (USP) . The results demonstrate the importance of the C-terminal part of the D-domain and helix 12 of EcR for hormone binding . Some amino acids are involved either in ligand binding to EcR (E476, M504, D572, I617, N626) or ligand-dependent heterodimerization as determined by gel mobility shift assays (A612, L615, T619), while others are involved in both functions (K497, E648) . Some amino acids are suboptimal for ligand binding (L615, T619), but mediate ligand-dependent dimerization . We conclude that the enhanced regulatory potential by ligand-dependent modulation of dimerization in the wild type is achieved at the expense of optimal ligand binding . Mutation of amino acids (K497, E648) involved in the salt bridge between helix 4 and 12 impair ligand binding to EcR more severely than hormone binding to the heterodimer, indicating that to some extent heterodimerization compensates for the deleterious effect of certain mutations . Different effects of the same point mutations on ligand binding to EcR and EcR/USP (R511, A612, L615, I617, T619, N626) indicate that the ligand-binding pocket is modified by heterodimerization.

Biol Chem, 2003 Jan, 384(1), 39 - 49
Hepatitis B virus HBx peptide 116-138 and proteasome activator PA28 compete for binding to the proteasome alpha4/MC6 subunit; Stohwasser R et al.; PA28 is a modulator of the 20S proteasome . The PA28 binding sites on the 20S proteasome are still not well defined . Using yeast two-hybrid interaction assays and proteasome inactivation kinetics we provide evidence that the proteasome alpha4 subunit is one of the PA28 binding sites . This finding is supported by the observation that a hepatitis B virus X protein-derived polypeptide habouring the alpha4 proteasome subunit binding motif impairs the activation of 20S proteasomes by PA28.

Cancer Biol Ther, 2003 Jan-Feb, 2(1), 92 - 9
A potential therapeutic strategy to combat leukemia virus infection; Pan J et al.; To test the concept that a replication-competent retrovirus carrying a suicide gene could have potential utility in the control of the natural virus infection in mammalian species, we constructed derivatives of a feline leukemia virus (FeLV) that is commonly associated with leukemia-lymphomas in this species . The FeLV, Rickard strain, subgroup A (FRA) genome contained at the 3' end of the envgene, an insert of an internal ribosomal entry site (IRES) linked to cDNA sequence of either herpes simplex virus thymidine kinase (HSV-TK) or a truncated HSV-TK (HSV-ATK) or yeast cytosine deaminase (CD) . These constructs were transfected into feline fibroblast cells (H927) . The viruses produced were determined to be replication-competent . The stable propagation of the full-length transgene was, however, dependent on the size of the insert, IRES-CD being the smallest in size (1031 bp) exhibiting maximal stability for at least up to six months . The protein products of the transgenes could be detected, despite the appearance of deleted proviruses at late passages . The transduced cells were susceptible to cytotoxic killing when the appropriate prodrug, ganciclovir (GCV), acyclovir (ACV) or 5-fluorocytosine (5-FC) was added to the culture medium . H927 cells, infected with another subgroup of FeLV, namely, FeLV-B or FeLV-C, could be superinfected by the FRA-suicide gene viruses and thus, subjected to killing . Interestingly, at an early stage of infection by the parental FRA, H927 cells could also be reinfected by the same subgroup FRA constructs to induce the suicide effect . Among the three constructs, the vector with the CD gene was determined to be superior to others in terms of stability, therapeutic index and bystander effect in the cell culture test system . While the in vivo correlates of the therapeutic effect in the feline model remain to be determined, our results do encourage investigation of the same concept in the control of HTLV and, perhaps even, HIV infection in humans.

Cancer Biol Ther, 2003 Jan-Feb, 2(1), 22 - 9
Chromatin remodeling and cancer; Davis PK et al.; The fate of the cell relies on a delicate balance between gene expression and repression . The transcriptional control of the genome is maintained not only by transcription factors but also chromatin remodeling proteins . The purpose of the chromatin remodeling proteins is to alter the nucleosome architecture such that genes are exposed to or hidden from the transcriptional machinery . The nucleosome can be restructured by two mechanisms: 1 . the movement of nucleosomes along DMA which is carried out by ATP-dependent chromatin remodeling complexes; and 2 . the modification of core histones by histone acetyltransferases, deactylases, methyltrans-ferases, and kinases . Since these chromatin remodeling proteins play an essential role in transcriptional regulation, it is not surprising that they have been linked to cancer . In this review, we provide a general overview on chromatin remodeling and describe known genetic alterations of chromatin remodeling proteins in human cancers . We also discuss potential other, as yet unexplored strategies that cancers might take to manipulate the chromatin remodeling machinery.

Proc Natl Acad Sci U S A, 2003 Apr 15, 100(8), 4527 - 32 Epub 2003 Apr 02.
Conducting nanowires built by controlled self-assembly of amyloid fibers and selective metal deposition; Scheibel T et al.; Recent research in the field of nanometer-scale electronics has focused on the operating principles of small-scale devices and schemes to realize useful circuits . In contrast to established "top-down" fabrication techniques, molecular self-assembly is emerging as a "bottom-up" approach for fabricating nanostructured materials . Biological macromolecules, especially proteins, provide many valuable properties, but poor physical stability and poor electrical characteristics have prevented their direct use in electrical circuits . Here we describe the use of self-assembling amyloid protein fibers to construct nanowire elements . Self-assembly of a prion determinant from Saccharomyces cerevisiae, the N-terminal and middle region (NM) of Sup35p, produced 10-nm-wide protein fibers that were stable under a wide variety of harsh physical conditions . Their lengths could be roughly controlled by assembly conditions in the range of 60 nm to several hundred micrometers . A genetically modified NM variant that presents reactive, surface-accessible cysteine residues was used to covalently link NM fibers to colloidal gold particles . These fibers were placed across gold electrodes, and additional metal was deposited by highly specific chemical enhancement of the colloidal gold by reductive deposition of metallic silver and gold from salts . The resulting silver and gold wires were approximately 100 nm wide . These biotemplated metal wires demonstrated the conductive properties of a solid metal wire, such as low resistance and ohmic behavior . With such materials it should be possible to harness the extraordinary diversity and specificity of protein functions to nanoscale electrical circuitry.

Physiol Genomics, 2003 Jun 24, 14(1), 35 - 46
Clustering gene expression data using adaptive double self-organizing map; Ressom H et al.; This paper presents a novel clustering technique known as adaptive double self-organizing map (ADSOM) . ADSOM has a flexible topology and performs clustering and cluster visualization simultaneously, thereby requiring no a priori knowledge about the number of clusters . ADSOM is developed based on a recently introduced technique known as double self-organizing map (DSOM) . DSOM combines features of the popular self-organizing map (SOM) with two-dimensional position vectors, which serve as a visualization tool to decide how many clusters are needed . Although DSOM addresses the problem of identifying unknown number of clusters, its free parameters are difficult to control to guarantee correct results and convergence . ADSOM updates its free parameters during training, and it allows convergence of its position vectors to a fairly consistent number of clusters provided that its initial number of nodes is greater than the expected number of clusters . The number of clusters can be identified by visually counting the clusters formed by the position vectors after training . A novel index is introduced based on hierarchical clustering of the final locations of position vectors . The index allows automated detection of the number of clusters, thereby reducing human error that could be incurred from counting clusters visually . The reliance of ADSOM in identifying the number of clusters is proven by applying it to publicly available gene expression data from multiple biological systems such as yeast, human, and mouse . ADSOM's performance in detecting number of clusters is compared with a model-based clustering method.

J Biol Chem, 2003 Jun 13, 278(24), 22090 - 101 Epub 2003 Apr 01.
Cu(I)-dependent biogenesis of the galactose oxidase redox cofactor; Whittaker MM et al.; Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272 . Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen . We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II) . Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7) . In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h) . EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction . The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction . The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading . The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2 . Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation . These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.

J Biol Chem, 2003 Jun 13, 278(24), 21361 - 9 Epub 2003 Apr 02.
Characterization of the auto-inhibitory sequence within the N-terminal domain of importin alpha; Harreman MT et al.; Protein cargoes that contain a classic nuclear localization signal (NLS) are transported into the nucleus through binding to a heterodimeric receptor comprised of importin/karyopherin alpha and beta . An evolutionarily conserved auto-inhibitory sequence within the N-terminal importin beta binding (IBB) domain of importin alpha regulates NLS-cargo binding to the NLS binding pocket on importin alpha . In this study, we have used site-directed mutagenesis coupled with in vitro binding assays and in vivo analyses to investigate the intramolecular interaction of the N-terminal IBB domain and the NLS binding pocket of Saccharomyces cerevisiae importin alpha, Srp1p . We find that mutations within the IBB domain that decrease the binding affinity of the auto-inhibitory sequence for the NLS binding pocket impact importin alpha function in vivo . In addition, the severity of the in vivo phenotype is directly correlated to the reduction of auto-inhibition measured in vitro, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function . We exploit a conditional auto-inhibitory mutant, srp1-55, to study the in vivo functional overlap between the N-terminal IBB domain of importin alpha and other factors implicated in NLS-cargo release, Cse1p and Nup2p . We propose that the N-terminal IBB domain of importin alpha and Cse1p function together in NLS-cargo release, whereas Nup2p contributes to cargo release/importin alpha recycling through a distinct mechanism.

Curr Opin Genet Dev, 2003 Apr, 13(2), 207 - 14
Looking at mRNA decay pathways through the window of molecular evolution; Culbertson MR et al.; In eukaryotes, mRNAs are monitored for errors in gene expression by RNA surveillance where untranslatable mRNAs are selectively degraded by the nonsense-mediated mRNA decay (NMD) pathway . Depending on the organism, three to seven genes are required for NMD . Besides RNA surveillance, the genes required for NMD serve a second purpose by controlling the overall abundance of a substantial fraction of the transcriptome.

Curr Opin Genet Dev, 2003 Apr, 13(2), 191 - 8
Stretching it: putting the CEN(P-A) in centromere; Mellone BG et al.; The centromere is the locus responsible for the segregation of chromosomes during mitosis and meiosis . The number of newly characterised centromere-associated proteins continues to increase . The kinetochore complex assembles at this site and in many organisms is visible as the primary constriction . In several systems the location of the site of kinetochore assembly is known to vary and the site is not specified by a strict cis-acting primary sequence . It is proposed that tension between bioriented sister centromeres may act to imprint the site.

Curr Opin Genet Dev, 2003 Apr, 13(2), 143 - 53
Collaborative spirit of histone deacetylases in regulating chromatin structure and gene expression; Yang XJ et al.; The flexible N-terminal tails of core histones are subject to dynamic, reversible lysine acetylation . At least 10 histone deacetylases have been identified in Saccharomyces cerevisiae and 19 in humans . Emerging themes regarding the function and regulation of these enzymes include the following: targeted and non-targeted chromatin deacetylation; their collaboration with each other and with other chromatin regulators to promote transcriptional repression and silencing; deacetylation of transcription factors and other non-histone proteins; and regulation by subcellular compartmentalization and subunit association . Histone deacetylases are important targets for drugs with potential therapeutic value in the treatment of cancer, neurodegenerative disorders, cardiac hypertrophy and other human diseases.

Int J Biochem Cell Biol, 2003 May, 35(5), 553 - 61
Role of the Apg12 conjugation system in mammalian autophagy; Mizushima N et al.; The Apg12 system is one of the ubiquitin-like protein conjugation systems conserved in eukaryotes . It was first discovered in yeast during systematic analyses of the apg mutants defective in autophagy, which is the intracellular bulk degradation system . Covalent attachment of Apg12-Apg5 is essential for autophagy . Enzymes catalyzing this conjugation reaction were also identified based on the apg mutant analyses . These are Apg7 and Apg10, corresponding to E1 and E2 enzymes, respectively . Studies using mammalian cells further revealed the function of the Apg12 system . The Apg12-Apg5 conjugate localizes to elongating autophagic isolation membranes . Apg12 conjugation of Apg5 is required for elongation of the isolation membrane to form a complete spherical autophagosome . Discovery of the Apg12 system has facilitated our understanding of the molecular mechanism of autophagosome formation.

Phytother Res, 2003 Mar, 17(3), 259 - 64
Antinociceptive and antipyretic activities of Pongamia pinnata leaves; Srinivasan K et al.; In the present study, the antinociceptive activity of a 70% ethanol extract of Pongamia pinnata leaves (PLE) was investigated in different models of pain in mice and rats . Further, PLE was also evaluated for its antipyretic activity in Brewer's yeast-induced pyrexia in rats . Per os (p.o.) administration of the PLE (100-1000 mg/kg) produced significant antinociceptive activity in the hotplate and tail flick (central) as well as in acetic acid writhing and Randall-Selitto (peripheral) nociceptive tests suggesting the involvement of both central and peripheral mechanisms in alleviating the pain response . In addition, PLE also exhibited a significant antipyretic response in Brewer's yeast-induced pyrexia in rats . These results demonstrated that PLE possesses marked antinociceptive as well as antipyretic activities and thus scientifically validated its use in the treatment of pain and pyretic disorders .

EMBO Rep, 2003 Apr, 4(4), 419 - 24
Self-assembly of minimal COPII cages; Antonny B et al.; The small G-protein Sar1 and the cytosolic complexes Sec23/24 and Sec13/31 associate sequentially on endoplasmic reticulum membranes to form a protein coat named COPII, which drives the formation of transport vesicles . Using dynamic light scattering, we show that Sec23/24 and Sec13/31 can self-assemble in a stoichiometric manner in solution to form particles with hydrodynamic radii in the range of 40-60 nm . Self-assembly is favoured by lowering the pH, the ionic strength and/or the temperature . Electron microscopy reveals the formation of spherical particles 60-120 nm in diameter with a tight, rough mesh on their surfaces . We suggest that these structures, which represent a minimal COPII cage, mimic the molecular organization of the membrane-associated COPII coat.

EMBO Rep, 2003 Apr, 4(4), 405 - 11
Glycopeptide specificity of the secretory protein folding sensor UDP-glucose glycoprotein:glucosyltransferase; Taylor SC et al.; Secretory and membrane N-linked glycoproteins undergo folding and oligomeric assembly in the endoplasmic reticulum with the aid of a folding mechanism known as the calnexin cycle . UDP-glucose glycoprotein:glucosyltransferase (UGGT) is the sensor component of the calnexin cycle, which recognizes these glycoproteins when they are incompletely folded, and transfers a glucose residue from UDP-glucose to N-linked Man9-GlcNAc2 glycans . To determine how UGGT recognizes incompletely folded glycoproteins, we used purified enzyme to glucosylate a set of Man9-GlcNAc2 glycopeptide substrates in vitro, and determined quantitatively the glucose incorporation into each glycan by mass spectrometry . A ranked order of glycopeptide specificity was found that provides the criteria for the recognition of substrates by UGGT . The preference for amino-acid residues close to N-linked glycans provides criteria for the recognition of glycopeptide substrates by UGGT.

EMBO Rep, 2003 Apr, 4(4), 387 - 93
Monitoring global messenger RNA changes in externally controlled microarray experiments; van de Peppel J et al.; Expression profiling is a universal tool, with a range of applications that benefit from the accurate determination of differential gene expression . To allow normalization using endogenous transcript levels, current microarray analyses assume that relatively few transcripts vary, or that any changes that occur are balanced . When normalization using endogenous genes is carried out, changes in expression levels are calculated relative to the behaviour of most of the transcripts . This does not reflect absolute changes if global shifts in messenger RNA populations occur . Using external RNA controls, we have set up microarray experiments to monitor global changes . The levels of most mRNAs were found to change during yeast stationary phase and human heat shock when external controls were included . Even small global changes had a significant effect on the number of genes reported as being differentially expressed . This suggests that global mRNA changes occur more frequently than is assumed at present, and shows that monitoring such effects may be important for the accurate determination of changes in gene expression.

J Biol Chem, 2003 Jun 27, 278(26), 23714 - 9 Epub 2003 Apr 01.
Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G and C subunits; Hirata T et al.; Vacuolar-type ATPases V1V0 (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells . In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase . Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit . V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase) . The rotation was inhibited by concanamycin and nitrate but not by azide . These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis.

Genes Dev, 2003 Apr 1, 17(7), 896 - 911
Human Sin3 deacetylase and trithorax-related Set1/Ash2 histone H3-K4 methyltransferase are tethered together selectively by the cell-proliferation factor HCF-1; Wysocka J et al.; The abundant and chromatin-associated protein HCF-1 is a critical player in mammalian cell proliferation as well as herpes simplex virus (HSV) transcription . We show here that separate regions of HCF-1 critical for its role in cell proliferation associate with the Sin3 histone deacetylase (HDAC) and a previously uncharacterized human trithorax-related Set1/Ash2 histone methyltransferase (HMT) . The Set1/Ash2 HMT methylates histone H3 at Lys 4 (K4), but not if the neighboring K9 residue is already methylated . HCF-1 tethers the Sin3 and Set1/Ash2 transcriptional regulatory complexes together even though they are generally associated with opposite transcriptional outcomes: repression and activation of transcription, respectively . Nevertheless, this tethering is context-dependent because the transcriptional activator VP16 selectively binds HCF-1 associated with the Set1/Ash2 HMT complex in the absence of the Sin3 HDAC complex . These results suggest that HCF-1 can broadly regulate transcription, both positively and negatively, through selective modulation of chromatin structure.

FEMS Microbiol Lett, 2003 Mar 28, 220(2), 241 - 6
Activation of chsC transcription by AbaA during asexual development of Aspergillus nidulans; Park BC et al.; The temporal and spatial regulation of chitin synthesis plays an important role in morphogenesis during fungal growth and development . Northern blot analysis showed that the transcription level of chsA, chsC, and chsD was significantly decreased in an Aspergillus nidulans abaA mutant . Electrophoretic mobility shift assays revealed that AbaA bound tightly to all three AREs (AbaA response elements) in the chsC promoter region . Experiments with the Saccharomyces cerevisiae heterologous expression system confirmed AbaA-dependent transcriptional activation of chsC . Taken together, these data suggest that AbaA plays an important role in chitin biosynthesis during conidiophore development by controlling the transcription level of certain chitin synthase genes.

Biochem Biophys Res Commun, 2003 Apr 11, 303(3), 978 - 84
High expression of Cks1 in human non-small cell lung carcinomas; Inui N et al.; Enhanced degradation of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is known to be a powerful prognostic marker in many types of human cancers . Human CDK subunit 1 (Cks1) and S-phase kinase associated protein 2 (Skp2) are components of the SCF(Skp2) complex, which acts as a ubiquitin ligase for p27(Kip1) . There are no reports about the involvement of Cks1 in the pathogenesis of human cancer . Here we show high expression of Cks1 in non-small cell lung cancers (NSCLCs) using Western blotting and quantitative real-time RT-PCR . The Skp2 mRNA expression level was high in squamous cell carcinomas and was inversely related with the p27(Kip1) protein level in individual clinical samples . In contrast, Cks1 mRNA expression had no such relationship with p27(Kip1), although Cks1 mRNA was significantly elevated in adenocarcinomas . These results suggest that high expression of Skp2 and Cks1 may be involved in the pathogenesis of NSCLCs via different mechanisms.

Plant Cell Physiol, 2003 Mar, 44(3), 223 - 32
The sucrose transporter gene family in rice; Aoki N et al.; In this paper we report the identification, cloning and expression analysis of four putative sucrose transporter (SUT) genes from rice, designated OsSUT2, 3, 4 and 5 . Three of the four genes were identified through extensive searches of the recently published draft sequence of the rice genome . Along with the previously reported OsSUT1 we propose that these five genes comprise the rice SUT gene family . Complementary DNA clones were isolated for the four newly identified genes . The deduced proteins of all five SUT genes were predicted to contain 12 membrane-spanning helices and a domain highly conserved throughout all known plant SUTs, suggesting the four additional OsSUT genes encode functional SUTs . Reverse transcription-PCR analysis was performed in order to investigate the expression pattern of each member of the SUT family in rice . A differing but overlapping expression pattern was observed for each member of the SUT family at different stages through plant development . These results, together with the structural variations apparent from the deduced protein sequences, suggest that the five SUTs possess diverse roles in both sink and source tissues . We also discuss the classification and evolution of the rice SUT gene family, using a comparison of the gene structures and deduced amino acid sequences with other known plant SUT genes.

J Biol Chem, 2003 Jun 20, 278(25), 22284 - 9 Epub 2003 Mar 31.
Regulation of Ste7 ubiquitination by Ste11 phosphorylation and the Skp1-Cullin-F-box complex; Wang Y et al.; Ste7 is a mitogen-activated protein kinase kinase that mediates pheromone signaling in Saccharomyces cerevisiae . We showed previously that Ste7 is ubiquitinated upon prolonged stimulation by pheromone and that accumulation of ubiquitinated Ste7 results in enhanced transcription and cell division arrest responses (Wang, Y., and Dohlman, H . G . (2002) J . Biol . Chem . 277, 15766-15772) . We now report that ubiquitination of Ste7 requires Ste11 kinase and Skp1/Cullin/F-box (SCF) ubiquitin-conjugating activities . Ste7 is not ubiquitinated in Ste11-deficient cells or when the Ste11 phosphorylation sites have been mutated . Ste7 ubiquitination and degradation (but not phosphorylation) is specifically blocked in mutants defective for the E2 ubiquitin-conjugating enzyme Cdc34 or the cullin homologue Cdc53 . Both are components of the SCF complex that ubiquitinates proteins during the G1-S transition of the cell cycle . Our findings suggest that SCF promotes the ubiquitination and degradation of Ste7, thereby favoring the resumption of cell division cycling after pheromone-induced growth arrest.

Arch Biochem Biophys, 2003 Apr 15, 412(2), 267 - 71
Hydrogen isotope tracing in the reaction of orotidine-5'-monophosphate decarboxylase; Smiley JA et al.; The mechanism of the enzyme orotidine-5(')-monophosphate decarboxylase (OMP decarboxylase, ODCase) is not fully characterized; some of the proposed mechanisms suggest the possibility of hydrogen rearrangement (shift from C5 to C6 or loss of H5 to solvent) during catalysis . In this study, we sought mechanistic information for the ODCase reaction by examining the extent of hydrogen exchange in the product uridine-5(')-monophosphate, in combination with ODCase, at the H5 and H6 positions . In a subsequent experiment, partially deuterated OMP was prepared, and the extent of 2H5 rearrangement or loss to solvent was examined by integration of 1H nuclear magnetic resonance signals in the substrate and the resulting enzymatically decarboxylated product . The absence of detectable hydrogen exchange in these experiments limits somewhat the possible mechanisms for ODCase catalysis.

Mol Cell, 2003 Mar, 11(3), 827 - 35
PP2C phosphatases Ptc2 and Ptc3 are required for DNA checkpoint inactivation after a double-strand break; Leroy C et al.; Saccharomyces cells suffering a DNA double-strand break (DSB) ultimately escape checkpoint-mediated G2/M arrest either by recovery once the lesion is repaired or by adaptation if the lesion proves irreparable . Cells lacking the PP2C-like phosphatases Ptc2 and Ptc3 are unable to adapt to a HO-induced DSB and are also defective in recovering from a repairable DSB . In contrast, overexpression of PTC2 rescues adaptation-defective yku80Delta and cdc5-ad mutants . These effects are not explained by alterations either in the processing of DSB ends or in DSB repair . In vivo and in vitro evidence suggests that phosphorylated forms of Ptc2 and Ptc3 specifically bind to the Rad53 FHA1 domain and inactivate Rad53-dependent pathways during adaptation and recovery by dephosphorylating Rad53.

Mol Cell, 2003 Mar, 11(3), 721 - 9
The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation; Krogan NJ et al.; Methylation of histone proteins is one of their many modifications that affect chromatin structure and regulate gene expression . Methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively, is required for silencing of expression of genes located near chromosome telomeres in yeast . We report that the Paf1 protein complex, which is associated with the elongating RNA polymerase II, is required for methylation of lysines 4 and 79 of histone H3 and for silencing of expression of a telomere-associated gene . We show that the Paf1 complex is required for recruitment of the COMPASS methyltransferase to RNA polymerase II and that the subunits of these complexes interact physically and genetically . Collectively, our results suggest that the Paf1 complex is required for histone H3 methylation, therefore linking transcriptional elongation to chromatin methylation.

Mol Cell, 2003 Mar, 11(3), 709 - 19
Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity; Ng HH et al.; Set1, the yeast histone H3-lysine 4 (H3-K4) methylase, is recruited by the Pol II elongation machinery to a highly localized domain at the 5' portion of active mRNA coding regions . Set1 association depends upon the TFIIH-associated kinase that phosphorylates the Pol II C-terminal domain (CTD) and mediates the transition between initiation and elongation, and Set1 interacts with the form of Pol II whose CTD is phosphorylated at serine 5 but not serine 2 . The Rtf1 and Paf1 components of the Pol II-associated Paf1 complex are also important for Set1 recruitment . Although the level of dimethylated H3-K4 is fairly uniform throughout the genome, the pattern of trimethylated H3-K4 strongly correlates with Set1 occupancy . Hypermethylated H3-K4 within the mRNA coding region persists for considerable time after transcriptional inactivation and Set1 dissociation from the chromatin, indicating that H3-K4 hypermethylation provides a molecular memory of recent transcriptional activity.

Mol Cell, 2003 Mar, 11(3), 635 - 46
Pex8p: an intraperoxisomal organizer of the peroxisomal import machinery; Agne B et al.; Peroxisomes transport folded and oligomeric proteins across their membrane . Two cytosolic import receptors, Pex5p and Pex7p, along with approximately 12 membrane-bound peroxins participate in this process . While interactions among individual peroxins have been described, their organization into functional units has remained elusive . We have purified and defined two core complexes of the peroxisomal import machinery: the docking complex comprising Pex14p and Pex17p, with the loosely associated Pex13p, and the RING finger complex containing Pex2p, Pex10p, and Pex12p . Association of both complexes into a larger import complex requires Pex8p, an intraperoxisomal protein . We conclude that Pex8p organizes the formation of the larger import complex from the trans side of the peroxisomal membrane and thus might enable functional communication between both sides of the membrane.

Biochemistry, 2003 Apr 8, 42(13), 3751 - 8
Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13; Anderson EM et al.; The essential Saccharomyces cerevisiae protein Cdc13 binds the conserved single-stranded overhang at the end of telomeres and mediates access of protein complexes involved in both end-capping and telomerase activity . The single-stranded DNA-binding domain (ssDBD) of Cdc13 exhibits both high affinity (K(d) of 3 pM) and sequence specificity for the GT-rich sequences present at yeast telomeres . We have used the ssDBD of Cdc13 to understand the sequence-specific recognition of extended single-stranded DNA (ssDNA) . The recent structure of the Cdc13 DNA-binding domain revealed that ssDNA is recognized by a large protein surface containing an oligonucleotide/oligosaccharide-binding fold (OB-fold) augmented by an extended 30-amino acid loop . Contacts to ssDNA occur via a contiguous surface of aromatic, hydrophobic, and basic residues . A complete alanine scan of the binding interface has been used to determine the contribution of each contacting side chain to binding affinity . Substitution of any aromatic or hydrophobic residue at the interface was deleterious to binding (20 to >700-fold decrease in binding affinity), while tolerance for replacement of basic residues was observed . The important aromatic and hydrophobic contacts are spread throughout the extended interface, indicating that the entire surface is both structurally and thermodynamically required for binding . While all of these contacts are important, several of the individual alanine substitutions that abolish binding cluster to one region of the protein surface . This region is vital for recognition of four bases at the 5' end of the DNA and constitutes a "hotspot" of binding affinity.

Biochemistry, 2003 Apr 8, 42(13), 3716 - 24
Biochemical and structural studies with prenyl diphosphate analogues provide insights into isoprenoid recognition by protein farnesyl transferase; Turek-Etienne TC et al.; Protein farnesyl transferase (PFTase) catalyzes the reaction between farnesyl diphosphate and a protein substrate to form a thioether-linked prenylated protein . The fact that many prenylated proteins are involved in signaling processes has generated considerable interest in protein prenyl transferases as possible anticancer targets . While considerable progress has been made in understanding how prenyl transferases distinguish between related target proteins, the rules for isoprenoid discrimination by these enzymes are less well understood . To clarify how PFTase discriminates between FPP and larger prenyl diphosphates, we have examined the interactions between the enzyme and several isoprenoid analogues, GGPP, and the farnesylated peptide product using a combination of biochemical and structural methods . Two photoactive isoprenoid analogues were shown to inhibit yeast PFTase with K(I) values as low as 45 nM . Crystallographic analysis of one of these analogues bound to PFTase reveals that the diphosphate moiety and the two isoprene units bind in the same positions occupied by the corresponding atoms in FPP when bound to PFTase . However, the benzophenone group protrudes into the acceptor protein binding site and prevents the binding of the second (protein) substrate . Crystallographic analysis of geranylgeranyl diphosphate bound to PFTase shows that the terminal two isoprene units and diphosphate group of the molecule map to the corresponding atoms in FPP; however, the first and second isoprene units bulge away from the acceptor protein binding site . Comparison of the GGPP binding mode with the binding of the farnesylated peptide product suggests that the bulkier isoprenoid cannot rearrange to convert to product without unfavorable steric interactions with the acceptor protein . Taken together, these data do not support the "molecular ruler hypotheses" . Instead, we propose a "second site exclusion model" in which PFTase binds larger isoprenoids in a fashion that prevents the subsequent productive binding of the acceptor protein or its conversion to product.

Biopolymers, 2003 Apr, 68(4), 528 - 38
Role of C-terminal domain phosphorylation in RNA polymerase II transcription through the nucleosome; Liu YV et al.; End-initiated transcription of a 256 base-pair (bp) template containing a single uniquely positioned nucleosome by yeast and calf thymus nuclear RNA polymerases II (pol II) was analyzed in vitro . The nucleosome-specific pausing pattern is similar to the pattern observed in the case of transcription of the same nucleosome by yeast RNA polymerase III . However, the pausing pattern is clearly different from the patterns observed previously during transcription by promoter-initiated and assembled pol II . This suggests that end-initiated and promoter-initiated RNA polymerases differ in the way they progress through the nucleosome . The rates of transcription through the nucleosome by pol II are significantly lower than the rates observed in the case of SP6 polymerase and RNA polymerase III . Using calf thymus pol II, we have investigated the possibility that phosphorylation of the C-terminal domain (CTD) facilitates transcription through the nucleosome . The rates of transcription through the nucleosome by phosphorylated (IIO) and nonphosphorylated (IIA) forms of calf thymus pol II are very similar . This suggests that CTD phosphorylation is not sufficient to facilitate transcription through the nucleosome by end-initiated pol II .

Mol Cell Biol, 2003 Apr, 23(8), 3008 - 12
Deoxynucleotide triphosphate binding mode conserved in Y family DNA polymerases; Johnson RE et al.; Although DNA polymerase eta (Pol eta) and other Y family polymerases differ in sequence and function from classical DNA polymerases, they all share a similar right-handed architecture with the palm, fingers, and thumb domains . Here, we examine the role in Saccharomyces cerevisiae Pol eta of three conserved residues, tyrosine 64, arginine 67, and lysine 279, which come into close contact with the triphosphate moiety of the incoming nucleotide, in nucleotide incorporation . We find that mutational alteration of these residues reduces the efficiency of correct nucleotide incorporation very considerably . The high degree of conservation of these residues among the various Y family DNA polymerases suggests that these residues are also crucial for nucleotide incorporation in the other members of the family . Furthermore, we note that tyrosine 64 and arginine 67 are functionally equivalent to the deoxynucleotide triphosphate binding residues arginine 518 and histidine 506 in T7 DNA polymerase, respectively.

Mol Cell Biol, 2003 Apr, 23(8), 2999 - 3007
Mechanical link between cohesion establishment and DNA replication: Ctf7p/Eco1p, a cohesion establishment factor, associates with three different replication factor C complexes; Kenna MA et al.; CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion . The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C {RFC} homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled-a link now confirmed by other studies . To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically associates with any components of the DNA replication machinery . Here, we report that Ctf7p/Eco1p associates with proteins that perform partially redundant functions in DNA replication . Chl12p/Ctf18p combines with Rfc2p to Rfc5p to form one of three independent RFC complexes . By chromatographic methods, Ctf7p/Eco1p was found to associate with Chl12/Ctf18p and with Rfc2p, Rfc3p, Rfc4p, and Rfc5p . The association between Ctf7p/Eco1p and this RFC complex is biologically relevant in that (i) Ctf7p/Eco1p cosediments with Chl12p/Ctf18p in vivo and (ii) rfc5-1 mutant cells exhibit precocious sister separation . Previous studies revealed that Rfc1p or Rad24p associates with Rfc2p to Rfc5p to form two other RFC complexes independent of Ctf18p-RFC complexes . These Rfc1p-RFC and Rad24p-RFC complexes function in DNA replication or repair and DNA damage checkpoint pathways . Importantly, Ctf7p/Eco1p also associates with Rfc1p and Rad24p, suggesting that these RFC complexes also play critical roles in cohesion establishment . The associations between Ctf7p/Eco1p and RFC subunits provide novel evidence regarding the physical linkage between cohesion establishment and DNA replication . Furthermore, the association of Ctf7p/Eco1p with each of three RFC complexes supplies new insights into the functional redundancy of RFC complexes in cohesion establishment.

Mol Cell Biol, 2003 Apr, 23(8), 2942 - 52
Novel SWI/SNF chromatin-remodeling complexes contain a mixed-lineage leukemia chromosomal translocation partner; Nie Z et al.; The SWI/SNF family of chromatin-remodeling complexes has been discovered in many species and has been shown to regulate gene expression by assisting transcriptional machinery to gain access to their sites in chromatin . Several complexes of this family have been reported for humans . In this study, two additional complexes are described that belong to the same SWI/SNF family . These new complexes contain as many as eight subunits identical to those found in other SWI/SNF complexes, and they possess a similar ATP-dependent nucleosome disruption activity . But unlike known SWI/SNFs, the new complexes are low in abundance and contain an extra subunit conserved between human and yeast SWI/SNF complexes . This subunit, ENL, is a homolog of the yeast SWI/SNF subunit, ANC1/TFG3 . Moreover, ENL is a fusion partner for the gene product of MLL that is a common target for chromosomal translocations in human acute leukemia . The resultant MLL-ENL fusion protein associates and cooperates with SWI/SNF complexes to activate transcription of the promoter of HoxA7, a downstream target essential for oncogenic activity of MLL-ENL . Our data suggest that human SWI/SNF complexes show considerable heterogeneity, and one or more may be involved in the etiology of leukemia by cooperating with MLL fusion proteins.

Mol Cell Biol, 2003 Apr, 23(8), 2844 - 58
Isotype-restricted corepressor recruitment: a constitutively closed helix 12 conformation in retinoic acid receptors beta and gamma interferes with corepressor recruitment and prevents transcriptional repression; Farboud B et al.; Retinoic acid receptors (RARs) are ligand-regulated transcription factors that play multiple roles in vertebrate development and differentiation . RARs as a class are capable of both repressing and activating target gene expression . Transcriptional repression is mediated through the recruitment of corepressor proteins such as SMRT . Notably, vertebrates encode three major forms of RARs, alpha, beta, and gamma, and these distinct RAR isotypes differ in the ability to recruit a corepressor . RAR alpha strongly interacts with SMRT and can repress target gene transcription, whereas RAR beta and -gamma interact with SMRT only weakly and fail to repress . We report here the use of a genetic suppressor approach, based on a yeast two-hybrid interaction assay using Saccharomyces cerevisiae, for the isolation of RAR beta mutants that have gained the RAR alpha-like corepressor phenotype, i.e., a strong interaction with SMRT and the ability to repress gene expression in vertebrate cells . Analysis of these gain-of-function mutants indicates that the different corepressor interaction properties of RAR alpha, -beta and -gamma are determined by a gating mechanism through which amino acid differences in the helix 3 region of these receptors influence the position of the receptor C-terminal helix 12 domain . As a consequence, the RAR beta and RAR gamma receptors appear to adopt a constitutively closed helix 12 conformation in the absence of hormone that may approximate the conformation of RAR alpha when bound to hormone agonist . This closed helix 12 conformation in RAR beta and RAR gamma blocks corepressor binding, prevents repression, and permits significant levels of target gene activation even in the absence of hormone . We refer to this phenomenon as a "gate-latch" model of corepressor regulation.

Mol Cell Biol, 2003 Apr, 23(8), 2800 - 20
A multiplicity of coactivators is required by Gcn4p at individual promoters in vivo; Swanson MJ et al.; Transcriptional activators interact with multisubunit coactivators that modify chromatin structure or recruit the general transcriptional machinery to their target genes . Budding yeast cells respond to amino acid starvation by inducing an activator of amino acid biosynthetic genes, Gcn4p . We conducted a comprehensive analysis of viable mutants affecting known coactivator subunits from the Saccharomyces Genome Deletion Project for defects in activation by Gcn4p in vivo . The results confirm previous findings that Gcn4p requires SAGA, SWI/SNF, and SRB mediator (SRB/MED) and identify key nonessential subunits of these complexes required for activation . Among the numerous histone acetyltransferases examined, only that present in SAGA, Gcn5p, was required by Gcn4p . We also uncovered a dependence on CCR4-NOT, RSC, and the Paf1 complex . In vitro binding experiments suggest that the Gcn4p activation domain interacts specifically with CCR4-NOT and RSC in addition to SAGA, SWI/SNF, and SRB/MED . Chromatin immunoprecipitation experiments show that Mbf1p, SAGA, SWI/SNF, SRB/MED, RSC, CCR4-NOT, and the Paf1 complex all are recruited by Gcn4p to one of its target genes (ARG1) in vivo . We observed considerable differences in coactivator requirements among several Gcn4p-dependent promoters; thus, only a subset of the array of coactivators that can be recruited by Gcn4p is required at a given target gene in vivo.

Mol Cell Biol, 2003 Apr, 23(8), 2633 - 44
The CBP bromodomain and nucleosome targeting are required for Zta-directed nucleosome acetylation and transcription activation; Deng Z et al.; The Epstein-Barr virus (EBV)-encoded lytic activator Zta is a bZIP protein that can stimulate nucleosomal histone acetyltransferase (HAT) activity of the CREB binding protein (CBP) in vitro . We now show that deletion of the CBP bromo- and C/H3 domains eliminates stimulation of nucleosomal HAT activity in vitro and transcriptional coactivation by Zta in transfected cells . In contrast, acetylation of free histones was not affected by the addition of Zta or by deletions in the bromo or C/H3 domain of CBP . Zta stimulated acetylation of oligonucleosomes assembled on supercoiled DNA and dinucleosomes assembled on linear DNA, but Zta-stimulated acetylation was significantly reduced for mononucleosomes . Western blotting and amino-terminal protein sequencing indicated that all lysine residues in the H3 and H4 amino-terminal tails were acetylated by CBP and enhanced by the addition of Zta . Histone acetylation was also dependent upon the Zta basic DNA binding domain, which could not be substituted with the homologous basic region of c-Fos, indicating specificity in the bZIP domain nucleosome binding function . Finally, we show that Zta and CBP colocalize to viral immediate-early promoters in vivo and that overexpression of Zta leads to a robust increase in H3 and H4 acetylation at various regions of the EBV genome in vivo . Furthermore, deletion of the CBP bromodomain reduced stable CBP-Zta complex formation and histone acetylation at Zta-responsive viral promoters in vivo . These results suggest that activator- and bromodomain-dependent targeting to oligonucleosomal chromatin is required for stable promoter-bound complex formation and transcription activity.

J Cell Sci, 2003 May 1, 116(Pt 9), 1719 - 31
Linear element formation and their role in meiotic sister chromatid cohesion and chromosome pairing; Molnar M et al.; Fission yeast does not form synaptonemal complexes in meiotic prophase . Instead, linear elements appear that resemble the axial cores of other eukaryotes . They have been proposed to be minimal structures necessary for proper meiotic chromosome functions . We examined linear element formation in meiotic recombination deficient mutants . The rec12, rec14 and meu13 mutants showed altered linear element formation . Examination of rec12 and other mutants deficient in the initiation of meiotic recombination revealed that occurrence of meiosis-specific DNA breaks is not a precondition for the formation of linear elements . The rec11 and rec8 mutants exhibited strongly impaired linear elements with morphologies specific for these meiotic cohesin mutants . The rec10 and rec16/rep1 mutants lack linear elements completely . The region specificity of loss of recombination in the rec8, rec10 and rec11 mutants can be explained by their defects in linear element formation . Investigation of the rec10 mutant showed that linear elements are basically dispensable for sister chromatid cohesion, but contribute to full level pairing of homologous chromosomes.

J Cell Sci, 2003 May 1, 116(Pt 9), 1679 - 88
Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate; Mizushima N et al.; Macroautophagy is the major intracellular degradation system delivering cytoplasmic components to the lysosome/vacuole . We have shown that, in yeast and mammalian cells, the Apg12-Apg5 protein conjugate, which is formed by a ubiquitin-like system, is essential for autophagosome formation . In yeast, the Apg12-Apg5 conjugate interacts with a small coiled-coil protein, Apg16, to form a approximately 350 kDa multimeric complex . We demonstrate that the mouse Apg12-Apg5 conjugate forms a approximately 800 kDa protein complex containing a novel WD-repeat protein . Because the N-terminal region of this novel protein shows homology with yeast Apg16, we have designated it mouse Apg16-like protein (Apg16L) . Apg16L, however, has a large C-terminal domain containing seven WD repeats that is absent from yeast Apg16 . Apg16L interacts with both Apg5 and additional Apg16L monomers; neither interaction, however, depends on the WD-repeat domain . In conjunction with Apg12-Apg5, Apg16L associates with the autophagic isolation membrane for the duration of autophagosome formation . Because these features are similar to yeast Apg16, we concluded Apg16L is the functional counterpart of the yeast Apg16 . We also found that membrane targeting of Apg16L requires Apg5 but not Apg12 . Because WD-repeat proteins provide a platform for protein-protein interactions, the approximately 800 kDa complex is expected to function in autophagosome formation, further interacting with other proteins in mammalian cells.

J Biol Chem, 2003 Jun 6, 278(23), 20954 - 60 Epub 2003 Mar 28.
The exocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum; Lipschutz JH et al.; We previously showed that the exocyst complex specifically affected the synthesis and delivery of secretory and basolateral plasma membrane proteins . Significantly, the entire spectrum of secreted proteins was increased when the hSec10 (human Sec10) component of the exocyst complex was overexpressed, suggestive of post-transcriptional regulation (Lipschutz, J . H., Guo, W., O'Brien, L . E., Nguyen, Y . H., Novick, P., and Mostov, K . E . (2000) Mol . Biol . Cell 11, 4259-4275) . Here, using an exogenously transfected basolateral protein, the polymeric immunoglobulin receptor (pIgR), and a secretory protein, gp80, we show that pIgR and gp80 protein synthesis and delivery are increased in cells overexpressing Sec10 despite the fact that mRNA levels are unchanged, which is highly indicative of post-transcriptional regulation . To test specificity, we also examined the synthesis and delivery of an exogenous apical protein, CNT1 (concentrative nucleoside transporter 1), and found no increase in CNT1 protein synthesis, delivery, or mRNA levels in cells overexpressing Sec10 . Sec10-GFP-overexpressing cell lines were created, and staining was seen in the endoplasmic reticulum . It was demonstrated previously in yeast that high levels of expression of SEB1, the Sec61beta homologue, suppressed sec15-1, an exocyst mutant (Toikkanen, J., Gatti, E., Takei, K., Saloheimo, M., Olkkonen, V . M., Soderlund, H., De Camilli, P., and Keranen, S . (1996) Yeast 12, 425-438) . Sec61beta is a member of the Sec61 heterotrimer, which is the main component of the endoplasmic reticulum translocon . By co-immunoprecipitation we show that Sec10, which forms an exocyst subcomplex with Sec15, specifically associates with the Sec61beta component of the translocon and that Sec10 overexpression increases the association of other exocyst complex members with Sec61beta . Proteosome inhibition does not appear to be the mechanism by which increased protein synthesis occurs in the face of equivalent amounts of mRNA . Although the exact mechanism remains to be elucidated, the exocyst/Sec61beta interaction represents an important link between the cellular membrane trafficking and protein synthetic machinery.

J Biol Chem, 2003 Jun 6, 278(23), 20652 - 8 Epub 2003 Mar 28.
A PHO80-like cyclin and a B-type cyclin control the cell cycle of the procyclic form of Trypanosoma brucei; Li Z et al.; Cyclins bind and activate cyclin-dependent kinases that regulate cell cycle progression in eukaryotes . Cell cycle control in Trypanosoma brucei was analyzed in the present study . Genes encoding four PHO80 cyclin homologues and three B-type cyclin homologues but no G1 cyclin homologues were identified in this organism . Through knocking down expression of the seven cyclin genes with the RNA interference technique in the procyclic form of T . brucei, we demonstrated that one PHO80 homologue (CycE1/CYC2) and a B-type cyclin homologue (CycB2) are the essential cyclins regulating G1/S and G2/M transitions, respectively . This lack of overlapping cyclin function differs significantly from that observed in the other eukaryotes . Also, PHO80 cyclin is known for its involvement only in phosphate signaling in yeast with no known function in cell cycle control . Both observations thus suggest the presence of simple and novel cell cycle regulators in trypanosomes . T . brucei cells deficient in CycE1/CYC2 displayed a long slender morphology, whereas those lacking CycB2 assumed a fat stumpy form . These cells apparently still can undergo cytokinesis generating small numbers of anucleated daughter cells, each containing a single kinetoplast known as a zoid . Two different types of zoids were identified, the slender zoid derived from reduced CycE1/CYC2 expression and the stumpy zoid from CycB2 deficiency . This observation indicates an uncoupling between the kinetoplast and the nuclear cycle, resulting in cell division driven by kinetoplast segregation with neither a priori S phase nor mitosis in the trypanosome.

Science, 2003 Mar 28, 299(5615), 2064 - 7
Crystal structure of the carboxyltransferase domain of acetyl-coenzyme A carboxylase; Zhang H et al.; Acetyl-coenzyme A carboxylases (ACCs) are required for the biosynthesis and oxidation of long-chain fatty acids . They are targets for therapeutics against obesity and diabetes, and several herbicides function by inhibiting their carboxyltransferase (CT) domain . We determined the crystal structure of the free enzyme and the coenzyme A complex of yeast CT at 2.7 angstrom resolution and found that it comprises two domains, both belonging to the crotonase/ClpP superfamily . The active site is at the interface of a dimer . Mutagenesis and kinetic studies reveal the functional roles of conserved residues here . The herbicides target the active site of CT, providing a lead for inhibitor development against human ACCs.

Science, 2003 Apr 18, 300(5618), 482 - 6 Epub 2003 Mar 27.
Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation; Lee BH et al.; Meiosis is a specialized cell division in which two chromosome segregation phases follow a single DNA replication phase . The budding yeast Polo-like kinase Cdc5 was found to be instrumental in establishing the meiosis I chromosome segregation program . Cdc5 was required to phosphorylate and remove meiotic cohesin from chromosomes . Furthermore, in the absence of CDC5 kinetochores were bioriented during meiosis I, and Mam1, a protein essential for coorientation, failed to associate with kinetochores . Thus, sister-kinetochore coorientation and chromosome segregation during meiosis I are coupled through their dependence on CDC5.

J Virol, 2003 Apr, 77(8), 4794 - 804
Role of ESCRT-I in retroviral budding; Martin-Serrano J et al.; Retroviral late-budding (L) domains are required for the efficient release of nascent virions . The three known types of L domain, designated according to essential tetrapeptide motifs (PTAP, PPXY, or YPDL), each bind distinct cellular cofactors . We and others have demonstrated that recruitment of an ESCRT-I subunit, Tsg101, a component of the class E vacuolar protein sorting (VPS) machinery, is required for the budding of viruses, such as human immunodeficiency virus type 1 (HIV-1) and Ebola virus, that encode a PTAP-type L domain, but subsequent events remain undefined . In this study, we demonstrate that VPS28, a second component of ESCRT-I, binds to a sequence close to the Tsg101 C terminus and is therefore recruited to the plasma membrane by HIV-1 Gag . In addition, we show that Tsg101 exhibits a multimerization activity . Using a complementation assay in which Tsg101 is artificially recruited to sites of HIV-1 assembly, we demonstrate that the integrity of the VPS28 binding site within Tsg101 is required for particle budding . In addition, mutation of a putative leucine zipper or residues important for Tsg101 multimerization also impairs the ability of Tsg101 to support HIV-1 budding . A minimal multimerizing Tsg101 domain is a dominant negative inhibitor of PTAP-mediated HIV-1 budding but does not inhibit YPDL-type or PPXY-type L-domain function . Nevertheless, YDPL-type L-domain activity is inhibited by expression of a catalytically inactive mutant of the class E VPS ATPase VPS4 . These results indicate that all three classes of retroviral L domains require a functioning class E VPS pathway in order to effect budding . However, the PTAP-type L domain appears to be unique in its requirement for an intact, or nearly intact, ESCRT-I complex.

Genetics, 2003 Mar, 163(3), 931 - 7
The Drosophila melanogaster sir2+ gene is nonessential and has only minor effects on position-effect variegation; Astrom SU et al.; Five Drosophila melanogaster genes belong to the highly conserved sir2 family, which encodes NAD(+)-dependent protein deacetylases . Of these five, dsir2(+) (CG5216) is most similar to the Saccharomyces cerevisiae SIR2 gene, which has profound effects on chromatin structure and life span . Four independent Drosophila strains were found with P-element insertions near the dsir2 transcriptional start site as well as extraneous linked recessive lethal mutations . Imprecise excision of one of these P elements (PlacW07223) from a chromosome freed of extraneous lethal mutations produced dsir2(17), a null intragenic deletion allele that generates no DSIR2 protein . Contrary to expectations from the report by Rosenberg and Parkhurst on their P-mobilization allele dSir2(ex10), homozygosity for dsir2(17) had no apparent deleterious effects on viability, developmental rate, or sex ratio, and it fully complemented sir2(ex10) . Moreover, through a genetic test, we ruled out the reported effect of dSir2(ex10) on Sex-lethal expression . We did observe a modest, strictly recessive suppression of white(m4) position-effect variegation and a shortening of life span in dsir2 homozygous mutants, suggesting that dsir2 has some functions in common with yeast SIR2.

Mol Cells, 2003 Feb 28, 15(1), 127 - 32
Molecular characterization of NbPAF encoding the alpha6 subunit of the 20S proteasome in Nicotiana benthamiana; Kim M et al.; The 26S proteasome involved in degradation of proteins covalently modified with polyubiquitin consists of the 20S proteasome and 19S regulatory complex . The NbPAF gene encoding the alpha6 subunit of the 20S proteasome was identified from Nicotiana benthamiana . NbPAF exhibits high sequence homology with the corresponding genes from Arabidopsis, human and yeast . The deduced amino acid sequence of NbPAF reveals that this protein contains the proteasome alpha-type subunits signature and nuclear localization signal at the N-terminus . The genomic Southern blot analysis suggests that the N . benthamiana genome contains one copy of NbPAF . The NbPAF mRNA was detected abundantly in flowers and weakly in roots and stems, but it was almost undetectable in mature leaves . In response to stresses, accumulation of the NbPAF mRNA was stimulated by methyl jasmonate, NaCl and salicylic acid, but not by abscisic acid and cold treatment in leaves . The NbPAF-GFP fusion protein was localized in the cytoplasm and nucleus.

Mol Cells, 2003 Feb 28, 15(1), 81 - 6
Dna2 requirement for normal reproduction of Caenorhabditis elegans is temperature-dependent; Lee KH et al.; To determine the function of Dna2 in a multicellular organism, the Caenorhabditis elegans Dna2 expression was probed and deletion mutant phenotypes were analyzed . Dna2 was localized to the nuclei of C . elegans oocytes and early embryos by immunostaining or green fluorescent protein-tagging . A homozygous dna2 deletion mutant showed a reduced brood size and embryonic lethality, and the phenotypes greatly depended on growth temperature and aggravated in the succeeding generation . The mutant embryos also showed delayed cell divisions, which together with temperature-dependence of the mutant phenotypes supported the well-conserved role of Dna2 in DNA replication.

J Biol Chem, 2003 May 30, 278(22), 20303 - 12 Epub 2003 Mar 25.
Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes; Furuta T et al.; DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (gammaH2AX) foci . Here we show that gammaH2AX formation is also a cellular response to topoisomerase I cleavage complexes known to induce DNA double-strand breaks during replication . In HCT116 human carcinoma cells exposed to the topoisomerase I inhibitor camptothecin, the resulting gammaH2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced gammaH2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine . This gammaH2AX formation is suppressed in ATR (ataxia telangiectasia and Rad3-related) deficient cells and markedly decreased in DNA-dependent protein kinase-deficient cells but is not abrogated in ataxia telangiectasia cells, indicating that ATR and DNA-dependent protein kinase are the kinases primarily involved in gammaH2AX formation at the sites of replication-mediated DNA double-strand breaks . Mre11- and Nbs1-deficient cells are still able to form gammaH2AX . However, H2AX-/- mouse embryonic fibroblasts exposed to camptothecin fail to form Mre11, Rad50, and Nbs1 foci and are hypersensitive to camptothecin . These results demonstrate a conserved gammaH2AX response for double-strand breaks induced by replication fork collision . gammaH2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.

EMBO J, 2003 Apr 1, 22(7), 1654 - 64
Rev1 is essential for DNA damage tolerance and non-templated immunoglobulin gene mutation in a vertebrate cell line; Simpson LJ et al.; The majority of DNA damage-induced mutagenesis in the yeast Saccharomyces cerevisiae arises as a result of translesion replication . This process is critically dependent on the deoxycytidyl transferase Rev1p, which forms a complex with the subunits of DNA polymerase zeta, Rev3p and Rev7p . To examine the role of Rev1 in vertebrate mutagenesis and the DNA damage response, we disrupted the gene in DT40 cells . Rev1-deficient DT40 grow slowly and are sensitive to a wide range of DNA-damaging agents . Homologous recombination repair is likely to be intact as basal and damage induced sister chromatid exchange and immunoglobulin gene conversion are unaffected . How ever, the mutant cells show a markedly reduced level of non-templated immunoglobulin gene mutation, indicating a defect in translesion bypass . Furthermore, ultraviolet exposure results in marked chromosome breakage, suggesting that replication gaps created in the absence of Rev1 cannot be efficiently repaired by recombination . Thus, Rev1-dependent translesion bypass and mutagenesis is likely to be a trade-off for the ability to complete replication of a damaged template and thereby maintain genome integrity.

Biochem Biophys Res Commun, 2003 Apr 4, 303(2), 594 - 9
Snapin interacts with the N-terminus of regulator of G protein signaling 7; Hunt RA et al.; The N-terminus of regulator of G protein signaling 7 (RGS7) contains a dishevelled/egl-10/pleckstrin (DEP) domain of unknown function . To gain insight into its function, we used yeast two-hybrid analysis to screen a human whole brain cDNA library in order to identify proteins that interact specifically with the N-terminus of human RGS7 (amino acid residues 1-248) . From this analysis, we identified snapin, a protein associated with the SNARE complex in neurons, as an interactor with the N-terminus of RGS7 . Deletion mutation analysis in yeast demonstrated that the interaction between RGS7 and snapin is specific and is mediated primarily by amino acid residues 1-69 of RGS7 (which contains the proximal portion of the DEP domain) . The interaction between RGS7 and snapin was also demonstrated in mammalian cells by coimmunoprecipitation and pull-down assays . Our results suggest that RGS7 could play a role in synaptic vesicle exocytosis through its interaction with snapin.

Biochem Biophys Res Commun, 2003 Apr 4, 303(2), 399 - 405
Identification of CGI-121, a novel PRPK (p53-related protein kinase)-binding protein; Miyoshi A et al.; PRPK (p53-related protein kinase) has been reported as a novel protein kinase which binds to the tumor suppressor protein p53 and induces phosphorylation of p53 at Ser 15 . To identify novel binding partners of PRPK, we performed a yeast two-hybrid screening and isolated an expressed sequence tag CGI-121 by which a 20-kDa protein was encoded . We demonstrated the protein-protein interaction of CGI-121 with PRPK in vivo and in vitro . The protein expression of CGI-121 was observed in many cell lines and was immunocytochemically identified in both the nucleus and cytosol . Although PRPK interacted with both CGI-121 and p53, several attempts to demonstrate an association between CGI-121 and p53 were unsuccessful . In addition, coprecipitation of p53 using recombinant PRPK was inhibited by adding recombinant CGI-121 in vitro, suggesting that CGI-121 could act as a potent inhibitor of the binding of PRPK to p53.

J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Apr 5, 787(1), 43 - 51
Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements; Janini GM et al.; We have developed an effective two-dimensional fractionation protocol of complex proteome mixtures that extends the ability to conduct more comprehensive proteome measurements . A sample containing intact proteins extracted from Saccharomyces cerevisiae was fractionated by liquid phase isoelectric focusing, followed by tryptic digestion and solid-phase extraction (SPE) clean-up and reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS) of the resultant peptides . The clean-up step is designed to desalt the fractions and rid them of urea and ampholytes prior to analysis by LC-MS-MS . Fifty milligrams of protein were separated into 20 fractions by liquid-phase isoelectric focusing, spanning a pH range of 3-10 . The effectiveness of the removal of ampholytes was monitored by capillary zone electrophoresis and LC-MS-MS . The ability to analyze all of the 20 fractions without any noticeable decrease in the separation efficiency demonstrates the overall effectiveness of the SPE clean-up step . The results show that the separation strategy is effective for high throughput characterization of proteins from complex proteomic mixtures .

Biochem J, 2003 Jul 1, 373(Pt 1), 167 - 78
Analysis of zinc-fingers and homeoboxes (ZHX)-1-interacting proteins: molecular cloning and characterization of a member of the ZHX family, ZHX3; Yamada K et al.; Human zinc-fingers and homeoboxes (ZHX) 1, a transcriptional repressor, was originally cloned as an interacting protein with the activation domain of the A subunit of nuclear factor-Y (NF-YA) . As the first step in investigating the mechanism by which ZHX1 acts as a transcriptional repressor, we conducted a search of ZHX1-interacting proteins using a yeast two-hybrid system . Nuclear proteins such as ZHX1, transcriptional co-factors and DNA-binding proteins, zyxin, androgen-induced aldose reductase and eleven-nineteen lysine-rich leukaemia gene, as well as some unknown proteins, were cloned . Molecular cloning and determination of the nucleotide sequence of the full-length cDNA encoding a novel protein revealed that it consists of 956 amino acid residues and contains two zinc-finger (Znf) motifs and five homeodomains (HDs) as well as ZHX1 . We concluded that the protein forms the ZHX family with ZHX1 and denoted it ZHX3 . ZHX3 not only dimerizes with both ZHX1 and ZHX3, but also interacts with the activation domain of the NF-YA . Further analysis revealed that ZHX3 is a ubiquitous transcriptional repressor that is localized in nuclei and functions as a dimer . Lastly, the dimerization domain, the interaction domain with NF-YA, and the repressor domain are mapped to a region including the HD1 region, and two nuclear localization signals are mapped to the N-terminal through Znf1 and the HD2 region, respectively.

Neuroreport, 2003 Mar 24, 14(4), 565 - 8
Inducible PC12 cell model of Huntington's disease shows toxicity and decreased histone acetylation; Igarashi S et al.; Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of a polyglutamine tract in the huntingtin protein . We have developed PC12 cell lines in which the expression of an N-terminal truncation of huntingtin (N63) with either wild type (23Q) or expanded polyglutamine (148Q) can be induced by the removal of doxycycline . Differentiated PC12 cells induced to express N63-148Q showed cellular toxicity reaching up to 50% at 6 days post-induction . Histone acetyltransferase (HAT) activity and global histone acetylation was significantly decreased in cells expressing truncated huntingtin with mutant but not normal huntingtin . These data suggest that altered chromatin modification via reduction in coactivator activity may cause neuronal transcriptional dysregulation and contribute to cellular toxicity.

J Biol Chem, 2003 May 30, 278(22), 19878 - 84 Epub 2003 Mar 25.
The Yip1p.Yif1p complex is required for the fusion competence of endoplasmic reticulum-derived vesicles; Barrowman J et al.; Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro . To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER) . These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles . We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding . Consistent with this proposal, we observe that the Yip1p.Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery.

J Biol Chem, 2003 May 30, 278(22), 19870 - 7 Epub 2003 Mar 25.
Biochemical characterization of sinapoylglucose:choline sinapoyltransferase, a serine carboxypeptidase-like protein that functions as an acyltransferase in plant secondary metabolism; Shirley AM et al.; Recently, serine carboxypeptidase-like (SCPL) proteins that catalyze transacylation reactions in plant secondary metabolism have been identified from wild tomato and Arabidopsis . These include sinapoylglucose: choline sinapoyltransferase (SCT), an enzyme that functions in Arabidopsis sinapate ester synthesis . SCT and the other known SCPL acyltransferases all share the conserved serine, aspartic acid, and histidine residues employed for catalysis by classical serine carboxypeptidases, although the importance of these residues and the mechanism by which this class of SCPL proteins catalyze acyltransferase reactions is unknown . To characterize further SCT and its catalytic mechanism, we have employed the Saccharomyces cerevisiae vacuolar protein localization 1 mutant, which secretes the serine carboxypeptidase, carboxypeptidase Y, and other proteins normally targeted to the vacuole . When expressed in this strain, SCT is similarly secreted . SCT has been purified from the yeast medium and used for kinetic characterization of the protein . Immunological analysis of SCT has revealed that the expected 50-kDa mature protein is proteolytically processed in yeast and in planta, most likely resulting in the production of a heterodimer derived from a 30- and 17-kDa polypeptide.

J Biol Chem, 2003 May 23, 278(21), 19347 - 51 Epub 2003 Mar 25.
Cleavage of 14-3-3 protein by caspase-3 facilitates bad interaction with Bcl-x(L) during apoptosis; Won J et al.; The 14-3-3 epsilon protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system . The cellular 14-3-3 epsilon protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells . Asp238 of the 14-3-3 epsilon protein was determined as the site of cleavage by caspase-3 . The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death-promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A) . However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of the 14-3-3 epsilon protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A) . The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell death by releasing the associated Bad from the 14-3-3 protein and facilitates Bad translocation to the mitochondria and its interaction with Bcl-x(L).

Gene, 2003 Mar 13, 306, 127 - 34
A highly efficient and sensitive screening method for trans-activation activity of estrogen receptors; Chen Z et al.; We describe a highly efficient and sensitive yeast-based screening method for isolating human estrogen receptor alpha (ERalpha) mutants with altered trans-activation activity . This method takes advantage of the fact that estrogen receptor is a ligand-activated transcription factor, and links the transactivation activity of estrogen receptor to the growth rate of yeast cells . We used this method to screen a library of human ERalpha mutants created by random mutagenesis of the ligand binding domain of human ERalpha in the presence of ligand 17beta-estradiol (E(2)) . We isolated several human ERalpha mutants with significantly altered trans-activation activity toward E(2) in yeast cells . We also used this method to screen a library of chemical compounds and showed that it can be used to rapidly identify estrogenic compounds and the different cell growth rates for these estrogenic compounds correlated well with their relative binding affinities . Thus, this method is suitable for selecting novel estrogenic compounds and estrogen receptor mutants . In principle, this method might also be used to isolate mutants of any nuclear receptors with altered trans-activation activity, which may greatly facilitate their structural and functional studies.

Biochem Soc Symp, 2002, (69), 117 - 34
The glycomes of Caenorhabditis elegans and other model organisms; Haslam SM et al.; There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research . However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited . This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced . To date, only limited structural data on the glycosylated molecules of this organism have been reported . Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C . elegans; high-mannose structures dominate, with only minor amounts of complex-type structures . Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fuc alpha 1-2 Gal 1-2 Man as peripheral structures . The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter . Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

Mol Genet Genomics, 2003 Mar, 268(6), 791 - 8 Epub 2003 Jan 25.
The different (sur)faces of Rap1p; Pina B et al.; The DNA-binding protein Rap1p fulfills many different functions in the yeast cell . It targets 5% of the promoters, acting both as a transcriptional activator and as a repressor, depending on the DNA sequence context . In addition, Rap1p is an essential structural component of yeast telomeres, where it contributes to telomeric silencing . Here we review the evidence indicating that Rap1p function is modulated by the precise architecture of the its binding site and its surroundings: long tracts of telomeric repeats for telomeric functions, specific sequences and orientation for maximal transcriptional activation, and specific DNA recognition sequences for complementary factors in other cases . Many of these functions are probably related to chromatin organization around Rap1p DNA binding sites, resulting from the very tight binding of Rap1p to DNA . We propose that Rap1p alters its structure to bind to different versions of its DNA binding sequence . These structural changes may modulate the function of Rap1p domains, providing different interacting surfaces for binding to specific co-operating factors, and thus contributing to the diversity of Rap1p function.

Mol Genet Genomics, 2003 Mar, 268(6), 750 - 60 Epub 2003 Feb 12.
Meiotic segregation of a homeologous chromosome pair; Maxfield Boumil R et al.; During meiosis, the alignment of homologous chromosomes facilitates their subsequent migration away from one another to opposite spindle poles at anaphase I . Recombination is part of the mechanism by which chromosomes identify their homologous partners, and serves to link the homologs in a way that, in some organisms, has been shown to promote proper attachment to the meiotic spindle . We have built a diploid strain that contains a pair of homeologous chromosomes V': one is derived from Saccharomyces cerevisiae and one originates from S . carlsbergensis . Sequence analysis reveals that these chromosomes share 71% sequence identity . The homeologs experience high levels of meiotic double-stranded breaks . Despite their relatedness and their competence to initiate recombination, the meiotic segregation behavior of the homeologous chromosomes suggests that, in most meioses, they are partitioned by a meiotic segregation system that has been shown previously to partition non-exchange chromosomes and pairs with no homology . Though the homeologous chromosomes show a degree of meiotic segregation fidelity similar to that of other non-exchange pairs, our data provide evidence that their limited sequence homology may provide some bias in meiotic partner choice.

Nucleic Acids Res, 2003 Apr 1, 31(7), 1877 - 87
The human Imp3 and Imp4 proteins form a ternary complex with hMpp10, which only interacts with the U3 snoRNA in 60-80S ribonucleoprotein complexes; Granneman S et al.; Ribosome biogenesis requires a vast number of trans-acting factors many of which are required for the chemical modification and processing of the pre-rRNA component . The U3 snoRNP complex is required for the early cleavage steps in pre-rRNA processing . We have cloned cDNAs encoding the human and mouse homologs of the yeast U3 snoRNP-associated proteins Imp3 and Imp4 . Both human proteins localize to nucleoli and interact with the U3 snoRNA . The results of complementation experiments show that, in contrast to mouse Imp4, mouse Imp3 can partially alleviate the growth defect of the corresponding yeast null strain, indicating that the role of Imp3 in pre-rRNA processing is evolutionarily conserved . The results of density gradient centrifugation experiments show that, in contrast to hU3-55K, the human Imp3 and Imp4 proteins predominantly interact with the U3 snoRNA in 60-80S ribonucleoprotein complexes . In addition, we have found that hImp3, hImp4 and hMpp10 can form a stable hetero-trimeric complex in vitro, which is generated by direct interactions of both hImp3 and hImp4 with hMpp10 . The analysis of hImp3 and hImp4 mutants indicated that their binding to hMpp10 correlates with their nucleolar accumulation, strongly suggesting that the formation of the ternary complex of hImp3, hImp4 and hMpp10 is required for their association with nucleolar components.

Nucleic Acids Res, 2003 Apr 1, 31(7), 1859 - 68
AINTEGUMENTA utilizes a mode of DNA recognition distinct from that used by proteins containing a single AP2 domain; Krizek BA; The Arabidopsis protein AINTEGUMENTA (ANT) is an important regulator of organ growth during flower development . ANT is a member of the AP2 subclass of the AP2/ERF family of plant-specific transcription factors . These proteins contain either one or two copies of a DNA-binding domain called the AP2 domain . Here, it is shown that ANT can act as a transcriptional activator in yeast through binding to a consensus ANT-binding site . This activity was used as the basis for a genetic screen to identify amino acids that are critical for the DNA binding ability of ANT . Mutants that showed reduced or no activation of a reporter gene under the control of ANT-binding sites were identified . The mutations identified in the screen as well as additional site-directed mutations suggest that the mode of DNA recognition by members of the AP2 subfamily is distinct from that of ERF proteins . Surprisingly, it appears that each AP2 domain of ANT uses different amino acids to contact DNA . Identification of several linker mutations argues that this sequence acts in the positioning of each AP2 domain on the DNA or makes direct DNA contacts.

Nucleic Acids Res, 2003 Apr 1, 31(7), 1853 - 8
Conditional gene expression by controlling translation with tetracycline-binding aptamers; Suess B et al.; We present a conditional gene expression system in Saccharomyces cerevisiae which exploits direct RNA-metabolite interactions as a mechanism of genetic control . We inserted preselected tetracycline (tc) binding aptamers into the 5'-UTR of a GFP encoding mRNA . While aptamer insertion generally reduces GFP expression, one group of aptamers displayed an additional, up to 6-fold, decrease in fluorescence upon tc addition . Regulation is observed for aptamers inserted cap-proximal or near the start codon, but is more pronounced from the latter position . Increasing the thermodynamic stability of the aptamer augments regulation but reduces expression of GFP . Decreasing the stability leads to the opposite effect . We defined nucleotides which influence the regulatory properties of the aptamer . Exchanging a nucleotide probably involved in tc binding only influences regulation, while mutations at another position alter expression in the absence of tc, without affecting regulation . Thus, we have developed and characterized a regulatory system which is easy to establish and controlled by a non-toxic, small ligand with good cell permeability.

Mol Biol Evol, 2003 Apr, 20(4), 484 - 90 Epub 2003 Mar 05.
Significantly different patterns of amino acid replacement after gene duplication as compared to after speciation; Seoighe C et al.; We have performed a large-scale analysis of amino acid sequence evolution after gene duplication by comparing evolution after gene duplication with evolution after speciation in over 1,800 phylogenetic trees constructed from manually curated alignments of protein domains downloaded from the PFAM database . The site-specific rate of evolution is significantly altered by gene duplication . A significant increase in the proportion of amino acid substitutions at constrained (slowly evolving) sites after duplication was observed . An increase in the proportion of replacements at normally constrained amino acid sites could result from relaxation of purifying selective pressure . However, the proportion of amino acid replacements involving radical changes in amino acid properties after duplication does not appear to be significantly increased by relaxed selective pressure . The increased proportion of replacements at constrained sites was observed over a relatively large range of protein change (up to 25% amino acid replacements per site) . These findings have implications for our understanding of the nature of evolution after duplication and may help to shed light on the evolution of novel protein functions through gene duplication.

J Biol Chem, 2003 May 30, 278(22), 20268 - 77 Epub 2003 Mar 24.
Interactions between Piccolo and the actin/dynamin-binding protein Abp1 link vesicle endocytosis to presynaptic active zones; Fenster SD et al.; Piccolo is a high molecular weight multi-domain protein shown to be a structural component of the presynaptic CAZ (cytoskeletal matrix assembled at active zones) . These features indicate that Piccolo may act to scaffold proteins involved in synaptic vesicle endo- and exocytosis near their site of action . To test this hypothesis, we have utilized a functional cell-based endocytosis assay and identified the N-terminal proline-rich Q domain in Piccolo as a region that interferes with clathrin-mediated endocytosis . Utilizing the Piccolo Q domain as bait in a yeast two-hybrid screen, we have identified the F-actin-binding protein Abp1 (also called SH3P7 or HIP-55) as a potential binding partner for this domain . The physiological relevance of this interaction is supported by in vitro binding studies, colocalization in nerve terminals, in vivo recruitment studies, and immunoprecipitation experiments . Intriguingly, Abp1 binds to both F-actin and the GTPase dynamin and has been implicated in linking the actin cytoskeleton to clathrin-mediated endocytosis . Our results suggest that Piccolo, as a structural protein of the CAZ, may serve to localize Abp1 at active zones where it can actively participate in creating a functional connection between the dynamic actin cytoskeleton and synaptic vesicle recycling.

J Biol Chem, 2003 May 30, 278(22), 19826 - 33 Epub 2003 Mar 24.
Vps9p CUE domain ubiquitin binding is required for efficient endocytic protein traffic; Davies BA et al.; Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway . The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p . Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction . Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole . In addition, Vps9p is itself monoubiquitylated . Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis . These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.

J Cell Biol, 2003 Mar 31, 160(7), 1069 - 81 Epub 2003 Mar 24.
The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity; Li SJ et al.; Protein modification by the ubiquitin-like SUMO protein contributes to many cellular regulatory mechanisms . In Saccharomyces cerevisiae, both sumoylating and desumoylating activities are essential for viability . Of its two known desumoylating enzymes, Ubl-specific protease (Ulp)1 and Ulp2/Smt4, Ulp1 is specifically required for cell cycle progression . A approximately 200-residue segment, the Ulp domain (UD), is conserved among Ulps and includes a core cysteine protease domain that is even more widespread . Here we demonstrate that the Ulp1 UD by itself can support wild-type growth rates and in vitro can cleave SUMO from substrates . However, in cells expressing only the UD of Ulp1, many SUMO conjugates accumulate to high levels, indicating that the nonessential Ulp1 NH2-terminal domain is important for activity against a substantial fraction of sumoylated targets . The NH2-terminal domain also includes sequences necessary and sufficient to concentrate Ulp1 at nuclear envelope sites . Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase . Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others . These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity.

Genes Dev, 2003 Apr 1, 17(7), 859 - 72 Epub 2003 Mar 21.
Translational control by TOR and TAP42 through dephosphorylation of eIF2alpha kinase GCN2; Cherkasova VA et al.; Yeast protein kinase GCN2 stimulates the translation of transcriptional activator GCN4 by phosphorylating eIF2alpha in response to amino acid starvation . Kinase activation requires binding of uncharged tRNA to a histidyl tRNA synthetase-related domain in GCN2 . Phosphorylation of serine 577 (Ser 577) in GCN2 by another kinase in vivo inhibits GCN2 function in rich medium by reducing tRNA binding activity . We show that rapamycin stimulates eIF2alpha phosphorylation by GCN2, with attendant induction of GCN4 translation, while reducing Ser 577 phosphorylation in nonstarved cells . The alanine 577 (Ala 577) mutation in GCN2 (S577A) dampened the effects of rapamycin on eIF2alpha phosphorylation and GCN4 translation, suggesting that GCN2 activation by rapamycin involves Ser 577 dephosphorylation . Rapamycin regulates the phosphorylation of Ser 577 and eIF2alpha by inhibiting the TOR pathway . Rapamycin-induced dephosphorylation of Ser 577, eIF2alpha phosphorylation, and induction of GCN4 all involve TAP42, a regulator of type 2A-related protein phosphatases . Our results add a new dimension to the regulation of protein synthesis by TOR proteins and demonstrate cross-talk between two major pathways for nutrient control of gene expression in yeast.

Anal Biochem, 2003 Mar 15, 314(2), 253 - 9
Series of vectors to evaluate the position and the order of two different affinity tags for purification of protein complexes; Kimura A et al.; A series of protein expression vectors with dual-affinity tags has been developed . With these constructed vectors, FLAG and hexahistidine tags were fused to a given protein at either the N- or the C-terminal ends or both, for a total of six combinations . Three auxotrophy markers were introduced into each construct, thus yielding 18 different vectors . These vectors allow evaluation of different positions and orders of two different tags . To confirm the efficacy of these vectors, we purified a histone acetyltransferase (Esa1p)-containing complex . First, an appropriate position of the tags was selected through small-scale purification . Next, large-scale purification was done for the selected construct, yielding an Esa1p-containing complex that was comparable to an Esa1p-containing complex (NuA4) obtained by a conventional activity-based purification . These vectors provide a convenient way to select the best position of tags for efficient purification of protein complexes also applicable in proteomics studies.

Cell, 2003 Mar 21, 112(6), 793 - 803
Structural basis for the function of the beta subunit of the eukaryotic signal recognition particle receptor; Schwartz T et al.; Protein translocation across and insertion into membranes is a process essential to all life forms . In higher eukaryotes, this process is initiated by targeting the translating ribosome to the endoplasmic reticulum via the signal recognition particle (SRP) and its membrane-associated heterodimeric receptor (SR) . This targeting step is regulated by three G proteins, SRP54, SR alpha, and SR beta, which act in concert . Little is known about the regulatory role of SR beta . Here, we present the 1.7 A crystal structure of the SR beta-GTP subunit in complex with the interaction domain of SR alpha . Strikingly, the binding interface overlaps largely with the switch 1 region of SR beta . This finding, together with additional biochemical data, shows that the eukaryotic SR is a conditional and not an obligate heterodimer . The results suggest that the GTP/GDP switch cycle of SR beta functions as a regulatory switch for the receptor dimerization . We discuss the implications for the translocation pathway.

Cell, 2003 Mar 21, 112(6), 765 - 77
Chromosomal cohesin forms a ring; Gruber S et al.; The cohesin complex is essential for sister chromatid cohesion during mitosis . Its Smc1 and Smc3 subunits are rod-shaped molecules with globular ABC-like ATPases at one end and dimerization domains at the other connected by long coiled coils . Smc1 and Smc3 associate to form V-shaped heterodimers . Their ATPase heads are thought to be bridged by a third subunit, Scc1, creating a huge triangular ring that could trap sister DNA molecules . We address here whether cohesin forms such rings in vivo . Proteolytic cleavage of Scc1 by separase at the onset of anaphase triggers its dissociation from chromosomes . We show that N- and C-terminal Scc1 cleavage fragments remain connected due to their association with different heads of a single Smc1/Smc3 heterodimer . Cleavage of the Smc3 coiled coil is sufficient to trigger cohesin release from chromosomes and loss of sister cohesion, consistent with a topological association with chromatin.

Cell Stress Chaperones, 2002 Oct, 7(4), 374 - 86
Analysis of interactions between domains of a small heat shock protein, Hsp30 of Neurospora crassa; Plesofsky N et al.; The alpha-crystallin-related, small heat shock proteins (sHsps), despite their overall variability in sequence, have discrete regions of conserved sequence that are involved in structural organization, as well as nonconserved regions that may perform similar roles in each protein . Recent X-ray diffraction analyses of an archeal and a plant sHsp have revealed both similarities and differences in how they are organized, suggesting that there is variability, particularly in the oligomeric organization of sHsps . As an adjunct to crystallographic analysis of sHsp structure, we employed the yeast 2-hybrid system to detect interactions between peptide regions of the sHsp of Neurospora crassa, Hsp30 . We found that the conserved alpha-crystallin domain can be divided into N-terminal and C-terminal subdomains that interact strongly with one another . This interaction likely represents the tertiary contacts of the monomer that were visualized in the crystallographic structures of MjHsp16.5 and wheat Hsp16.9 . The conserved sHsp monomeric fold is apparently determined by these regions of conserved sequence . We found that the C-terminal portion of the alpha-crystallin domain also interacts with itself in 2-hybrid assays; however, this interaction requires peptide extension into the semiconserved carboxyl tail . This C-terminal association may represent a principal contact site between dimers that contributes to higher-order assembly, as seen for the crystallized sHsps.

J Biomol NMR, 2003 Mar, 25(3), 235 - 42
A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes; Gross JD et al.; A method for measuring intermolecular NOEs in protein complexes based on asymmetric sample deuteration is described . (13)C/(1)H-I,L,V-methyl, U-(2)H labeled protein is produced using the biosynthetic precursors {gamma-(13)C}-alpha-ketobutyrate and {gamma,gamma'-(13)C(2)}-alpha-ketoisovalerate . The labeled protein is mixed with its unlabeled binding partner and a 3D (13)C-HMQC-NOESY is recorded, yielding unambiguous intermolecular aromatic/methyl NOEs . A simple synthesis of the biosynthetic precursors via reaction of diethyl oxalate with alkyl Grignard compounds is reported . The method is demonstrated for a 35 kDa heterodimeric protein complex dissolved in a CHAPS micelle . This approach will facilitate the solution structure determination of protein/protein, protein/ligand or protein/nucleic acid complexes.

Annu Rev Phytopathol, 2003, 41, 399 - 427 Epub 2003 Mar 10.
Of smuts, blasts, mildews, and blights: cAMP signaling in phytopathogenic fungi; Lee N et al.; cAMP regulates morphogenesis and virulence in a wide variety of fungi including the plant pathogens . In saprophytic yeasts such as Saccharomyces cerevisiae, cAMP signaling plays an important role in nutrient sensing . In filamentous saprophytes, the cAMP pathway appears to play an integral role in vegetative growth and sporulation, with possible connections to mating . Infection-related morphogenesis includes sporulation (conidia and teliospores), formation of appressoria, infection hyphae, and sclerotia . Here, we review studies of cAMP signaling in a variety of plant fungal pathogens . The primary fungi to be considered include Ustilago maydis, Magnaporthe grisea, Cryphonectria parasitica, Colletotrichum and Fusarium species, and Erisyphe graminis . We also include related information on Trichoderma species that act as mycoparasites and biocontrol agents of phytopathogenic fungi . We point out similarities in infection mechanisms, conservation of signaling components, as well as instances of cross-talk with other signaling pathways.

Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 3965 - 70 Epub 2003 Mar 21.
The retinitis pigmentosa GTPase regulator (RPGR)- interacting protein: subserving RPGR function and participating in disk morphogenesis; Zhao Y et al.; Retinitis pigmentosa is a photoreceptor degenerative disease leading to blindness in adulthood . Leber congenital amaurosis (LCA) describes a more severe condition with visual deficit in early childhood . Defects in the retinitis pigmentosa GTPase regulator (RPGR) and an RPGR-interacting protein (RPGRIP) are known causes of retinitis pigmentosa and LCA, respectively . Both proteins localize in the photoreceptor connecting cilium (CC), a thin bridge linking the cell body and the light-sensing outer segment . We show that RPGR is absent in the CC of photoreceptors lacking RPGRIP, but not vice versa . Mice lacking RPGRIP elaborate grossly oversized outer segment disks resembling a cytochalasin D-induced defect and have a more severe disease than mice lacking RPGR . Mice lacking both proteins are phenotypically indistinguishable from mice lacking RPGRIP alone . In vitro, RPGRIP forms homodimer and elongated filaments via interactions involving its coiled-coil and C-terminal domains . We conclude that RPGRIP is a stable polymer in the CC where it tethers RPGR and that RPGR depends on RPGRIP for subcellular localization and normal function . Our data suggest that RPGRIP is also required for disk morphogenesis, putatively by regulating actin cytoskeleton dynamics . The latter hypothesis may be consistent with a distant homology between the C-terminal domain of RPGRIP and an actin-fragmin kinase, predicted by fold recognition algorithms . A defect in RPGRIP encompasses loss of both functions, hence the more severe clinical manifestation as LCA.

Anesth Analg, 2003 Apr, 96(4), 1019 - 26, table of contents
Blockade of voltage-operated neuronal and skeletal muscle sodium channels by S(+)- and R(-)-ketamine; Haeseler G et al.; Besides its general anesthetic effect, ketamine has local anesthetic-like actions . We studied the voltage- and use-dependent interaction of S(+)- and R(-)-ketamine with two different isoforms of voltage-operated sodium channels, with a special emphasis on the difference in affinity between resting and inactivated channel states . Rat brain IIa and human skeletal muscle sodium channels were heterologously expressed in human embryonic kidney 293 cells . S(+)- and R(-)-ketamine reversibly suppressed whole-cell sodium inward currents; the 50% inhibitory concentration values at -70 mV holding potential were 240 +/- 60 microM and 333 +/- 93 microM for the neuronal isoform and 59 +/- 10 microM and 181 +/- 49 microM for the skeletal muscle isoform . S(+)-ketamine was significantly more potent than R(-)-ketamine in the skeletal muscle isoform only . Ketamine had a higher affinity to inactivated than to resting channels . However, the estimated difference in affinity between inactivated and resting channels was only 8- to 10-fold, and the time course of drug equilibration between inactivated and resting channels was too fast to cause use-dependent block at 10 Hz up to a concentration of 300 microM . These results suggest that ketamine is less effective than lidocaine-like local anesthetics in stabilizing the inactivated channel state . IMPLICATIONS: Blockade of sodium channels by ketamine shows voltage dependency, an important feature of local anesthetic action . However, ketamine is less effective than lidocaine-like local anesthetics in stabilizing the inactivated state . Because it does not elicit phasic blockade at small concentrations, its ability to reduce the firing frequency of action potentials may be small.

Biochem J, 2003 Jul 1, 373(Pt 1), 221 - 9
Properties of phagocyte NADPH oxidase p47-phox mutants with unmasked SH3 (Src homology 3) domains: full reconstitution of oxidase activity in a semi-recombinant cell-free system lacking arachidonic acid; Peng G et al.; In an early step in the assembly of the phagocyte NADPH oxidase, p47-phox translocates from the cytosol to the membrane, mediated by engagement of the N-termini of two p47-phox Src homology 3 (SH3) domains with a proline-rich region (PRR) in the p22-phox subunit of cytochrome b (558) . In response to phagocyte activation, several serine residues in a C-terminal arginine/lysine-rich domain of p47-phox are phosphorylated, leading to changes in the conformation of p47-phox and exposure of its N-terminal SH3 domain that is normally masked by internal association with the arginine/lysine-rich domain . We report that triple alanine substitutions at Asp-217, Glu-218 and Glu-223 in a short sequence that links the tandem p47-phox SH3 domains unmasked the N-terminal SH3 domain, similar to the effects of aspartic acid substitutions at Ser-310 and Ser-328 in the arginine/lysine-rich region . Recombinant p47-phox proteins with mutations in either the linker region or the arginine/lysine-rich domain were active in the absence of arachidonic acid stimulation in a cell-free NADPH oxidase system consisting of recombinant p67-phox, Rac1-guanosine 5'-{gamma-thio}triphosphate and neutrophil membranes . Supplementing neutrophil membranes with phosphoinositides or other negatively charged phospholipids markedly enhanced cell-free superoxide generation by these p47-phox mutants in the absence of arachidonic acid, to levels equivalent to those generated by wild-type p47-phox following arachidonic acid activation . This enhancement may be related to recruitment to the membrane of p47-phox mediated by a novel secondary phox homology (PX) domain binding site that broadly recognizes phospholipids . No specific enhancement by specific phosphorylated phosphatidylinositols was found to suggest a dominant role for the p47-phox primary PX domain binding site . Truncated p47-phox S310D S328D lacking the C-terminal PRR was inactive in the cell-free system without arachidonic acid, but was fully active with arachidonic acid . This suggests that activation of NADPH oxidase in an arachidonate-free cell-free system requires association of the p47-phox C-terminal PRR with the p67-phox C-terminal SH3 domain.

RNA, 2003 Apr, 9(4), 493 - 501
Lentivirus-delivered stable gene silencing by RNAi in primary cells; Stewart SA et al.; Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems . Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes . RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics . Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors . Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts . This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.

Biochem Biophys Res Commun, 2003 Mar 21, 302(4), 646 - 52
Molecular cloning, biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue; Nandan D et al.; The Src-homology 2 domain containing protein tyrosine phosphatase-1 (SHP-1) is involved in the pathogenesis of infection with Leishmania . Recently, we identified elongation factor-1 alpha (EF-1 alpha) from Leishmania donovani as a SHP-1 binding and activating protein {J . Biol . Chem . 277 (2002) 50190} . To characterize this apparent Leishmania virulence factor further, the cDNA encoding L . donovani EF-1 alpha was cloned and sequenced . Whereas nearly complete sequence conservation was observed amongst EF-1 alpha proteins from trypanosomatids, the deduced amino acid sequence of EF-1 alpha of L . donovani when compared to mammalian EF-1 alpha sequences showed a number of significant changes . Protein structure modeling-based upon the known crystal structure of EF-1 alpha for Saccharomyces cerevisiae-identified a hairpin loop present in mammalian EF-1 alpha and absent from the Leishmania protein which corresponded to a 12 amino acid deletion . Consistent with these structural differences, the sub-cellular distributions of L . donovani EF-1 alpha and host EF-1 alpha were strikingly different . Interestingly, infection of macrophages with L . donovani caused redistribution of host as well as pathogen EF-1 alpha . Since EF-1 alpha is essential for survival, the distinct biochemical and structural properties of Leishmania EF-1 alpha may provide a novel target for drug development.

Biochem Biophys Res Commun, 2003 Mar 21, 302(4), 635 - 45
U-box proteins as a new family of ubiquitin ligases; Hatakeyama S et al.; Ubiquitin-protein ligases (E3s) determine the substrate specificity of ubiquitylation and, until recently, had been classified into two families, the HECT and RING-finger families . The U-box is a domain of approximately 70 amino acids that is present in proteins from yeast to humans . The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates . We recently showed that mammalian U-box proteins, in conjunction with an E1 and an E2, mediate polyubiquitylation in the absence of a HECT type or RING-finger type E3 . U-box proteins have thus been defined as a third family of E3s . We here review recent progress in the characterization of U-box proteins and of their role in the quality control system that underlies the cellular stress response to the intracellular accumulation of abnormal proteins.

Biochem Biophys Res Commun, 2003 Mar 28, 303(1), 370 - 8
Characterization of Grp1p, a novel cis-Golgi matrix protein; Kim DW; A high copy suppressor screen with sec34-2, a temperature-sensitive mutant defective in the late stages of ER to Golgi transport, has resulted in the identification of a novel gene called GRP1 (also called RUD3) . GRP1 encodes a hydrophilic yeast protein related to the mammalian Golgi matrix protein golgin-160 . A large portion of the protein is predicted to form a coiled-coil structure . Although GRP1 is not essential for growth, the loss of Grp1p results in a growth defect at high temperature . GRP1 genetically interacts with several genes involved in vesicle targeting/fusion stages of ER to Golgi transport . Despite these interactions, pulse chase analysis using Grp1p-depleted cells did not reveal a significant delay in the transit of the vacuolar protease carboxypeptidase Y . Grp1p-depleted cells efficiently secreted invertase which was underglycosylated, suggesting some disturbance of Golgi function . Grp1p-GFP predominantly colocalizes with the cis-Golgi marker Och1p . Despite lacking a signal peptide and a significant stretch of hydrophobic amino acids, Grp1p pellets with membranes . It is extracted with 1M NaCl or 0.1M Na(2)CO(3) (pH 11.0), but is surprisingly insoluble in 1% Triton X-100 . Grp1p does not recycle to the ER when forward transport is blocked and a cis-Golgi marker (Och1p-HA), but not a trans-Golgi marker (Chs5p-HA), became dispersed in grp1 Delta cells after 1.5h incubation at 38.5 degrees C . Together, these data suggest that Grp1p is a novel matrix protein that is involved in the structural organization of the cis-Golgi.

Biochem Biophys Res Commun, 2003 Mar 28, 303(1), 46 - 51
Function of the loop residue Thr792 in human DNA topoisomerase II alpha; Suda N et al.; We studied the mutation effect of one of the putative loop residues Thr792 in human DNA topoisomerase II alpha (TOP2 alpha) . Thr792 mutants were expressed from high or low copy plasmids in a temperature sensitive yeast strain deficient in TOP2 (top2-1) . When expressed from a high copy plasmid, mutants with small side chains complemented the yeast defect; however, from a low copy plasmid, only wild-type, Ser, and Cys substitution mutants complemented the yeast defect . Interestingly, at the permissive temperature other mutants (e.g., Val, Gly, and Glu substitutions) showed the dominant negative effect to the top2-1 allele, which was not observed by the control alpha 4-helix mutants . T792E mutant was 10-fold less active than wild-type and the T792P had no decatenation activity in vitro . These results suggest that Thr792 in human TOP2 alpha is involved in enzyme catalysis.

Curr Biol, 2003 Mar 18, 13(6), R229 - 30
Gene duplications: the gradual evolution of functional divergence; Brookfield JF; Budding yeast provides a useful resource for studies of gene function . A new analysis of the fitness effects of deletion mutations in budding yeast reveals that genes that have duplicates create lower fitness losses when inactivated than do genes that are singletons.

Curr Biol, 2003 Mar 18, 13(6), 455 - 63
A WASp homolog powers actin polymerization-dependent motility of endosomes in vivo; Chang FS et al.; BACKGROUND: WASp/SCAR proteins activate the Arp2/3 complex to nucleate actin filament assembly and are thought to have important roles in endocytosis . WASp is required for efficient endocytosis of antigen receptors, N-WASp promotes actin polymerization-dependent movement of endomembrane vesicles, and Las17 (a yeast WASp homolog) is required for endocytic internalization . However, it is unknown whether movement of endosomes or other organelles requires activation of the Arp2/3 complex by members of the WASp/SCAR family . RESULTS: Fluorescence video microscopy of yeast cells expressing a GFP-tagged G protein-coupled receptor (Ste2-GFP) as an endocytic marker revealed that endosomes and the lysosome-like vacuole are highly motile . Endosome/vacuole motility required actin polymerization, as indicated by sensitivity to latrunculin A, whereas microtubules were uninvolved . Endosome/vacuole motility did not require actin cables or myosin V (a MYO2 gene product), which moves secretory vesicles and the Golgi apparatus and mediates vacuole segregation . However, endosome motility required Las17, a WASp homolog . In contrast to other processes involving Las17, endosome/vacuole motility required the WCA domain of Las17, which is necessary and sufficient to activate the Arp2/3 complex . CONCLUSIONS: Endosome/vacuole motility in vivo requires actin polymerization stimulated by the WASp homolog Las17 . WASp/SCAR family members in mammalian cells may have similar functions . Defects in endosome/lysosome motility may contribute to deficits in lymphocyte or macrophage function observed in human patients lacking WASp or developmental defects in N-WASp-deficient mice.

J Proteome Res, 2002 May-Jun, 1(3), 211 - 5
Code developments to improve the efficiency of automated MS/MS spectra interpretation; Sadygov RG et al.; We report the results of our work to facilitate protein identification using tandem mass spectra and protein sequence databases . We describe a parallel version of SEQUEST (SEQUEST-PVM) that is tolerant toward arithmetic exceptions . The changes we report effectively separate search processes on slave nodes from each other . Therefore, if one of the slave nodes drops out of the cluster due to an error, the rest of the cluster will carry the search process to the end . SEQUEST has been widely used for protein identifications . The modifications made to the code improve its stability and effectiveness in a high-throughput production environment . We evaluate the overhead associated with the parallelization of SEQUEST . A prior version of software to preprocess LC/MS/MS data attempted to differentiate the charge states of ions . Singly charged ions can be accurately identified, but the software was unable to reliably differentiate tandem mass spectra of +2 and +3 charge states . We have designed and implemented a computational approach to narrow charge states of precursor ions from nominal resolution ion-trap tandem mass spectra . The preprocessing code, 2to3, determines the charge state of the precursor ion using its mass-to-charge ratio (m/z) and fragment ions contained in the tandem mass spectrum . For each possible charge state the program calculates the expected fragment ions that account for precursor ion m/z values . If any one of the numbers is less than an empirically determined threshold value then the spectrum corresponding to that charge state is removed . If both numbers are higher than the threshold value then +2 and +3 copies of the spectrum are kept . We present the comparison of results from protein identification experiments with and without using 2 to 3 . It is shown that by determining the charge state and eliminating poor quality spectra 2to3 decreases the number of spectral files to be searched without affecting the search results . The decrease reduces computer requirements and researcher efforts for analysis of the results.

J Biol Chem, 2003 Mar 14, 278(11), 9258 - 66
The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type); Cohen R et al.; Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion . Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains . In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release . For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells . Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission . We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics . Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit . Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity . This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit . The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel . It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.

Yi Chuan Xue Bao, 2002, 29(11), 953 - 8
{LIM protein KyoT2 interacts with human tight junction protein ZO-2-i3}; Huang HY et al.; It was reported that LIM protein KyoT2 negatively regulated transcription by association with the RBP-J DNA-binding protein . Using yeast two-hybrid system with LIM protein KyoT2 as a bait, we have isolated an alternatively spliced form of human tight junction protein 2--ZO-2-i3 . Sequence analysis indicated that ZO-2-i3 is composed of 19 exons, and selected usage of exons led to an alteration in the region following the kinase domain as compared with the published sequence . To identify the interaction between LIM protein KyoT2 and ZO-2-i3, yeast two-hybrid system, purification of KyoT2 protein, and GST pull-down assay were performed in the experiments . After KyoT2 and ZO-2-i3 changed vectors, positive two-hybrid yeast was obtained . Using KyoT2 protein and antibody in GST pull-down assay positive result was also obtained . Therefore we conformed KyoT2 interacted ZO-2-i3 in vitro . Furthermore it was identified in yeast that KyoT2 associated with ZO-2-i3 through its LIM2 domain.

Chromosoma, 2003 Mar, 111(6), 369 - 76 Epub 2003 Jan 25.
Mammalian meiosis involves DNA double-strand breaks with 3' overhangs; Zenvirth D et al.; Meiotic recombination in yeast is initiated at DNA double-strand breaks (DSBs), processed into 3' single-strand overhangs that are active in homology search, repair and formation of recombinant molecules . Are 3' overhangs recombination intermediaries in mouse germ cells too? To answer this question we developed a novel approach based on the properties of the Klenow enzyme . We carried out two different, successive in situ Klenow enzyme-based reactions on sectioned preparations of testicular tubules . Signals showing 3' overhangs were observed during wild-type mouse spermatogenesis, but not in Spo11(-/-) males, which lack meiotic DSBs . In Atm(-/-) mice, abundant positively stained spermatocytes were present, indicating an accumulation of non-repaired DSBs, suggesting the involvement of ATM in repair of meiotic DSBs . Thus the processing of DSBs into 3' overhangs is common to meiotic cells in mammals and yeast, and probably in all eukaryotes.

FEMS Microbiol Lett, 2003 Mar 14, 220(1), 57 - 65
HIV Tat, its TARgets and the control of viral gene expression; Brigati C et al.; The human immunodeficiency virus (HIV-1) (transactivator of transcription (Tat)) protein is a pleiotropic factor that induces a broad range of biological effects in numerous cell types . At the HIV promoter, Tat is a powerful transactivator of gene expression, which acts by both inducing chromatin remodeling and by recruiting elongation-competent transcriptional complexes onto the viral LTR . Besides these transcriptional activities, Tat is released outside the cells and interacts with different cell membrane-associated receptors . Finally, extracellular Tat can be internalized by cells through an active endocytosis process . Here we discuss some of the molecular mechanisms involved in intracellular and extracellular Tat function.

J Agric Food Chem, 2003 Mar 26, 51(7), 2089 - 95
Study of low molecular weight phenolic compounds during the aging of sparkling wines manufactured with red and white grape varieties; Pozo-Bayon MA et al.; Thirty-two phenolic compounds of low molecular weight were identified in 36 white, blanc de noir, and rose sparkling wines by using HPLC with photodiode array and mass spectrometry detection . Some of the identified compounds, such as cis- and trans-ethylcaftaric, cis- and trans-ethylcaffeic, and cis- or trans-ethyl-p-coumaric acids, 2R,3R-dihydroquercetin, 2R,3R-dihydrokaempferol 3-O-beta-d-glucoside, and a lignan derivative are described for the first time in sparkling wines manufactured with grapes of red varieties . This is also the first time that cis- or trans-diethylfertaric acids have been identified in wines . When cluster analysis was applied to the data of 19 of the 32 identified compounds, the greatest differences found in the low molecular weight phenolic compounds in sparkling wines were due to the grape variety from which they were manufactured, whereas aging time did not significantly influence phenolic composition . Nine phenolic compounds, that is, trans-p-coumaric and trans-caftaric acids, trans-resveratrol glucoside, cis-coutaric, trans-coutaric, cis-p-coumaric, and cis-caftaric acids, tryptophol, and syringic acid, permit the wines to be classified correctly in accordance with the grape variety from which they were manufactured.

J Agric Food Chem, 2003 Mar 26, 51(7), 1850 - 7
Estrogenic activity in white and red wine extracts; Klinge CM et al.; Red wine is enriched in resveratrol, trans-3,5,4'-trihydroxystilbene, a compound in grape skin that inhibits the development of pre-neoplastic lesions in mouse mammary tumor cells in culture and inhibits cancer cell proliferation in vitro . Grapes also contain other bioactive compounds including flavonoids, flavans, and anthocyanins . The estrogenic activities of extracts prepared from one white (Freie Weingartner Wachau, Gruner Veltliner, Austria) and two red wines (Woodbridge, Cabernet Sauvignon, California; and Lenz Moser Prestige, Blaufrankisch Barrique, Austria) were examined and compared with those induced by estradiol (E(2)) and trans-resveratrol . First, the estrogenic activity of the wine extracts was evaluated in a yeast estrogen screen (YES) assay, in which yeast express copper-inducible estrogen receptor alpha (ERalpha) and an estrogen-response-element (ERE)-driven beta-galactosidase reporter . In YES, the white wine extract showed no estrogenic activity . In contrast, both of the red wine extracts showed estrogenic activity equivalent to that of 0.2 nM E(2) . Similarly, the white wine extract showed no transcriptional activity with either ERalpha and ERbeta in transiently transfected CHO-K1 cells . In contrast, both red wine extracts stimulated ERE-reporter activity in a concentration-dependent manner that was inhibited by 4-hydroxytamoxifen (4-OHT), indicating that the observed transcriptional activity was ER-mediated . The red wine extracts showed significantly higher ERbeta versus ERalpha agonist activity . Resveratrol showed no agonist activity in YES but activated ERalpha and ERbeta in CHO-K1 cells in a concentration-dependent manner that was inhibited by 4-OHT . This indicates that resveratrol requires mammalian cell components that are absent in yeast for estrogen agonist activity, whereas the estrogenic activity of wine extracts is directly through ERalpha and does not require mammalian cell factors such as coactivators . The estrogenic activity in red wine found by using YES indicates that estrogenic compounds other than resveratrol are present . Chemical analysis clearly showed that the trans-resveratrol content of the red wine extracts was 1 order of magnitude below the detection limit for YES assay.

Exp Mol Med, 2003 Feb 28, 35(1), 1 - 7
Conjugation and deconjugation of ubiquitin regulating the destiny of proteins; Baek KH; The homeostasis for a number of cellular proteins is regulated by not only phosphorylation and dephosphorylation, but also ubiquitination and deubiquitination . A number of proteins involved in the degradation of polypeptides have been isolated in various eukaryotic organisms from Saccharomyces cerevisiae to human . Recently, several deubiquitinating enzymes, classified into either the Ub C-terminal hydrolase (UCH) or the Ub-specific processing protease (UBP), have been reported . It has been shown that they contain conserved domains including Cys, His, and Asp residues throughout the enzyme . These proteins have been demonstrated that Cys and His domains are critical for deubiquitinating enzymatic activity . Recently, we have shown that the Asp domain localized between Cys and His domains is also essential for cleaving the ubiquitin from protein substrates . Mouse deubiquitinating enzymes including DUB-1, DUB-2, and DUB-2A have been isolated and they showed the expression specificity . Of these, DUB- 1 and DUB-2 are expressed in lymphocytes depending on the presence of cytokines (interleukin-3 in B-lymphocytes and interleukin-2 in T- lymphocytes, respectively), indicating that they are involved in cytokine signaling pathways . Isolation of all putative DUBs will help to identify their substrates and to regulate the homeostasis of cellular proteins, especially in proliferative cells.

J Cell Biol, 2003 Mar 17, 160(6), 887 - 97
Identification of an organelle-specific myosin V receptor; Ishikawa K et al.; Class V myosins are widely distributed among diverse organisms and move cargo along actin filaments . Some myosin Vs move multiple types of cargo, where the timing of movement and the destinations of selected cargoes are unique . Here, we report the discovery of an organelle-specific myosin V receptor . Vac17p, a novel protein, is a component of the vacuole-specific receptor for Myo2p, a Saccharomyces cerevisiae myosin V . Vac17p interacts with the Myo2p cargo-binding domain, but not with vacuole inheritance-defective myo2 mutants that have single amino acid changes within this region . Moreover, a region of the Myo2p tail required specifically for secretory vesicle transport is neither required for vacuole inheritance nor for Vac17p-Myo2p interactions . Vac17p is localized on the vacuole membrane, and vacuole-associated Myo2p increases in proportion with an increase in Vac17p . Furthermore, Vac17p is not required for movement of other cargo moved by Myo2p . These findings demonstrate that Vac17p is a component of a vacuole-specific receptor for Myo2p . Organelle-specific receptors such as Vac17p provide a mechanism whereby a single type of myosin V can move diverse cargoes to distinct destinations at different times.

Proc R Soc Lond B Biol Sci, 2003 Mar 7, 270(1514), 457 - 66
How the global structure of protein interaction networks evolves; Wagner A; Two processes can influence the evolution of protein interaction networks: addition and elimination of interactions between proteins, and gene duplications increasing the number of proteins and interactions . The rates of these processes can be estimated from available Saccharomyces cerevisiae genome data and are sufficiently high to affect network structure on short time-scales . For instance, more than 100 interactions may be added to the yeast network every million years, a fraction of which adds previously unconnected proteins to the network . Highly connected proteins show a greater rate of interaction turnover than proteins with few interactions . From these observations one can explain (without natural selection on global network structure) the evolutionary sustenance of the most prominent network feature, the distribution of the frequency P(d) of proteins with d neighbours, which is broad-tailed and consistent with a power law, that is: P(d) proportional, variant d (-gamma).

Biochemistry, 2003 Mar 25, 42(11), 3189 - 202
Sterol carrier protein-2 functions in phosphatidylinositol transfer and signaling; Schroeder F et al.; Over 20 years ago, it was reported that liver cytosol contains at least two distinct proteins that transfer phosphatidylinositol in vitro, phosphatidylinositol transfer protein (PITP) and a pH 5.1 supernatant fraction containing sterol carrier protein-2 (SCP-2) . In contrast to PITP, there has been minimal progress on the structural and functional significance of SCP-2 in phosphatidylinositol transport . As shown herein, highly purified, recombinant SCP-2 stimulated up to 13-fold the rapid (s) transfer of radiolabeled phosphatidylinositol (PI) from microsomal donor membranes to highly curved acceptor membranes . SCP-2 bound to microsomes in vitro and overexpression of SCP-2 in transfected L-cells resulted in the following: (i) redistribution of phosphatidylinositols from intracellular membranes (mitochondria and microsomes) to the plasma membrane; (ii) enhancement of insulin-mediated inositol-triphosphate production; and (iii) 5.5-fold down regulation of PITP . Like PITP, SCP-2 binds two ligands required for vesicle budding from the Golgi, PI, and fatty acyl CoA . Double immunolabeling confocal microscopy showed SCP-2 significantly colocalized with caveolin-1 in the cytoplasm (punctate) and plasma membrane of SCP-2 overexpressing hepatoma cells (72%), HT-29 cells (58%), and SCP-2 overexpressing L-cells (37%) . Taken together, these data show for the first time that SCP-2 plays a hitherto unrecognized role in intracellular phosphatidylinositol transfer, distribution, and signaling.

Anal Chem, 2003 Mar 1, 75(5), 1155 - 63
Statistical characterization of ion trap tandem mass spectra from doubly charged tryptic peptides; Tabb DL et al.; Collision-induced dissociation (CID) is a common ion activation technique used to energize mass-selected peptide ions during tandem mass spectrometry . Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence . The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID . An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process . While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms . Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities . Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond . Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn . These trends are partially responsible for the complexity of peptide tandem mass spectra.

Nat Genet, 2003 Apr, 33(4), 522 - 6 Epub 2003 Mar 17.
Sir3p phosphorylation by the Slt2p pathway effects redistribution of silencing function and shortened lifespan; Ray A et al.; An organism's lifespan is modulated by environmental conditions . When nutrients are abundant, the metabolism of many organisms shifts to growth or reproduction at the expense of longer lifespan, whereas a scarcity of nutrients reverses this shift . These correlations suggest that organisms respond to environmental changes by altering their metabolism to promote either reproduction and growth or long life . The only previously reported signaling mechanism involved in this response is the nutrient-responsive insulin/insulin-like growth factor-1 receptor pathway . Here we report another pathway that controls the length of yeast lifespan . Commitment to cell growth activates the Slt2p MAP kinase pathway, which phosphorylates the transcriptional silencing protein Sir3p, resulting in a shorter lifespan . Elimination of the Sir3p phosphorylation site at Ser275 extended lifespan by 38% . Lifespan extension occurs by a mechanism that is independent of suppressing rDNA recombination . Thus, Slt2p is an enzymatic regulator of silencing function that couples commitment to cell growth and shorter lifespan.

Mol Cell Biol, 2003 Apr, 23(7), 2239 - 50
Mitochondrial protein import: recognition of internal import signals of BCS1 by the TOM complex; Stan T et al.; BCS1, a component of the inner membrane of mitochondria, belongs to the group of proteins with internal, noncleavable import signals . Import and intramitochondrial sorting of BCS1 are encoded in the N-terminal 126 amino acid residues . Three sequence elements were identified in this region, namely, the transmembrane domain (amino acid residues 51 to 68), a presequence type helix (residues 69 to 83), and an import auxiliary region (residues 84 to 126) . The transmembrane domain is not required for stable binding to the TOM complex . The Tom receptors (Tom70, Tom22 and Tom20), as determined by peptide scan analysis, interact with the presequence-like helix, yet the highest binding was to the third sequence element . We propose that the initial recognition of BCS1 precursor at the surface of the organelle mainly depends on the auxiliary region and does not require the transmembrane domain . This essential region represents a novel type of signal with targeting and sorting functions . It is recognized by all three known mitochondrial import receptors, demonstrating their capacity to decode various targeting signals . We suggest that the BCS1 precursor crosses the TOM complex as a loop structure and that once the precursor emerges from the TOM complex, all three structural elements are essential for the intramitochondrial sorting to the inner membrane.

J Cell Sci, 2003 Apr 15, 116(Pt 8), 1563 - 77
Genome-wide expression screens indicate a global role for protein kinase CK2 in chromatin remodeling; Barz T et al.; Protein kinase CK2, a vital, pleiotropic and highly conserved serine/threonine phosphotransferase is involved in transcription-directed signaling, gene control and cell cycle regulation and is suspected to play a role in global processes . Searching for these global roles, we analyzed the involvement of CK2 in gene expression at cell cycle entry by using genome-wide screens . Comparing expression profiles of Saccharomyces cerevisiae wild-type strains with strains with regulatory or catalytic subunits of CK2 deleted, we found significant alterations in the expression of genes at all cell cycle phases and often in a subunit- and isoform-specific manner . Roughly a quarter of the genes known to be regulated by the cell cycle are affected . Functionally, the genes are involved with cell cycle entry, progression and exit, including spindle pole body formation and dynamics . Strikingly, most CK2-affected genes exhibit no common transcriptional control features, and a considerable proportion of temporarily altered genes encodes proteins involved in chromatin remodeling and modification, including chromatin assembly, (anti-)silencing and histone (de-)acetylation . In addition, various metabolic pathway and nutritional supply genes are affected . Our data are compatible with the idea that CK2 acts at different levels of cellular organization and that CK2 has a global role in transcription-related chromatin remodeling.

Science, 2003 Mar 14, 299(5613), 1747 - 51
Protein insertion into the mitochondrial inner membrane by a twin-pore translocase; Rehling P et al.; The mitochondrial inner membrane imports numerous proteins that span it multiple times using the membrane potential Deltapsi as the only external energy source . We purified the protein insertion complex (TIM22 complex), a twin-pore translocase that mediated the insertion of precursor proteins in a three-step process . After the precursor is tethered to the translocase without losing energy from the Deltapsi, two energy-requiring steps were needed . First, Deltapsi acted on the precursor protein and promoted its docking in the translocase complex . Then, Deltapsi and an internal signal peptide together induced rapid gating transitions in one pore and closing of the other pore and drove membrane insertion to completion . Thus, protein insertion was driven by the coordinated action of a twin-pore complex in two voltage-dependent steps.

Mol Endocrinol, 2003 Jun, 17(6), 1095 - 105 Epub 2003 Mar 13.
Cellular context of coregulator and adaptor proteins regulates human adenovirus 5 early region 1A-dependent gene activation by the thyroid hormone receptor; Meng X et al.; In mammalian cells, the human adenovirus type 5 early region 1A (E1A) oncoprotein functions as a thyroid hormone (TH)-dependent activator of the thyroid hormone receptor (TR) . Interestingly, in the cellular context of the yeast Saccharomyces cerevisiae, E1A acts as a TR-specific constitutive coactivator that is down-regulated by TH . TH reduces the interaction of E1A with the TR in yeast but not HeLa cells . The N-terminal 82 amino acids of E1A are sufficient for coactivation in yeast and residues 4-29 are essential . In yeast, expression of the nuclear receptor corepressor (N-CoR) could down-regulate constitutive transcriptional activation of the TR by E1A, whereas expression of the glucocorticoid receptor interacting protein 1 (GRIP-1) coactivator reconstituted the E1A-induced pattern of enhanced TH-dependent gene activation by TR observed in mammalian cells . We further show that the mating type switching gene (SWI)/sucrose nonfermenting (SNF) gene chromatin remodeling complex is required for both TH/GRIP-1- and E1A-dependent coactivator function, whereas the general control nonrepressed protein (GCN5)/alteration/deficiency in activation protein (ADA2) components of the SPT, ADA, GCN5, acetylation (SAGA) transcriptional adaptor complex are required for TH/GRIP-1, but not E1A-dependent activation of the TR . Taken together, these studies demonstrate that the novel TR-specific coactivator function of E1A in yeast depends on the SWI/SNF chromatin remodeling complex and can be further influenced by changes in the cellular complement of transcriptional coregulatory proteins.

J Biol Chem, 2003 May 16, 278(20), 17912 - 7 Epub 2003 Mar 10.
The role of transcription initiation factor IIIB subunits in promoter opening probed by photochemical cross-linking; Kassavetis GA et al.; The core transcription initiation factor (TF) IIIB recruits its conjugate RNA polymerase (pol) III to the promoter and also plays an essential role in promoter opening . TFIIIB assembled with certain deletion mutants of its Brf1 and Bdp1 subunits is competent in pol III recruitment, but the resulting preinitiation complex does not open the promoter . Whether Brf1 and Bdp1 participate in opening the promoter by direct DNA interaction (as sigma subunits of bacterial RNA polymerases do) or indirectly by their action on pol III has been approached by site-specific photochemical protein-DNA cross-linking of TFIIIB-pol III-U6 RNA gene promoter complexes . Brf1, Bdp1, and several pol III subunits can be cross-linked to the nontranscribed strand of the U6 promoter at base pair -9/-8 and +2/+3 (relative to the transcriptional start as +1), respectively the upstream and downstream ends of the DNA segment that opens up into the transcription bubble . Cross-linking of Bdp1 and Brf1 is detected at 0 degrees C in closed preinitiation complexes and at 30 degrees C in complexes that are partly open, but also it is detected in mutant TFIIIB-pol III-DNA complexes that are unable to open the promoter . In contrast, promoter opening-defective TFIIIB mutants generate significant changes of cross-linking of polymerase subunits . The weight of this evidence argues in favor of an indirect mode of action of TFIIIB in promoter opening.

J Biol Chem, 2003 May 16, 278(20), 18303 - 12 Epub 2003 Mar 13.
NTP-driven translocation by human RNA polymerase II; Nedialkov YA et al.; We report a "running start, two-bond" protocol to analyze elongation by human RNA polymerase II (RNAP II) . In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism . Formation of two bonds was tracked to ensure that at least one translocation event was analyzed . By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP . Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP . The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis delta antigen and transcription factor IIF . Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure.

Biochim Biophys Acta, 2003 Mar 21, 1646(1-2), 57 - 66
Tryptophan-tagged cutinase studied by steady state fluorescence for understanding of tag interactions in aqueous two-phase systems; Nilsson A et al.; Genetic engineering has been used to construct fusion proteins of Fusarium solani pisi cutinase and tryptophan-based tags, expressed in Saccharomyces cerevisiae, to increase the partitioning in aqueous two-phase systems . The separation systems were composed of thermoseparating polymers (random copolymers of ethylene oxide and propylene oxide, EOPO) and detergents (C(12)EO(n)) . In this study, the fluorescence behaviour of the peptide-tagged protein, free peptide tag and tryptophan was investigated . The tryptophan-tagged proteins, cutinase-(WP)(4) and cutinase-TGGSGG-(WP)(4), showed emission spectra similar to the free peptides and tryptophan, indicating solvent exposure of the tag . The influence of polymers and detergents on the fluorescence of tagged proteins was examined . When peptides and tagged proteins were exposed to polymer, a slight blue shift of the emission maximum was observed . Larger blue shifts of the emission maximum were observed when C(12)EO(n) detergents were utilised . The results correlate with aqueous two-phase partitioning where addition of C(12)EO(n) detergents results in more extreme partitioning compared to systems containing only polymers . Dynamic light scattering (DLS) measurements of the EOPO copolymers were carried out, showing that the polymers did not aggregate at concentrations used in aqueous two-phase systems . Quenching of fluorescence with iodide for both proteins and peptide tags was studied . Plots according to the Stern-Volmer equation resulted in a linear fit, indicating exposed tryptophan residues for both free peptides and fusion proteins . The quenching constants were similar for both tagged protein and free peptide tag . The fluorescence results indicated that the tryptophan residues in the tag were exposed to the solvent and could interact with detergents and polymers in the two-phase systems.

Dev Cell, 2003 Mar, 4(3), 345 - 57
Phosphorylation-dependent regulation of septin dynamics during the cell cycle; Dobbelaere J et al.; Septins are GTPases involved in cytokinesis . In yeast, they form a ring at the cleavage site . Using FRAP, we show that septins are mobile within the ring at bud emergence and telophase and are immobile during S, G2, and M phases . Immobilization of the septins is dependent on both Cla4, a PAK-like kinase, and Gin4, a septin-dependent kinase that can phosphorylate the septin Shs1/Sep7 . Induction of septin ring dynamics in telophase is triggered by the translocation of Rts1, a kinetochore-associated regulatory subunit of PP2A phosphatase, to the bud neck and correlates with Rts1-dependent dephosphorylation of Shs1 . In rts1-Delta cells, the actomyosin ring contracts properly but cytokinesis fails . Together our results implicate septins in a late step of cytokinesis and indicate that proper regulation of septin dynamics, possibly through the control of their phosphorylation state, is required for the completion of cytokinesis.

Biosci Rep, 2002 Oct-Dec, 22(5-6), 529 - 40
Phagocytosis and phagosome maturation are regulated by calcium in J774 macrophages interacting with unopsonized prey; Tejle K et al.; Phagocytosis by neutrophils, macrophages, and other professional phagocytes requires rapid remodeling of actin . Early phagosomes are surrounded by a rim of F-actin that is disassembled during phagosomoal maturation . Breakdown of periphagosomal F-actin and phagolysosome fusion are calcium dependent processes in neutrophils interacting with serum-opsonized prey, but appears to be calcium independent in macrophages interacting with serum- or IgG-opsonized prey . In the present study, we found that calcium was necessary for phagocytosis, breakdown of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey . We also observed that lipophosphoglycan (LPG) from Leishmania donovani promastigotes required calcium to exert its inhibitory effect on macrophage phagocytosis and periphagosomal F-actin breakdown . We conclude that calcium is essential for phagocytosis, depolymerization of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey, as well as for proper functioning of LPG.

EMBO Rep, 2003 Mar, 4(3), 274 - 7
The soybean retroelement SIRE1 uses stop codon suppression to express its envelope-like protein; Havecker ER et al.; The soybean SIRE1 family of Ty1/copia retrotransposons encodes an envelope-like gene (env-like) . We analysed the DNA sequences of nine SIRE1 insertions and observed that the gag/pol and env-like genes are in the same reading frame and separated by a single UAG stop codon . The six nucleotides immediately downstream of the stop codon conform to a degenerate nucleotide motif, CARYYA, which is sufficient to facilitate stop codon suppression in tobacco mosaic virus . In vivo stop codon suppression assays indicate that SIRE1 sequences confer leakiness to the UAG stop codon at an efficiency of 5% . These data suggest that SIRE1 retro-elements use translational suppression to express their envelope-like protein; this is in contrast with all characterized retroviruses, which express the envelope protein from a spliced genomic messenger RNA.

EMBO Rep, 2003 Mar, 4(3), 246 - 51
The Ark1/Prk1 family of protein kinases . Regulators of endocytosis and the actin skeleton; Smythe E et al.; The Ark/Prk serine/threonine kinases initiate phosphorylation cycles that control the endocytic machinery in mammalian cells and in yeast, and the actin cytoskeleton in yeast . The members of this protein family are unified by homologies in their kinase domain, but are generally diverse in their other domains . The evolution of Ark/Prk family members in different organisms may have allowed the conserved role of the kinase domain, which is required for the phosphorylation of both endocytic and cytoskeletal components, to be coupled to other functional domains.

Phys Rev Lett . 2003 Feb 28;90(8):088102 . Epub 2003 Feb 26.
Zipf's law in gene expression; Furusawa C et al.; Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1; i.e., they obey Zipf's law . Furthermore, by simulations of a simple model with an intracellular reaction network, we found that Zipf's law of chemical abundance is a universal feature of cells where such a network optimizes the efficiency and faithfulness of self-reproduction . These findings provide novel insights into the nature of the organization of reaction dynamics in living cells.

Mol Biol Cell, 2003 Mar, 14(3), 958 - 72
Global transcription analysis of Krebs tricarboxylic acid cycle mutants reveals an alternating pattern of gene expression and effects on hypoxic and oxidative genes; McCammon MT et al.; To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction . mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes . The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction . Many genes displayed a common response to TCA cycle dysfunction indicative of a shift away from oxidative metabolism . Another set of genes displayed a pairwise, alternating pattern of expression in response to contiguous TCA cycle enzyme defects: expression was elevated in aconitase and isocitrate dehydrogenase mutants, diminished in alpha-ketoglutarate dehydrogenase and succinyl-CoA ligase mutants, elevated again in succinate dehydrogenase and fumarase mutants, and diminished again in malate dehydrogenase and citrate synthase mutants . This pattern correlated with previously defined TCA cycle growth-enhancing mutations and suggested a novel metabolic signaling pathway monitoring TCA cycle function . Expression of hypoxic/anaerobic genes was elevated in alpha-ketoglutarate dehydrogenase mutants, whereas expression of oxidative genes was diminished, consistent with a heme signaling defect caused by inadequate levels of the heme precursor, succinyl-CoA . These studies have revealed extensive responses to changes in TCA cycle function and have uncovered new and unexpected metabolic networks that are wired into the TCA cycle.

Mol Biol Cell, 2003 Mar, 14(3), 939 - 57
Peroxisome biogenesis occurs in an unsynchronized manner in close association with the endoplasmic reticulum in temperature-sensitive Yarrowia lipolytica Pex3p mutants; Bascom RA et al.; Pex3p is a peroxisomal integral membrane protein required early in peroxisome biogenesis, and Pex3p-deficient cells lack identifiable peroxisomes . Two temperature-sensitive pex3 mutant strains of the yeast Yarrowia lipolytica were made to investigate the role of Pex3p in the early stages of peroxisome biogenesis . In glucose medium at 16 degrees C, these mutants underwent de novo peroxisome biogenesis and exhibited early matrix protein sequestration into peroxisome-like structures found at the endoplasmic reticulum-rich periphery of cells or sometimes associated with nuclei . The de novo peroxisome biogenesis seemed unsynchronized, with peroxisomes occurring at different stages of development both within cells and between cells . Cells with peripheral nascent peroxisomes and cells with structures morphologically distinct from peroxisomes, such as semi/circular tubular structures that immunostained with antibodies to peroxisomal matrix proteins and to the endoplasmic reticulum-resident protein Kar2p, and that surrounded lipid droplets, were observed during up-regulation of peroxisome biogenesis in cells incubated in oleic acid medium at 16 degrees C . These structures were not detected in wild-type or Pex3p-deficient cells . Their role in peroxisome biogenesis remains unclear . Targeting of peroxisomal matrix proteins to these structures suggests that Pex3p directly or indirectly sequesters components of the peroxisome biogenesis machinery . Such a role is consistent with Pex3p overexpression producing cells with fewer, larger, and clustered peroxisomes.

Mol Biol Cell, 2003 Mar, 14(3), 836 - 47
Sac3 is an mRNA export factor that localizes to cytoplasmic fibrils of nuclear pore complex; Lei EP et al.; In eukaryotes, mRNAs are transcribed in the nucleus and exported to the cytoplasm for translation to occur . Messenger RNAs complexed with proteins referred to as ribonucleoparticles are recognized for nuclear export in part by association with Mex67, a key Saccharomyces cerevisiae mRNA export factor and homolog of human TAP/NXF1 . Mex67, along with its cofactor Mtr2, is thought to promote ribonucleoparticle translocation by interacting directly with components of the nuclear pore complex (NPC) . Herein, we show that the nuclear pore-associated protein Sac3 functions in mRNA export . Using a mutant allele of MTR2 as a starting point, we have identified a mutation in SAC3 in a screen for synthetic lethal interactors . Loss of function of SAC3 causes a strong nuclear accumulation of mRNA and synthetic lethality with a number of mRNA export mutants . Furthermore, Sac3 can be coimmunoprecipitated with Mex67, Mtr2, and other factors involved in mRNA export . Immunoelectron microscopy analysis shows that Sac3 localizes exclusively to cytoplasmic fibrils of the NPC . Finally, Mex67 accumulates at the nuclear rim when SAC3 is mutated, suggesting that Sac3 functions in Mex67 translocation through the NPC.

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3351 - 6 Epub 2003 Mar 11.
Generalized singular value decomposition for comparative analysis of genome-scale expression data sets of two different organisms; Alter O et al.; We describe a comparative mathematical framework for two genome-scale expression data sets . This framework formulates expression as superposition of the effects of regulatory programs, biological processes, and experimental artifacts common to both data sets, as well as those that are exclusive to one data set or the other, by using generalized singular value decomposition . This framework enables comparative reconstruction and classification of the genes and arrays of both data sets . We illustrate this framework with a comparison of yeast and human cell-cycle expression data sets.

Oncogene, 2003 Mar 13, 22(10), 1536 - 45
Wnt signaling in B-cell neoplasia; Qiang YW et al.; Wnts comprise a family of secreted proteins that interact with receptors consisting of a Frizzled (Fz) family member alone or complexed with LDL receptor-related proteins (LRP5/6) . Wnt signaling plays a crucial role in both development and differentiation, and activation of a 'canonical' Wnt pathway resulting in beta-catenin stabilization is associated with several types of human cancers . To date, little is known about potential Wnt signaling in mature lymphocytes or lymphoid neoplasia . Herein, we have analysed Wnt signaling in mature B cells (lymphomas) and plasma cells (multiple myeloma) . Both Fz and LRP5/6 mRNAs were expressed in myeloma lines, but LRP5/6 were not observed in lymphomas . In myelomas, a canonical Wnt signaling pathway was activated following treatment with Wnt-3a as assessed by accumulation of beta-catenin, but beta-catenin levels actually decreased in lymphoma cells . Wnt-3a treatment further led to striking morphological changes in myeloma cells accompanied by rearrangement of the actin cytoskeleton . Morphological changes were associated with a second Wnt pathway dependent on Rho activation . These results suggest that Wnt responsiveness is a stage-specific phenomenon in B-cell development and that the morphological changes associated with Wnt signaling may play a role in the motility and metastatic potential of myeloma cells.

J Urol, 2003 Apr, 169(4), 1541 - 5
Hypermethylation of an E-cadherin (CDH1) promoter region in high grade transitional cell carcinoma of the bladder comprising carcinoma in situ; Horikawa Y et al.; PURPOSE: We elucidated the role of methylation in the promoter region of the 1 gene in bladder carcinogenesis, particularly in those comprising carcinoma in situ . MATERIALS AND METHODS: A total of 49 cases of transitional cell carcinoma of the bladder obtained from transurethral resection were examined . Methylation status of the 1 promoter region was analyzed by methylation specific polymerase chain reaction from chemically modified DNA after Na-bisulfite treatment . Loss of heterozygosity on 16q was examined by blunt end single strand DNA conformation polymorphism using 4 tetranucleotide repeat microsatellite markers assigned on 16q13 to 22.1 . E-cadherin expression was evaluated by immunostaining on formalin fixed, paraffin embedded tissue sections using anti E-cadherin murine monoclonal antibody, HECD1 and standard avidin-biotin immunoperoxidase complex technique . RESULTS: Analysis of the 49 bladder transitional cell carcinoma samples showed 1 promoter methylation in 23 (47%) . Methylation of the 1 gene did not correlate with tumor stage (p = 0.2097) but with high grade transitional cell carcinoma (p = 0.0416) . 1 promoter methylation was observed at a significantly higher frequency in the carcinoma in situ positive group than in the carcinoma in situ negative group (16 of 18 cases or 89% versus 7 of 31 or 23%, p <0.0001) and it strongly correlated with abnormal E-cadherin expression (p <0.0001) . We found 16q loss of heterozygosity in 16 of 47 cases (34%), which correlated with higher histological grade (p = 0.0069) but not with the presence of the carcinoma in situ component (p = 0.1235) . CONCLUSIONS: This study showed that 1 gene promoter methylation is strongly associated with bladder transitional cell carcinoma comprising carcinoma in situ.

EMBO J, 2003 Mar 17, 22(6), 1370 - 80
The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes; Schafer T et al.; Recent reports have increased our knowledge of the consecutive steps during 60S ribosome biogenesis substantially, but 40S subunit formation is less well understood . Here, we investigate the maturation of nucleolar 90S pre-ribosomes into cytoplasmic 40S pre-ribosomes . During the transition from 90S to 40S particles, the majority of non-ribosomal proteins (approximately 30 species) dissociate, and significantly fewer factors associate with 40S pre-ribosomes . Notably, some of these components are part of both early 90S and intermediate 40S pre-particles in the nucleolus (e.g . Enp1p, Dim1p and Rrp12p), whereas others (e.g . Rio2p and Nob1p) are found mainly on late cytoplasmic pre-40S subunits . Finally, temperature-sensitive mutants mapping either in earlier (enp1-1) or later (rio2-1) components exhibit defects in the formation and nuclear export of pre-40S subunits . Our data provide an initial biochemical map of the pre-40S ribosomal subunit on its path from the nucleolus to the cytoplasm . This pathway involves fewer changes in composition than seen during 60S biogenesis.

EMBO J, 2003 Mar 17, 22(6), 1235 - 44
Mrs2p is an essential component of the major electrophoretic Mg2+ influx system in mitochondria; Kolisek M et al.; Steady-state concentrations of mitochondrial Mg(2+) previously have been shown to vary with the expression of Mrs2p, a component of the inner mitochondrial membrane with two transmembrane domains . While its structural and functional similarity to the bacterial Mg(2+) transport protein CorA suggested a role for Mrs2p in Mg(2+) influx into the organelle, other functions in cation homeostasis could not be excluded . Making use of the fluorescent dye mag-fura 2 to measure free Mg(2+) concentrations continuously, we describe here a high capacity, rapid Mg(2+) influx system in isolated yeast mitochondria, driven by the mitochondrial membrane potential Deltapsi and inhibited by cobalt(III)hexaammine . Overexpression of Mrs2p increases influx rates 5-fold, while the deletion of the MRS2 gene abolishes this high capacity Mg(2+) influx . Mg(2+) efflux from isolated mitochondria, observed with low Deltapsi only, also requires the presence of Mrs2p . Cross-linking experiments revealed the presence of Mrs2p-containing complexes in the mitochondrial membrane, probably constituting Mrs2p homo- oligomers . Taken together, these findings characterize Mrs2p as the first molecularly identified metal ion channel protein in the inner mitochondrial membrane.

Bioorg Med Chem, 2003 Apr 3, 11(7), 1593 - 6
DNA damaging activity of ellagic acid derivatives; Xu YM et al.; A strain of yeast rendered repair deficient by the conditional expression of the RAD52 locus was used to search for natural products capable of damaging DNA . Four ellagic acid derivatives, namely 3,3'-dimethyl-4'-O-beta-D-glucopyranosyl ellagic acid (1), 3,3',4-trimethyl-4'-O-beta-D-glucopyranosyl ellagic acid (2), 3'-methyl-3,4-O,O-methylidene ellagic acid (3) and 3'-methyl-3,4-O,O-methylidene-4'-O-beta-D-glucopyranosyl ellagic acid (4), were identified by this assay as DNA damaging natural principles from several plants, including Alangium javanicum, Anisophyllea apetala, Crypteronia paniculata, Mouririi sp . and Scholtzia parviflora . Although none of the isolated principles mediated frank strand scission of DNA in vitro, all of them potently inhibited the growth of yeast in the absence of expression of RAD52.

Bioorg Med Chem, 2003 Apr 3, 11(7), 1531 - 50
In vitro antifungal activity of new series of homoallylamines and related compounds with inhibitory properties of the synthesis of fungal cell wall polymers; Vargas M LY et al.; The synthesis, in vitro antifungal evaluation and SAR studies of 101 compounds of the 4-aryl-, 4-alkyl-, 4-pyridyl or -quinolinyl-4-N-arylamino-1-butenes series and related compounds, are reported here . Active structures showed to inhibit (1,3)-beta-D-glucan and mainly chitin synthases, enzymes that catalyze the synthesis of the major fungal cell wall polymers.

Bioorg Med Chem, 2003 Apr 3, 11(7), 1215 - 25
Inhibition of protein kinase C by synthetic xanthone derivatives; Saraiva L et al.; The modulatory activity of two xanthones (3,4-dihydroxyxanthone and 1-formyl-4-hydroxy-3-methoxyxanthone) on isoforms alpha, betaI, delta, eta and zeta of protein kinase C (PKC) was evaluated using an in vivo yeast phenotypic assay . Both xanthones caused an effect compatible with PKC inhibition, similar to that elicited by known PKC inhibitors (chelerythrine and NPC 15437) . PKC inhibition caused by xanthones was confirmed using an in vitro kinase assay . The yeast phenotypic assay revealed that xanthones present differences on their potency towards the distinct PKC isoforms tested . It is concluded that 3,4-dihydroxyxanthone and 1-formyl-4-hydroxy-3-methoxyxanthone may become useful PKC inhibitors and xanthone derivatives can be explored to develop new isoform-selective PKC inhibitors.

Trends Cell Biol, 2003 Mar, 13(3), 110 - 3
Down to the origin: Cdc6 protein and the competence to replicate; Pelizon C; In eukaryotes, DNA replication requires the regulated assembly of pre-replicative complexes (pre-RCs) onto DNA during G1 phase . Pre-RCs render the chromatin competent to replicate, yet it is only at the G1-S phase transition that protein-kinase complexes trigger the transition to DNA replication . Central to the formation of pre-RCs and regulation of DNA replication is the Cdc6 protein . Two recent studies have shown that Cdc6 is the long-sought factor that confers the competence to replicate in unfertilized Xenopus eggs.

Cell, 2003 Mar 7, 112(5), 725 - 36
Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of silent heterochromatin; Meneghini MD et al.; Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin . Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes . Microarray analysis revealed that HTZ1-activated genes cluster near telomeres . The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing . Other Htz1-activated genes flank the silent HMR mating-type locus . Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR . In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation . Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin . Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state.

Cell, 2003 Mar 7, 112(5), 711 - 23
Structure of the catalytic domain of human DOT1L, a non-SET domain nucleosomal histone methyltransferase; Min J et al.; Dot1 is an evolutionarily conserved histone methyltransferase that methylates lysine-79 of histone H3 in the core domain . Unlike other histone methyltransferases, Dot1 does not contain a SET domain, and it specifically methylates nucleosomal histone H3 . We have solved a 2.5 A resolution structure of the catalytic domain of human Dot1, hDOT1L, in complex with S-adenosyl-L-methionine (SAM) . The structure reveals a unique organization of a mainly alpha-helical N-terminal domain and a central open alpha/beta structure, an active site consisting of a SAM binding pocket, and a potential lysine binding channel . We also show that a flexible, positively charged region at the C terminus of the catalytic domain is critical for nucleosome binding and enzymatic activity . These structural and biochemical analyses, combined with molecular modeling, provide mechanistic insights into the catalytic mechanism and nucleosomal specificity of Dot1 proteins.

Biochemistry, 2003 Mar 18, 42(10), 3089 - 95
Arabidopsis thaliana Ogg1 protein excises 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine from oxidatively damaged DNA containing multiple lesions; Morales-Ruiz T et al.; A functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thalianahas recently been cloned, isolated, and characterized {Garcia-Ortiz, M . V., Ariza, R . R., and Roldan-Arjona, T . (2001) Plant Mol . Biol . 47, 795-804} . This enzyme (AtOgg1) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster, and human Ogg1 proteins . We investigated the substrate specificity and kinetics of AtOgg1 for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine-derived lesions . Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose . Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples . Of the 17 modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates . This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins that had previously been studied under identical conditions . Excision depended on incubation time, enzyme concentration, and substrate concentration and followed Michaelis-Menten kinetics . A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases . A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins . We also investigated the effect of base-pairing on the excision using double-stranded oligodeoxynucleotides that contained 8-OH-Gua paired with each of the four DNA bases . The activity of AtOgg1 was most effective on the 8-OH-Gua:C pair with some or very low activity on other pairs in agreement with the activity of other Ogg1 proteins . The results unequivocally show that AtOgg1 possesses common substrates with other eukaryotic Ogg1 proteins albeit significant differences between their excision kinetics.

Biochemistry, 2003 Mar 18, 42(10), 2941 - 51
Active site loop motion in triosephosphate isomerase: T-jump relaxation spectroscopy of thermal activation; Desamero R et al.; As for many enzymes, the enzymatic pathway of triosephosphate isomerase (TIM) includes the partially rate determining motion of an active site loop (loop 6, residues 166-176), which must remain closed during chemistry but must open just before product release . The motion of this loop was monitored using laser induced temperature-jump relaxation spectroscopy at nanosecond to millisecond time resolution . Trp168 in the hinge of the mobile loop served as a fluorophore reporter in a mutant of the yeast enzyme . The opening rate was studied as a function of the concentration of glycerol 3-phosphate, a substrate surrogate . Monoexponential kinetics were observed; assuming a simple two-step ligand release mechanism involving an encounter complex intermediate, the time scales of loop opening and closing were derived . The opening rate of the loop at 25 degrees C was determined to be 2500 +/- 1000 s(-1), in remarkable agreement with solution and solid state NMR measurements . The closing rate at the same temperature was 46,700 +/- 1800 s(-1) . The rates were also studied as a function of the sample temperature following the jump . Enthalpies of activation of the loop motion, DeltaH(close) and DeltaH(open), were estimated to be 13.8 and 14.1 kcal/mol, respectively . The enthalpy of dissociation estimated from the kinetic studies is in reasonable agreement with steady-state values . Moreover, the enthalpy was dissected, for the first time, into components associated with ion binding and with protein conformational change . The enthalpy of the release reaction appeared to have a substantial contribution from the dissociation of the ligand from the encounter complex, found to be endothermic at 6 kcal/mol . In contrast, the population ratio of the open to closed loop conformations is found to favor the closed conformation but to be substantially less temperature dependent than the release step . Preliminary data of other ligands show that G3P behavior resembles that of the substrate but differs from 2-phosphoglycolate, a tight binding inhibitor, and phosphate . This study represents one of the first detailed comparisons between NMR and fluorescence based probes of protein motion and results in good agreement between the methods . The data in aggregate support a model in which the rate of the loop opening for TIM is dependent on the ligand and results in opening rates in the presence of the product that are comparable to enzymatic throughput, kcat.

Biochemistry, 2003 Mar 18, 42(10), 2816 - 24
Design of a ruthenium-labeled cytochrome c derivative to study electron transfer with the cytochrome bc1 complex; Engstrom G et al.; A new ruthenium-cytochrome c derivative was designed to study electron transfer from cytochrome bc1 to cytochrome c (Cc) . The single sulfhydryl on yeast H39C;C102T iso-1-Cc was labeled with Ru(2,2'-bipyrazine)2(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form Ru(z)-39-Cc . The Ru(z)-39-Cc derivative has the same steady-state activity with yeast cytochrome bc1 as wild-type yeast iso-1-Cc, indicating that the ruthenium complex does not interfere in the binding interaction . Laser excitation of reduced Ru(z)-39-Cc results in electron transfer from heme c to the excited state of ruthenium with a rate constant of 1.5 x 10(6) x s(-1) . The resulting Ru(I) is rapidly oxidized by atmospheric oxygen in the buffer . The yield of photooxidized heme c is 20% in a single flash . Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc1 and Ru(z)-39-Cc at low ionic strength leads to rapid photooxidation of heme c, followed by intracomplex electron transfer from cytochrome c1 to heme c with a rate constant of 1.4 x 10(4) x s(-1) . As the ionic strength is raised above 100 mM, the intracomplex phase disappears, and a new phase appears due to the bimolecular reaction between solution Ru-39-Cc and cytochrome bc1 . The interaction of yeast Ru-39-Cc with yeast cytochrome bc1 is stronger than that of horse Ru-39-Cc with bovine cytochrome bc1, suggesting that nonpolar interactions are stronger in the yeast system.

Nat Struct Biol, 2003 Apr, 10(4), 250 - 5
Structural insights into the U-box, a domain associated with multi-ubiquitination; Ohi MD et al.; The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR . The conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box . These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box . A conservative Val-->Ile point mutation in the Prp19p U-box domain leads to pre-mRNA splicing defects in vivo . NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain . Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface . Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo . These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3339 - 44 Epub 2003 Mar 07.
Integrating regulatory motif discovery and genome-wide expression analysis; Conlon EM et al.; We propose motif regressor for discovering sequence motifs upstream of genes that undergo expression changes in a given condition . The method combines the advantages of matrix-based motif finding and oligomer motif-expression regression analysis, resulting in high sensitivity and specificity . motif regressor is particularly effective in discovering expression-mediating motifs of medium to long width with multiple degenerate positions . When applied to Saccharomyces cerevisiae, motif regressor identified the ROX1 and YAP1 motifs from Rox1p and Yap1p overexpression experiments, respectively; predicted that Gcn4p may have increased activity in YAP1 deletion mutants; reported a group of motifs (including GCN4, PHO4, MET4, STRE, USR1, RAP1, M3A, and M3B) that may mediate the transcriptional response to amino acid starvation; and found all of the known cell-cycle regulation motifs from 18 expression microarrays over two cell cycles.

Nucleic Acids Res . 2003 Mar 15;31(6):e29.
Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning; Leem SH et al.; Transformation-associated recombination (TAR) cloning in yeast is used to isolate a desired chromosomal region or gene from a complex genome without construction of a genomic library . The technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector containing short 5' and 3' gene-specific targeting hooks . Efficient gene capture requires a high yield of transformants, and we demonstrate here that the transformant yield increases approximately 10-fold when the genomic DNA is sheared to 100-200 kb before being presented to the spheroplasts . Here we determine the most effective concentration of genomic DNA, and also show that the targeted sequences recombine much more efficiently with the vector's targeting hooks when they are located at the ends of the genomic DNA fragment . We demonstrate that the yield of gene-positive clones increases approximately 20-fold after endonuclease digestion of genomic DNA, which caused double strand breaks near the targeted sequences . These findings have led to a greatly improved protocol.

Nucleic Acids Res . 2003 Mar 15;31(6):e28.
Adaptation of the Ras-recruitment system to the analysis of interactions between membrane-associated proteins; Kohler F et al.; Interactions of membrane-associated proteins play important roles in many cellular processes . The yeast two-hybrid assay is of limited utility for the analysis of such interactions, due to the need for soluble protein partners, whose interaction is assessed in the nucleus . The advent of the Ras-recruitment system (RRS) has enabled the study of membrane-associated proteins interacting with cytoplasmic proteins fused to Ras . Constitutive membrane association of the Ras fusion protein is expected to complement the growth defect of the yeast strain CDC25-2, assayed in the RRS, independent from the interaction with a membrane-bound partner . We describe the adaptation of the RRS to the analysis of interactions between two membrane-associated proteins using a model system . These results may facilitate the study of protein-protein interactions between membrane-bound proteins and further increase the utility of the RRS.

Nucleic Acids Res, 2003 Mar 15, 31(6), 1744 - 52
Functional dissection of the zinc finger and flanking domains of the Yth1 cleavage/polyadenylation factor; Tacahashi Y et al.; Yth1, a subunit of yeast Cleavage Polyadenylation Factor (CPF), contains five CCCH zinc fingers . Yth1 was previously shown to interact with pre-mRNA and with two CPF subunits, Brr5/Ysh1 and the polyadenylation-specific Fip1, and to act in both steps of mRNA 3' end processing . In the present study, we have identified new domains involved in each interaction and have analyzed the consequences of mutating these regions on Yth1 function in vivo and in vitro . We have found that the essential fourth zinc finger (ZF4) of Yth1 is critical for interaction with Fip1 and RNA, but not for cleavage, and a single point mutation in ZF4 impairs only polyadenylation . Deletion of the essential N-terminal region that includes the ZF1 or deletion of ZF4 weakened the interaction with Brr5 in vitro . In vitro assays showed that the N-terminus is necessary for both processing steps . Of particular importance, we find that the binding of Fip1 to Yth1 blocks the RNA-Yth1 interaction, and that this inhibition requires the Yth1-interacting domain on Fip1 . Our results suggest a role for Yth1 not only in the execution of cleavage and poly(A) addition, but also in the transition from one step to the other.

Microbiol Mol Biol Rev, 2003 Mar, 67(1), 1 - 15, table of contents
Leucine biosynthesis in fungi: entering metabolism through the back door; Kohlhaw GB; After exploring evolutionary aspects of branched-chain amino acid biosynthesis, the review focuses on the extended leucine biosynthetic pathway as it operates in Saccharomyces cerevisiae . First, the genes and enzymes specific for the leucine pathway are considered: LEU4 and LEU9 (encoding the alpha-isopropylmalate synthase isoenzymes), LEU1 (isopropylmalate isomerase), and LEU2 (beta-isopropylmalate dehydrogenase) . Emphasis is given to the unusual distribution of the branched-chain amino acid pathway enzymes between mitochondrial matrix and cytosol, on the newly defined role of Leu5p, and on regulatory mechanisms governing gene expression and enzyme activity, including new evidence for the metabolic importance of the regulation of alpha-isopropylmalate synthase by coenzyme A . Next, structure-function relationships of the transcriptional regulator Leu3p are addressed, defining its dual role as activator and repressor and discussing evidence in support of the self-masking model . Recent data pointing at a more extended Leu3p regulon are discussed . An overview of the layered controls of the extended leucine pathway is provided that includes a description of the newly recognized roles of Ilv5p and Bat1p in maintaining mitochondrial integrity . Finally, branched-chain amino acid biosynthesis and its regulation in other fungi are summarized, the question of leucine as metabolic signal is addressed, and possible directions of future research in this area are outlined.

J Immunol, 2003 Mar 15, 170(6), 2971 - 6
The cyclic adenosine 5'-monophosphate response element modulator suppresses IL-2 production in stimulated T cells by a chromatin-dependent mechanism; Tenbrock K et al.; The production of IL-2 is tightly controlled by several transcription factors that bind to the IL-2 promoter . The cAMP response element modulator (CREM) is known to form complexes with CREB and bind to the -180 site of the IL-2 promoter in anergic and in systemic lupus erythematosus T cells . In this study we show that CREM is transcriptionally induced in T cells following stimulation through CD3 and CD28, binds to the IL-2 promoter in vivo, and suppresses IL-2 production . Transfection of an antisense CREM plasmid into T cells blocked the expression and binding of CREM to the IL-2 promoter and the decrease of IL-2 production, which follows the early increase after T cell stimulation with CD3 and CD28 . In addition, as assessed by chromatin immunoprecipitation experiments, antisense CREM prevented the binding of protein 300 and cAMP response element binding protein and promoted the acetylation of histones . Antisense CREM also enhanced the accessibility of the IL-2 promoter to endonucleases and prevented the condensation of chromatin in vivo . Our data suggest that upon T cell activation, CREM gradually replaces phosphorylated CREB at the -180 site of the IL-2 promoter . CREM, in turn, binds protein 300 and cAMP response element binding protein, but CREM is unable to activate its histone acetyltransferase activity, which results in condensation of chromatin and down-regulation of IL-2 production.

J Biol Chem, 2003 May 9, 278(19), 16887 - 92 Epub 2003 Mar 06.
Sas4 and Sas5 are required for the histone acetyltransferase activity of Sas2 in the SAS complex; Sutton A et al.; The SAS2 gene is involved in transcriptional silencing in Saccharomyces cerevisiae . Based on its primary sequence, the Sas2 protein is predicted to be a member of the MYST family of histone acetyltransferases (HATs) . Sas2 forms a complex with Sas4 and Sas5, which are required for its silencing function . Here we show that recombinant Sas2 has HAT activity that absolutely requires Sas4 and is stimulated by Sas5 . The recombinant SAS complex acetylates H4 lysine 16 and H3 lysine 14 . Furthermore, a purified SAS complex from yeast shows similar activity and specificity . In contrast to other MYST HATs, neither the recombinant nor the native SAS complex acetylated nucleosomal histones under conditions that were optimum for acetylating free histones . Finally, although the SAS subunits interact genetically and physically with Asf1, a histone deposition factor, association of H3 and H4 with Asf1 blocks their acetylation by the SAS complex, raising the possibility that the SAS HAT complex may acetylate free histones prior to their deposition onto DNA by Asf1 or CAF-I.

J Biol Chem, 2003 May 16, 278(20), 17845 - 51 Epub 2003 Mar 07.
Conserved Asp684 in transmembrane segment M6 of the plant plasma membrane P-type proton pump AHA2 is a molecular determinant of proton translocation; Buch-Pedersen MJ et al.; The mechanism of proton pumping by P-type H(+)-ATPases is still unclear . In the plant P-type plasma membrane H(+)-ATPase AHA2, two charged residues, Arg(655) and Asp(684), are conserved in transmembrane segments M5 and M6, respectively, a region that has been shown be contribute to ion coordination in related P-type ATPases . Substitution of Arg(655) with either alanine or aspartate resulted in mutant enzymes exhibiting a significant shift in the P-type ATPase E(1)P-E(2)P conformational equilibrium . The mutant proteins accumulated in the E(1)P conformation, but were capable of conducting proton transport . This points to an important role of Arg(655) in the E(1)P-E(2)P conformational transition . The presence of a carboxylate moiety at position Asp(684) proved essential for coupling between initial proton binding and proton pumping . The finding that the carboxylate side chain of Asp(684) contributes to the proton-binding site and appears to function as an absolutely essential proton acceptor along the proton transport pathway is discussed in the context of a possible proton pumping mechanism of P-type H(+)-ATPases.

J Biol Chem, 2003 May 16, 278(20), 17636 - 45 Epub 2003 Mar 07.
Macroautophagy is required for multicellular development of the social amoeba Dictyostelium discoideum; Otto GP et al.; Macroautophagy is a mechanism employed by eukaryotic cells to recycle non-essential cellular components during starvation, differentiation, and development . Two conjugation reactions related to ubiquitination are essential for autophagy: Apg12p conjugation to Apg5p, and Apg8p conjugation to the lipid phosphatidylethanolamine . These reactions require the action of the E1-like enzyme, Apg7p, and the E2-like enzymes, Apg3p and Apg10p . In Dictyostelium, development is induced by starvation, conditions under which autophagy is required for survival in yeast and plants . We have identified Dictyostelium homologues of 10 budding yeast autophagy genes . We have generated mutations in apg5 and apg7 that produce defects typically associated with an abrogation of autophagy . Mutants are not grossly affected in growth, but survival during nitrogen starvation is severely reduced . Starved mutant cells show little turnover of cellular constituents by electron microscopy, whereas wild-type cells show significant cytoplasmic degradation and reduced organelle number . Bulk protein degradation during starvation-induced development is reduced in the autophagy mutants . Development is aberrant; the autophagy mutants do not aggregate in plaques on bacterial lawns, but they do proceed further in development on nitrocellulose filters, forming defective fruiting bodies . The autophagy mutations are cell autonomous, because wild-type cells in a chimaera do not rescue development of the autophagy mutants . We have complemented the mutant phenotypes by expression of the cognate gene fused to green fluorescent protein . A green fluorescent protein fusion of the autophagosome marker Apg8 mislocalizes in the two autophagy mutants . We show that the Apg5-Apg12 conjugation system is conserved in Dictyostelium.

Technol Cancer Res Treat, 2002 Oct, 1(5), 329 - 40
Digression on membrane electroporation for drug and gene delivery; Neumann E et al.; Membrane electroporation (ME) defines an electrical technique to render lipid membranes porous and permeable, transiently and reversibly, by external voltage pulses . Although there are numerous applications of ME to manipulate cells, organelles and tissues in cell biology, biotechnology and medicine, yet the molecular mechanism of ME is only slowly being understood . A general chemical- thermodynamical approach for the quantitative description of cell membrane electroporation has been developed to provide the framework to quantitatively rationalize electroporative cell transformation and electroporative uptake of drug-like dyes into cells, as well as electrolyte efflux from salt-filled electroporated vesicles . Mechanistically, the electroporative transfer of gene and drug-like dyes involves the coupling between an interactive contact formation of the permeates with the cell surface membrane and the structural electroporation-resealing cycle C <--> (P) where C is the closed and (P) represents a number of different porated membrane states, respectively . The experimentally accessible concentration fraction f(p) = {(P)} / ({C} + {(P)}) of porous states is related to thermodynamic and electro-mechanic parameters such as temperature and the electric field strength, membrane rigidity or curvature . The results of the theoretical approach, mainly based on electrooptical data of lipid vesicles, have been successfully used to analyze single cells and to specify conditions for the practical purpose of direct electroporative gene transfer and drug delivery, in particular in the new medical disciplines of electroporative chemotherapy and electroporative gene vaccination.

J Biol Chem, 2003 May 9, 278(19), 16878 - 86 Epub 2003 Mar 05.
Domains of Gln3p interacting with karyopherins, Ure2p, and the target of rapamycin protein; Carvalho J et al.; Gln3p is a GATA-type transcription factor responsive to the quality of nitrogen and carbon . In preferred nitrogen such as glutamine, Gln3p is phosphorylated and sequestered in the cytoplasm in a manner that is dependent on the target of rapamycin (TOR) protein and Ure2p . In nonpreferred nitrogen or nitrogen starvation, Gln3p is dephosphorylated and imported into the nucleus via karyopherin alpha/Srp1p . Upon reintroduction of preferred nitrogen, Gln3p is exported from the nucleus by Crm1p/Xpo1p . Although recent work has provided insights into Gln3p, a more detailed understanding is needed to elucidate the mechanism of its localization and function . In this study, we show that Gln3p contains canonical nuclear localization signal and nuclear export signal sequences necessary for its localization and interaction with its relevant karyopherins . In addition, we identify an N-terminal domain of Gln3p interacting with Ure2p and a C-terminal region for binding to TOR . Finally, we find a lysine/arginine-rich domain essential for the rapamycin-sensitive function, but dispensable for its localization . Our results reveal key domains of Gln3p important for its function and regulation.

Mol Microbiol, 2003 Mar, 47(6), 1577 - 88
The MAPKK kinase SteC regulates conidiophore morphology and is essential for heterokaryon formation and sexual development in the homothallic fungus Aspergillus nidulans; Wei H et al.; Environmental signals can be transduced into intracellular responses by the action of MAP kinase cascades . Sequential phosphorylation results in the transient activation of a MAP kinase, which in turn activates certain transcription factors and thus a set of pathway-specific genes . Many steps in this cascade are conserved, and homologues have been discovered from yeast to human . We have characterized the MAPKK kinase, SteC, a homologue of Saccharomyces cerevisiae Ste11, in the filamentous fungus Aspergillus nidulans . The 886-amino-acid-long protein shares the highest similarity to Neurospora crassa Nrc-1 . Deletion of the gene in A . nidulans results in a slower growth rate, the formation of more branched hyphae, altered conidiophore morphology, an inhibition of heterokaryon formation and a block of cleistothecium development . The gene is transcriptionally activated during asexual development and controls the phosphorylation of two putative MAP kinases.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Mar, 35(3), 289 - 95
{Detection of 2'-O-ribose Methylation Sites on Rice 25 S rRNA}; Li W et al.; Ribose methylation is a widespread type of nucleotide modification in rRNA . In order to map the methylation sites of rice 25 S rRNA, a series of primers complementary to both yeast 28 S and rice 25 S rRNA simultaneously were synthesized . Primer extensions at different dNTP concentrations were carried out to detect the methylation sites of both yeast and rice rRNAs . The data showed that over 80% of the methylation sites in yeast 28 S rRNA was also detected in rice . In addition, compared with the known methylation sites of Arabidopsis 25 S rRNA, other 54 sites probably methylated in rice were found in Arabidopsis . Thus, there are 85 methylation sites detected altogether; the distribution of the methyl sites in rice 25 S rRNA was determined . The results show that most of the sites are conserved among different species, especially between closely related species . And remarkably, there are much more ribose methylation sites in plant rRNA, and the propinquous methylation sites in plants are more frequent than those in other eukaryotes . Moreover, the data provide the most important clue for searching new box C/D snoRNAs.

J Biol Chem, 2003 May 23, 278(21), 18714 - 9 Epub 2003 Mar 05.
Activation of transforming growth factor-beta signaling by SUMO-1 modification of tumor suppressor Smad4/DPC4; Lin X et al.; Smads are important intracellular effectors in signaling pathways of the transforming growth factor-beta (TGF-beta) superfamily . Upon activation by TGF-beta, receptor-phosphorylated Smads form a complex with tumor suppressor Smad4/DPC4, and the Smad complexes then are imported into the nucleus . Although diverse pathways regulate the activity and expression of receptor-phosphorylated and inhibitory Smads, cellular factors modulating the activity of the common Smad4 remain unidentified . Here we describe the involvement of the small ubiquitin-like modifier-1 (SUMO-1) conjugation pathway in regulating the growth inhibitory and transcriptional responses of Smad4 . The MH1 domain of Smad4 was shown to associate physically with Ubc9, the ubiquitin carrier protein (E2) conjugating enzyme in sumoylation . In cultured cells, Smad4 is modified by SUMO-1 at the endogenous level . The sumoylation sites were identified as two evolutionarily conserved lysine residues, Lys-113 and Lys-159, in the MH1 domain . We found that the mutations at Lys-113 and Lys-159 did not alter the ability of Smad4 to form a complex with Smad2 and FAST on the Mix.2 promoter . Importantly, SUMO-1 overexpression enhanced TGF-beta-induced transcriptional responses . These findings identify sumoylation as a unique mechanism to modulate Smad4-dependent cellular responses.

Phytochemistry, 2003 Feb, 62(3), 491 - 500
Selective desensitization of jasmonate- and pH-dependent signaling in the induction of benzophenanthridine biosynthesis in cells of Eschscholzia californica; Farber K et al.; The biosynthesis of benzophenanthridine alkaloids, phytoalexins of Eschscholzia californica, in cultured cells can be induced by a glycoprotein preparation from yeast, methyljasmonate, artificial acidification with permeant acids, or mild osmotic stress . Each of these stimuli strongly attenuated the subsequent response to the same stimulus (homologous desensitization) . Elicitor contact and artificial acidification mutually desensitized the cells for either signal . In contrast, elicitor-treated cells maintained their responsiveness to methyljasmonate or hyperosmolarity (sorbitol) . Elicitor concentrations that nearly saturated the alkaloid response did not cause a detectable increase of jasmonate content . Transient acidification of the cytoplasm is a necessary step of signaling by low elicitor concentrations but was not detectable after jasmonate treatment . Seen together, the data indicate the existence of a jasmonate-dependent and jasmonate-independent (Delta pH controlled) signal pathway towards the expression of benzophenanthridine biosynthesis . Selective desensitization allows either stimulus to activate a distinct share of the biosynthetic capacity of the cell and limits the accumulation of toxic defense metabolites.

Fungal Genet Biol, 2003 Mar, 38(2), 175 - 86
Specific induction and carbon/nitrogen repression of arginine catabolism gene of Aspergillus nidulans--functional in vivo analysis of the otaA promoter; Dzikowska A et al.; The arginine catabolism gene otaA encoding ornithine transaminase (OTAse) is specifically induced by arginine and is under the control of the broad-domain carbon and nitrogen repression systems . Arginine induction is mediated by a product of arcA gene coding for Zn(2)C(6) activator . We have identified a region responsible for arginine induction in the otaA promoter (AnUAS(arg)) . Deletions within this region result in non-inducibility of OTAse by arginine, whether in an arcA(+) strain or in the presence of the arcA(d)47 gain of function allele . AnUAS(arg) is very similar to the Saccharomyces cerevisiae UAS(arg), a sequence bound by the Zn(2)C(6) activator (ArgRIIp), acting in a complex with two MADS-box proteins (McmIp and ArgRIp).We demonstrate here that two CREA in vitro binding sites in the otaA promoter are functional in vivo . CREA is directly involved in carbon repression of the otaA gene and it also reduces its basal level of expression . Although AREA binds to the otaA promoter in vitro, it probably does not participate in nitrogen metabolite repression of the gene in vivo . We show here that another putative negatively acting GATA factor AREB participates directly or indirectly in otaA nitrogen repression . We also demonstrate that the high levels of OTAse activity are an important factor in the suppression of proline auxotrophic mutations . This suppression can be achieved neither by growing of the proline auxotroph under carbon/nitrogen derepressing conditions nor by introducing of a creA(d) mutation.

Mol Cell, 2003 Feb, 11(2), 437 - 44
The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase; North BJ et al.; The silent information regulator 2 protein (Sir2p) of Saccharomyces cerevisiae is an NAD-dependent histone deacetylase that plays a critical role in transcriptional silencing . Here, we report that a human ortholog of Sir2p, sirtuin type 2 (SIRT2), is a predominantly cytoplasmic protein that colocalizes with microtubules . SIRT2 deacetylates lysine-40 of alpha-tubulin both in vitro and in vivo . Knockdown of SIRT2 via siRNA results in tubulin hyperacetylation . SIRT2 colocalizes and interacts in vivo with HDAC6, another tubulin deacetylase . Enzymatic analysis of recombinant SIRT2 in comparison to a yeast homolog of Sir2 protein (Hst2p) shows a striking preference of SIRT2 for acetylated tubulin peptide as a substrate relative to acetylated histone H3 peptide . These observations establish SIRT2 as a bona fide tubulin deacetylase.

Mol Cell, 2003 Feb, 11(2), 425 - 35
Ribosome structure and activity are altered in cells lacking snoRNPs that form pseudouridines in the peptidyl transferase center; King TH et al.; One of the oldest questions in RNA science is the role of nucleotide modification . Here, the importance of pseudouridine formation (Psi) in the peptidyl transferase center of rRNA was examined by depleting yeast cells of 1-5 snoRNAs that guide a total of six Psi modifications . Translation was impaired substantially with loss of a conserved Psi in the A site of tRNA binding . Depletion of other Psis had subtle or no apparent effect on activity; however, synergistic effects were observed in some combinations . Pseudouridines are proposed to enhance ribosome activity by altering rRNA folding and interactions, with some Psis having greater effects than others . The possibility that modifying snoRNPs might affect ribosome structure in other ways is also discussed.

Mol Cell, 2003 Feb, 11(2), 391 - 403
SWI/SNF unwraps, slides, and rewraps the nucleosome; Kassabov SR et al.; The structure of the SWI/SNF-remodeled nucleosome was characterized with single base-pair resolution by mapping the contacts of specific histone fold residues with nucleosomal DNA . We demonstrate that SWI/SNF peels up to 50 bp of DNA from the edge of the nucleosome, translocates the histone octamer beyond the DNA ends via a DNA bulge propagation mechanism, and promotes the formation of an intramolecular DNA loop between the nucleosomal entry and exit sites . This stable altered nucleosome conformation also exhibits alterations in the distance between contacts of specific histone residues with DNA and higher electrophoretic and sedimentation mobility, consistent with a more compact molecular shape . SWI/SNF converts a nucleosome to the altered state in less than 1 s, hydrolyzing fewer than 10 ATPs per event.

Mol Cell, 2003 Feb, 11(2), 365 - 76
Bromodomains mediate an acetyl-histone encoded antisilencing function at heterochromatin boundaries; Ladurner AG et al.; Bromodomains bind acetylated histone H4 peptides in vitro . Since many chromatin remodeling complexes and the general transcription factor TFIID contain bromodomains, they may link histone acetylation to increased transcription . Here we show that yeast Bdf1 bromodomains recognize endogenous acetyl-histone H3/H4 as a mechanism for chromatin association in vivo . Surprisingly, deletion of BDF1 or a Bdf1 mutation that abolishes histone binding leads to transcriptional downregulation of genes located at heterochromatin-euchromatin boundaries . Wild-type Bdf1 protein imposes a physical barrier to the spreading of telomere- and mating-locus-proximal SIR proteins . Biochemical experiments indicate that Bdf1 competes with the Sir2 deacetylase for binding to acetylated histone H4 . These data suggest an active role for Bdf1 in euchromatin maintenance and antisilencing through a histone tail-encoded boundary function.

Mol Cell, 2003 Feb, 11(2), 353 - 63
Different sensitivities of bromodomain factors 1 and 2 to histone H4 acetylation; Matangkasombut O et al.; The histone code hypothesis proposes that covalently modified histone tails are binding sites for specific proteins . In vitro evidence suggests that factors containing bromodomains read the code by binding acetylated histone tails . Bromodomain Factor 1 (Bdf1), a protein that associates with TFIID, binds histone H4 with preference for multiply acetylated forms . In contrast, the closely related protein Bdf2 shows no preference for acetylated forms . A deletion of BDF1 but not BDF2 is lethal when combined with a mutant allele of ESA1 (a histone H4 acetyltransferase) or with nonacetylatable histone H4 variants . Bromodomain point mutations that block Bdf1 binding to histones disrupt transcription and reduce Bdf1 association with chromatin in vivo . Therefore, bromodomains with different specificity generate further complexity of the histone code.

Mol Cell, 2003 Feb, 11(2), 329 - 40
Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17; Lee J et al.; Claspin is required for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA . We show here that Claspin associates with chromatin in a regulated manner during S phase . Binding of Claspin to chromatin depends on the pre-replication complex (pre-RC) and Cdc45 but not on replication protein A (RPA) . These dependencies suggest that binding of Claspin occurs around the time of initial DNA unwinding at replication origins . By contrast, both ATR and Rad17 require RPA for association with DNA . Claspin, ATR, and Rad17 all bind to chromatin independently . These findings suggest that Claspin plays a role in monitoring DNA replication during S phase . Claspin, ATR, and Rad17 may collaborate in checkpoint regulation by detecting different aspects of a DNA replication fork.

Mol Cell, 2003 Feb, 11(2), 289 - 91
Enforced proximity in the function of a famous scaffold; Ferrell JE Jr et al.; Recent studies by Park, Zarrinipar, and Lim with reengineered Ste5 scaffold proteins underscore the fundamental importance of proximity in enzyme regulation and of keeping a proper distance for maintaining signaling specificity.

Mol Cell, 2003 Feb, 11(2), 283 - 4
Histone deposition at the replication fork: a matter of urgency; Verreault A; In this issue of Molecular Cell, Ye et al . provide a biological rationale for rapid histone deposition behind the replication fork . They show that defects in nucleosome assembly lead to DNA double-strand breaks and S phase arrest . Their results have important implications for the maintenance of genome integrity in proliferating cells.

Curr Biol, 2003 Mar 4, 13(5), 405 - 10
The ARF-like GTPases Arl1p and Arl3p act in a pathway that interacts with vesicle-tethering factors at the Golgi apparatus; Panic B et al.; The ARLs are a diverse family of GTPases that are related to ADP-ribosylation factors (ARFs), but whose function is poorly understood . There are at least ten ARLs in humans, two of which have homologs in the yeast Saccharomyces cerevisiae (ARL1/Arl1p and ARFRP1/Arl3p) . The function of ARFRP1 is unknown, but mammalian ARL1 has recently been found to interact with a number of effectors including the GRIP domain that is present in a family of Golgi-localized long coiled-coil proteins . We find that in yeast, the intracellular targeting of Imh1p, the only yeast GRIP domain protein, is dependent on both Arl1p and Arl3p, but not on the ARF proteins . A recombinant form of the Imh1p GRIP domain binds to Arl1p in a GTP-dependent manner, but not to Arl3p . Yeast also contain a relative of SCOCO, a protein proposed to bind human ARL1, but this yeast protein, Slo1p, appears to bind Arl3p rather than Arl1p in vitro . However, Imh1p is not the sole effector of Arl1p since affinity chromatography of cytosol with immobilized Arl1p:GTP revealed an interaction with the GARP/VFT complex that is thought to act in the tethering of vesicles to the Golgi apparatus . Finally, we find that Arl3p is required in vivo for the targeting of Arl1p, explaining its requirement for the normal distribution of Imh1p.

Curr Biol, 2003 Mar 4, 13(5), 401 - 4
Golgi recruitment of GRIP domain proteins by Arf-like GTPase 1 is regulated by Arf-like GTPase 3; Setty SR et al.; Golgins are Golgi-localized proteins present in all molecularly characterized eukaryotes that function in Golgi transport and maintenance of Golgi structure . Some peripheral membrane Golgins, including the yeast Imh1 protein, contain the recently described GRIP domain that can independently mediate Golgi localization by an unknown mechanism . To identify candidate Golgi receptors for GRIP domain proteins, a collection of Saccharomyces cerevisiae deletion mutants was visually screened by using yeast, mouse, and human GFP-GRIP domain fusion proteins for defects in Golgi localization . GFP-GRIP reporters were localized to the cytosol in cells lacking either of two ARF-like (ARL) GTPases, Arl1p and Arl3p . In vitro binding experiments demonstrated that activated Arl1p-GTP binds specifically and directly to the Imh1p GRIP domain . Arl1p colocalized with Imh1p-GRIP at the Golgi, and Golgi localization of Arl1p was regulated by the GTPase cycle of Arl3p . These results suggest a cascade in which the GTPase cycle of Arl3p regulates Golgi localization of Arl1p, which in turn binds to the GRIP domain of Imh1p and recruits it to the Golgi . The similar requirements for localization of GRIP domains from yeast, mouse, and human when expressed in yeast, and the presence of Arl1p and Arl3p homologs in these species, suggest that this is an evolutionarily conserved mechanism.

Genome Biol . 2003;4(2):204 . Epub 2003 Jan 30.
DNA replication: telling time with microarrays; McCune HJ et al.; A long-standing hypothesis about eukaryotic DNA replication is that the late-replicating regions are transcriptionally inert and that repressing transcription delays replication initiation . But do contrasting results from yeast and a recent study in Drosophila imply that replication timing and transcriptional activity are differentially regulated in yeast and higher eukaryotes?

Genome Biol . 2003;4(3):R23 . Epub 2003 Feb 27.
The GRID: the General Repository for Interaction Datasets; Breitkreutz BJ et al.; We have developed a relational database, called the General Repository for Interaction Datasets (The GRID) to archive and display physical, genetic and functional interactions . The GRID displays data-rich interaction tables for any protein of interest, combines literature-derived and high-throughput interaction datasets, and is readily accessible via the web . Interactions parsed in The GRID can be viewed in graphical form with a versatile visualization tool called Osprey.

Genes Chromosomes Cancer, 2003 Apr, 36(4), 402 - 5
t(10;16)(q22;p13) and MORF-CREBBP fusion is a recurrent event in acute myeloid leukemia; Vizmanos JL et al.; Recently, it was shown that t(10;16)(q22;p13) fuses the MORF and CREBBP genes in a case of childhood acute myeloid leukemia (AML) M5a, with a complex karyotype containing other rearrangements . Here, we report a new case with the MORF-CREBBP fusion in an 84-year-old patient diagnosed with AML M5b, in which the t(10;16)(q22;p13) was the only cytogenetic aberration . This supports that this is a recurrent pathogenic translocation in AML .

J Biol Chem, 2003 May 9, 278(19), 17012 - 20 Epub 2003 Mar 04.
The dynamin-like GTPase DLP1 is essential for peroxisome division and is recruited to peroxisomes in part by PEX11; Li X et al.; Peroxisome division involves the conserved PEX11 peroxisomal membrane proteins and in yeast has been shown to require Vps1p, a dynamin-like protein . We show here that DLP1, the human homolog of the yeast DNM1 and VPS1 genes, plays an important role in peroxisome division in human cells . Disruption of DLP1 function by either RNA interference or overexpressing dominant negative DLP1 mutants causes a dramatic reduction in peroxisome abundance, although overexpression of functional DLP1 has no effect on peroxisome abundance . Overexpression of PEX11 induces peroxisome division in a multistep process involving elongation of preexisting peroxisomes followed by their division . We find that DLP1 is dispensable for the first phase of this process but essential for the second . Furthermore, we show that DLP1 associates with peroxisomes and that PEX11 overexpression recruits DLP1 to peroxisome membranes . However, we were unable to detect physical interaction between PEX11 and DLP1, and the stoichiometry of PEX11 and peroxisome-associated DLP1 was far less than 1:1 . Based on these and other aspects, we propose that DLP1 performs an essential but transient role in peroxisome division and that PEX11 promotes peroxisome division by recruiting DLP1 to peroxisome membranes through an indirect mechanism.

J Biol Chem, 2003 Apr 18, 278(16), 13611 - 4 Epub 2003 Mar 03.
Identification of acyl coenzyme A:monoacylglycerol acyltransferase 3, an intestinal specific enzyme implicated in dietary fat absorption; Cheng D et al.; Acyl coenzyme A:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol using 2-monoacylglycerol and fatty acyl coenzyme A . This enzymatic reaction is believed to be an essential and rate-limiting step for the absorption of fat in the small intestine . Although the first MGAT-encoding cDNA, designated MGAT1, has been recently isolated, it is not expressed in the small intestine and hence cannot account for the high intestinal MGAT enzyme activity that is important for the physiology of fat absorption . In the current study, we report the identification of a novel MGAT, designated MGAT3, and present evidence that it fulfills the criteria to be the elusive intestinal MGAT . MGAT3 encodes a approximately 36-kDa transmembrane protein that is highly homologous to MGAT1 and -2 . In humans, expression of MGAT3 is restricted to gastrointestinal tract with the highest level found in the ileum . At the cellular level, recombinant MGAT3 is localized to the endoplasmic reticulum . Recombinant MGAT3 enzyme activity produced in insect Sf9 cells selectively acylates 2-monoacylglycerol with higher efficiency than other stereoisomers . The molecular identification of MGAT3 will facilitate the evaluation of using intestinal MGAT as a potential point of intervention for antiobesity therapies.

Biochim Biophys Acta, 2003 Mar 17, 1603(2), 47 - 82
GAPs galore! A survey of putative Ras superfamily GTPase activating proteins in man and Drosophila; Bernards A; Typical members of the Ras superfamily of small monomeric GTP-binding proteins function as regulators of diverse processes by cycling between biologically active GTP- and inactive GDP-bound conformations . Proteins that control this cycling include guanine nucleotide exchange factors or GEFs, which activate Ras superfamily members by catalyzing GTP for GDP exchange, and GTPase activating proteins or GAPs, which accelerate the low intrinsic GTP hydrolysis rate of typical Ras superfamily members, thus causing their inactivation . Two among the latter class of proteins have been implicated in common genetic disorders associated with an increased cancer risk, neurofibromatosis-1, and tuberous sclerosis . To facilitate genetic analysis, I surveyed Drosophila and human sequence databases for genes predicting proteins related to GAPs for Ras superfamily members . Remarkably, close to 0.5% of genes in both species (173 human and 64 Drosophila genes) predict proteins related to GAPs for Arf, Rab, Ran, Rap, Ras, Rho, and Sar family GTPases . Information on these genes has been entered into a pair of relational databases, which can be used to identify evolutionary conserved proteins that are likely to serve basic biological functions, and which can be updated when definitive information on the coding potential of both genomes becomes available .

Mech Ageing Dev, 2003 Jan, 124(1), 9 - 16
Systems biology: integrating technology, biology, and computation; Hood L; The Human Genome Project has changed the worlds of biology and medicine-helping to catalyze two major paradigm changes: systems biology and predictive, preventive and personalized medicine . These two themes will dominate 21st century biology and medicine . I will discuss these changes and indicate how they may interface with with the process of aging.

Best Pract Res Clin Gastroenterol, 2003 Feb, 17(1), 19 - 35
Serological markers in inflammatory bowel diseases; Reumaux D et al.; This chapter is an overview of the literature on serological markers of inflammatory bowel diseases (IBD), focusing on anti-neutrophil cytoplasm autoantibodies (ANCA) and anti- Saccharomyces cerevisiae mannan antibodies (ASCA) . The methodology for ANCA and ASCA testing is first introduced . The value of these markers as diagnostic tools is then discussed . Other chapters are devoted to the potential role of ANCA and ASCA in disease monitoring, disease stratification and as subclinical markers in families . Finally reviewed are other antibodies recently tested in clinical trials such as pancreatic antibodies and antibodies directed against bacterial antigens . The role of these antibodies in the pathophysiology of IBD still needs to be assessed . We also need to identify the ASCA immunogen(s) eliciting the antibody response.

Metab Eng, 2002 Jul, 4(3), 193 - 201
Amplification of HMG-CoA reductase production enhances carotenoid accumulation in Neurospora crassa; Wang GY et al.; Neurospora crassa, a filamentous fungus, naturally produces the carotenoids lycopene and neurosporaxanthin . To increase the carbon flux through the carotenoid biosynthetic pathway, the 1658-bp region of the HMG1 gene encoding the catalytic domain (cHMG1) of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Saccharomyces cerevisiae was expressed in N . crassa under control of the strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and the inducible alcohol dehydrogenase (alcA) promoter . Overexpressing cHMG1 under control of the GPD promoter increased lycopene and neurosporaxanthin production 6- and 1.5-fold, respectively, relative to the wild-type strain . Over-expression of cHMG1 under control of the alcA promoter increased production of lycopene and neurosporaxanthin 3- and 2-fold, respectively.

Proc Natl Acad Sci U S A, 2003 Mar 18, 100(6), 3345 - 50 Epub 2003 Mar 03.
Novel functions of the phosphatidylinositol metabolic pathway discovered by a chemical genomics screen with wortmannin; Zewail A et al.; We report a novel connection between the phosphatidylinositol (PI) metabolic pathway and the DNA replication and damage checkpoint pathway discovered from an unbiased chemical genomics screen . Substrates and products of PI kinases are important signaling molecules that affect a wide range of biological processes . The full collection of yeast deletion strains was screened to identify genes that confer altered sensitivity to the natural product wortmannin, a PI kinase inhibitor . These experiments have allowed us to explore metabolomic and proteomic implications of PI synthesis and turnover . This study also uncovers other biological processes affected by wortmannin treatment, including proteasome-mediated degradation and chromatin remodeling . Bioinformatic analyses were used to reveal the relative distances among cellular processes affected by wortmannin and protein-protein interactions in the wortmannin-sensitive proteomic subnetwork . These results illustrate the great utility of using a whole-genome approach in annotating the biological effects of small molecules and have clear implications for pharmacogenomics . Furthermore, our discovery points to a route to overcoming genome instability, a result of defective DNA damage signaling/repair and a hallmark of cancer.

J Cell Sci, 2003 Apr 1, 116(Pt 7), 1249 - 60
Control of morphogenesis and actin localization by the Penicillium marneffei RAC homolog; Boyce KJ et al.; Rac proteins control polarized growth in many organisms but the specific function of these proteins remains undefined . In this study, we describe the cloning and functional characterization of a RAC homolog, cflB, from the dimorphic fungus Penicillium marneffei . P . marneffei produces asexual spores on complex structures (conidiophores) and switches between hyphal and yeast growth . CflB colocalizes with actin at the tips of vegetative hyphal cells and at sites of cell division . Deletion of cflB results in cell division (septation) and growth defects in both vegetative hyphal and conidiophore cell types such that cells become depolarized, exhibit inappropriate septation and the actin cytoskeleton is severely disrupted . This data suggests that Rac proteins play a crucial role in actin dependent polarized growth and division . The CDC42 ortholog in P . marneffei, cflA, controls vegetative hyphal and yeast growth polarization but does not affect asexual development . By contrast, CflB affects cellular polarization during asexual development and hyphal growth but not during yeast growth . This shows that these two GTPases have both overlapping and distinct roles during growth and development . RAC orthologs are not found in less morphologically complex eukaryotes such as Saccharomyces cerevisiae, suggesting that RAC genes might have evolved with increasing cellular complexity.

Plant Cell, 2003 Mar, 15(3), 719 - 31
Characterization of the last subunit of the Arabidopsis COP9 signalosome: implications for the overall structure and origin of the complex; Serino G et al.; The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that resembles the lid subcomplex of proteasomes . Through its ability to regulate specific proteasome-mediated protein degradation events, CSN controls multiple aspects of development . Here, we report the cloning and characterization of AtCSN2, the last uncharacterized CSN subunit from Arabidopsis . We show that the AtCSN2 gene corresponds to the previously identified FUS12 locus and that AtCSN2 copurifies with CSN, confirming that AtCSN2 is an integral component of CSN . AtCSN2 is not only able to interact with the SCF(TIR1) subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCF(TIR1), but also interacts with AtCUL3, suggesting that CSN is able to regulate the activity of other cullin-based E3 ligases through conserved interactions . Phylogenetic analysis indicated that the duplication and subsequent divergence events that led to the genes that encode CSN and lid subunits occurred before the divergence of unicellular and multicellular eukaryotic organisms and that the CSN subunits were more conserved than the lid subunits during evolution . Comparative analyses of the subunit interaction of CSN revealed a set of conserved subunit contacts and resulted in a model of CSN subunit topology, some aspects of which were substantiated by in vivo cross-link tests.

J Struct Biol, 2003 Feb, 141(2), 132 - 42
The native-like conformation of Ure2p in fibrils assembled under physiologically relevant conditions switches to an amyloid-like conformation upon heat-treatment of the fibrils; Bousset L et al.; The {URE3} phenotype in the yeast Saccharomyces cerevisiae is inherited by a prion mechanism involving self-propagating Ure2p aggregates . It is believed that assembly of intact Ure2p into fibrillar polymers that bind Congo Red and show yellow-green birefringence upon staining and are resistant to proteolysis is the consequence of a major change in the conformation of the protein . We recently dissected the assembly process of Ure2p and showed the protein to retain its native alpha-helical structure upon assembly into protein fibrils that are similar to amyloids in that they are straight, bind Congo red and show green-yellow birefringence and have an increased resistance to proteolysis () . Here we further show using specific ligand binding, FTIR spectroscopy and X-ray fiber diffraction that Ure2p fibrils assembled under physiologically relevant conditions are devoid of a cross-beta core . The X-ray fiber diffraction pattern of these fibrils reveals their well-defined axial supramolecular order . By analyzing the effect of heat-treatment on Ure2p fibrils we bring evidences for a large conformational change that occurs within the fibrils with the loss of the ligand binding capacity, decrease of the alpha helicity, the formation of a cross-beta core and the disappearance of the axial supramolecular order . The extent of the conformational change suggests that it is not limited to the N-terminal part of Ure2p polypeptide chain . We show that the heat-treated fibrils that possess a cross-beta core are unable to propagate their structural characteristic while native-like fibrils are . Finally, the potential evolution of native-like fibrils into amyloid fibrils is discussed.

Mol Biochem Parasitol, 2003 Feb, 126(2), 193 - 200
The second largest subunit of Trypanosoma brucei's multifunctional RNA polymerase I has a unique N-terminal extension domain; Schimanski B et al.; In the protist parasite Trypanosoma brucei, RNA polymerase (pol) I transcribes the large ribosomal RNA gene unit and, in addition, variant surface glycoprotein gene expression sites and procyclin gene transcription units . The multifunctional role of RNA pol I in this organism is unique among eukaryotes, but only its largest subunit TbRPA1 has been characterized thus far . We have recently established the procyclic cell line RPIC which exclusively expresses RNA pol I tagged with the protein C epitope at the TbRPA1 C-terminus . In the present study, we prepared RPIC cell extracts and immunopurified RNA pol I using anti-protein C affinity matrix under high stringency conditions . We were able to identify five specific polypeptides on a silver-stained polyacrylamide-SDS gel with apparent molecular weights of 200, 180, 55, 29, and 22 kDa . Interestingly, the second largest subunit, TbRPA2, is 42-58 kDa larger than counterparts of other organisms . We have cloned and sequenced the complete TbRPA2 cDNA and found an open reading frame for a polypeptide of 179.5 kDa . The deduced amino acid sequence of TbRPA2 contains a unique N-terminal domain of approximately 250 amino acids . By raising a polyclonal antibody against a N-terminal peptide sequence of TbRPA2, we could specifically detect this polypeptide in immunoblots showing that it co-purifies with epitope-tagged TbRPA1 . Moreover, we identified the homologous gene sequence LmRPA2 in Leishmania major and found that it encodes a homologous extension domain . Therefore, the N-terminal extra domain in trypanosomatid RPA2 polypeptides may serve a parasite-specific function .

Biochem Biophys Res Commun, 2003 Mar 14, 302(3), 484 - 8
Two forms of RNA polymerase II holoenzyme display different abundance during the cell cycle; Holland L et al.; We analyzed the composition and abundance of two forms of RNA polymerase II (pol II) holoenzyme in synchronized HeLa cells . We did not detect significant changes in pol II holoenzyme composition, but we noticed differences in the abundance of the two complexes at different stages of the cell cycle . Summarized data from several independent experiments demonstrate that pol II holoenzyme, which is purified by GST-TFIIS affinity chromatography, is more abundant during G1/S and S phases . Another form of pol II holoenzyme, which is purified by anti-CDK7 antibodies, shows relatively higher amounts in G2/M and early G1 phases.

Biochem Biophys Res Commun, 2003 Mar 14, 302(3), 454 - 61
C-terminal phosphorylation of MRP2 modulates its interaction with PDZ proteins; Hegedus T et al.; MRP2, a member of the ABC protein superfamily, functions as an ATP-dependent export pump for anionic conjugates in the apical membranes of epithelial cells . It has been reported that the trafficking of MRP2 is modulated by PKC . Adjacent to the C-terminal PDZ binding motif, which may be involved in the targeting of MRP2, we found a potential PKC phosphorylation site (Ser(1542)) . Therefore, we examined the interaction of MRP2 and its phosphorylation-mimicking mutants with different PDZ proteins (EBP50, E3KARP, PDZK1, IKEPP, beta2-syntrophin, and SAP-97) . The binding of these PDZ proteins to CFTR and ABCA1, other ABC proteins, possessing PDZ binding motif, was also studied . We observed a strong binding of apically localized PDZ proteins to both MRP2 and CFTR, whereas beta2-syntrophin exhibited binding only to ABCA1 . The phosphorylation-mimicking MRP2 mutant and a phosphorylated C-terminal MRP2 peptide showed significantly increased binding to IKEPP, EBP50, and both individual PDZ domains of EBP50 . Our results suggest that phosphorylation of the MRP2 PDZ binding motif has a profound effect on the PDZ binding of MRP2.

Immunol Rev, 2003 Feb, 191, 183 - 95
Role of Shc in T-cell development and function; Zhang L et al.; Shc is a prototype adapter protein that is expressed from the earliest stages of T-cell development . Shc becomes rapidly tyrosine phosphorylated after T-cell receptor (TCR) engagement . Expression of dominant negative forms of Shc in T-cell lines had also suggested a role for this adapter downstream of the TCR . However, until recently, the relative significance of Shc compared to several other adapters in T cells was unclear . Mice lacking Shc expression specifically in the T-cell lineage together with inducible expression of dominant negative Shc in transgenic mice have revealed an essential and nonredundant role for Shc in thymic T-cell development . Functional defects in a Jurkat T-cell line lacking Shc expression also suggest a role for Shc in mature T-cell functions . While the requirement of Shc in T-cell signaling is now established, precisely what signaling pathways downstream of Shc make this adapter unique are less clear . Although the Shc-mediated activation of the extracellular signal regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathway could be one component, Shc likely signals to other pathways in T cells that are not yet discovered . A better molecular understanding of Shc function in the future could provide insights into how multiple adapters coordinate the various outcomes downstream of the TCR.

Biochemistry, 2003 Mar 11, 42(9), 2656 - 63
Transcriptional regulation of the nuclear gene encoding the alpha-subunit of the mammalian mitochondrial F1F0 ATP synthase complex: role for the orphan nuclear receptor, COUP-TFII/ARP-1; Jordan EM et al.; Our laboratory has been studying the transcriptional regulation of the nuclear gene (ATPA) that encodes the alpha-subunit of the mammalian mitochondrial F1F0 ATP synthase complex . We have previously determined that the regulatory factor, upstream stimulatory factor 2 (USF2), can stimulate transcription of the ATPA gene through the cis-acting regulatory element 1 in the upstream promoter of this gene . In this study, we used the yeast one-hybrid screening method to identify another factor, COUP-TFII/ARP-1, which also binds to the ATPA cis-acting regulatory element 1 . Binding of the orphan nuclear receptor, COUP-TFII/ARP-1, to the ATPA regulatory element 1 was confirmed using electrophoretic mobility shift experiments, and COUP-TFII/ARP-1-containing complexes were detected in HeLa cell nuclear extracts . A mutational analysis indicated that the binding site for COUP-TFII/ARP-1 in the ATPA regulatory element 1 is an imperfect direct repeat of a nuclear receptor response element (A/GGGTCA) with a spacer of three nucleotides . Functional assays in HeLa cells showed that COUP-TFII/ARP-1 represses the ATPA promoter activity in a dose- and sequence-dependent manner . Furthermore, cotransfection assays demonstrated that COUP-TFII/ARP-1 inhibits the USF2-mediated activation of the wild-type ATPA gene promoter but not a mutant promoter that is defective in COUP-TFII/ARP-1-binding . Overexpression of USF2 reversed the COUP-TFII/ARP-1-mediated repression of the ATPA promoter . Mobility shift assays revealed that COUP-TFII/ARP-1 and USF2 compete for binding to the ATPA regulatory element 1 . Thus, the ATPA gene is regulated by a multifunctional binding site through which the transcription factors, COUP-TFII/ARP-1 and USF2, bind and exert their antagonistic effects.

Nat Rev Mol Cell Biol, 2003 Mar, 4(3), 181 - 91
Quality control in the endoplasmic reticulum; Ellgaard L et al.; The endoplasmic reticulum (ER) has a quality-control system for 'proof-reading' newly synthesized proteins, so that only native conformers reach their final destinations . Non-native conformers and incompletely assembled oligomers are retained, and, if misfolded persistently, they are degraded . As a large fraction of ER-synthesized proteins fail to fold and mature properly, ER quality control is important for the fidelity of cellular functions . Here, we discuss recent progress in understanding the conformation-specific sorting of proteins at the level of ER retention and export.

EMBO Rep, 2003 Feb, 4(2), 184 - 8
Non-reciprocal regulation of the redox state of the glutathione-glutaredoxin and thioredoxin systems; Trotter EW et al.; Our studies in yeast show that there is an essential requirement for either an active thioredoxin or an active glutathione (GSH)-glutaredoxin system for cell viability . Glutathione reductase (Glr1) and thioredoxin reductase (Trr1) are key regulatory enzymes that determine the redox state of the GSH-glutaredoxin and thioredoxin systems, respectively . Here we show that Trr1 is required during normal cell growth, whereas there is no apparent requirement for Glr1 . Analysis of the redox state of thioredoxins and glutaredoxins in glr1 and trr1 mutants reveals that thioredoxins are maintained independently of the glutathione system . In contrast, there is a strong correlation between the redox state of glutaredoxins and the oxidation state of the GSSG/2GSH redox couple . We suggest that independent redox regulation of thioredoxins enables cells to survive in conditions under which the GSH-glutaredoxin system is oxidized.

Mol Cell Biol, 2003 Mar, 23(6), 2109 - 22
A novel RING finger protein, human enhancer of invasion 10, alters mitotic progression through regulation of cyclin B levels; Toby GG et al.; The process of cellular morphogenesis is highly conserved in eukaryotes and is dependent upon the function of proteins that are centrally involved in specification of the cell cycle . The human enhancer of invasion clone 10 (HEI10) protein was identified from a HeLa cell library based on its ability to promote yeast agar invasion and filamentation . Through two-hybrid screening, the mitotic cyclin B1 and an E2 ubiquitin-conjugating enzyme were isolated as HEI10-interacting proteins . Mutation of the HEI10 divergent RING finger motif (characteristic of E3 ubiquitin ligases) and Cdc2/cyclin binding and phosphorylation sites alter HEI10-dependent yeast phenotypes, including delay in G(2)/M transition . In vertebrates, the addition of HEI10 inhibits nuclear envelope breakdown and mitotic entry in Xenopus egg extracts . Mechanistically, HEI10 expression reduces cyclin B levels in cycling Xenopus eggs and reduces levels of the cyclin B ortholog Clb2p in yeast . HEI10 is itself a specific in vitro substrate of purified cyclin B/cdc2, with a TPVR motif as primary phosphorylation site . Finally, HEI10 is itself ubiquitinated in egg extracts and is also autoubiquitinated in vitro . These and other points lead to a model in which HEI10 defines a divergent class of E3 ubiquitin ligase, functioning in progression through G(2)/M.

Mol Cell Biol, 2003 Mar, 23(6), 2042 - 54
Intersection of the Kap123p-mediated nuclear import and ribosome export pathways; Sydorskyy Y et al.; Kap123p is a yeast beta-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles . Surprisingly, Kap123p is not essential for growth, under normal conditions . To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123 . Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p . We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import . In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing . Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes . Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits . Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a DeltaRAI1-induced decrease in the overall biogenesis efficiency.

Mol Cell Biol, 2003 Mar, 23(6), 2009 - 16
Rfm1, a novel tethering factor required to recruit the Hst1 histone deacetylase for repression of middle sporulation genes; McCord R et al.; Transcriptional repression is often correlated with the alteration of chromatin structure through modifications of the nucleosomes in the promoter region, such as by deacetylation of the N-terminal histone tails . This is presumed to make the promoter region inaccessible to other regulatory factors and the general transcription machinery . To accomplish this, histone deacetylases are recruited to specific promoters via DNA-binding proteins and tethering factors . We have previously reported the requirement for the NAD(+)-dependent histone deacetylase Hst1 and the DNA-binding protein Sum1 for vegetative repression of many middle sporulation genes in Saccharomyces cerevisiae . Here we report the identification of a novel tethering factor, Rfm1, that is required for Hst1-mediated repression . Rfm1 interacts with both Sum1 and Hst1 and is required for the Sum1-Hst1 interaction . DNA microarray and Northern blot analyses showed that Rfm1 is required for repression of the same subset of Sum1-repressed genes that require Hst1 . These results suggest that Rfm1 is a specificity factor that targets the Hst1 deacetylase to a subset of Sum1-regulated genes.

Mol Cell Biol, 2003 Mar, 23(6), 1910 - 21
Regulation of TATA-binding protein binding by the SAGA complex and the Nhp6 high-mobility group protein; Yu Y et al.; Transcriptional activation of the yeast HO gene involves the sequential action of DNA-binding and chromatin-modifying factors . Here we examine the role of the SAGA complex and the Nhp6 architectural transcription factor in HO regulation . Our data suggest that these factors regulate binding of the TATA-binding protein (TBP) to the promoter . A gcn5 mutation, eliminating the histone acetyltransferase present in SAGA, reduces the transcription of HO, but expression is restored in a gcn5 spt3 double mutant . We conclude that the major role of Gcn5 in HO activation is to overcome repression by Spt3 . Spt3 is also part of SAGA, and thus two proteins in the same regulatory complex can have opposing roles in transcriptional regulation . Chromatin immunoprecipitation experiments show that TBP binding to HO is very weak in wild-type cells but markedly increased in an spt3 mutant, indicating that Spt3 reduces HO expression by inhibiting TBP binding . In contrast, it has been shown previously that Spt3 stimulates TBP binding to the GAL1 promoter as well as GAL1 expression, and thus, Spt3 regulates these promoters differently . We also find genetic interactions between TBP and either Gcn5 or the high-mobility-group protein Nhp6, including multicopy suppression and synthetic lethality . These results suggest that, while Spt3 acts to inhibit TBP interaction with the HO promoter, Gcn5 and Nhp6 act to promote TBP binding . The result of these interactions is to limit TBP binding and HO expression to a short period within the cell cycle . Furthermore, the synthetic lethality resulting from combining a gcn5 mutation with specific TBP point mutations can be suppressed by the overexpression of transcription factor IIA (TFIIA), suggesting that histone acetylation by Gcn5 can stimulate transcription by promoting the formation of a TBP/TFIIA complex.

Mol Cell Biol, 2003 Mar, 23(6), 1863 - 73
Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex; Narita T et al.; The multisubunit transcription elongation factor NELF (for negative elongation factor) acts together with DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF)/human Spt4-Spt5 to cause transcriptional pausing of RNA polymerase II (RNAPII) . NELF activity is associated with five polypeptides, A to E . NELF-A has sequence similarity to hepatitis delta antigen (HDAg), the viral protein that binds to and activates RNAPII, whereas NELF-E is an RNA-binding protein whose RNA-binding activity is critical for NELF function . To understand the interactions of DSIF, NELF, and RNAPII at a molecular level, we identified the B, C, and D proteins of human NELF . NELF-B is identical to COBRA1, recently reported to associate with the product of breast cancer susceptibility gene BRCA1 . NELF-C and NELF-D are highly related or identical to the protein called TH1, of unknown function . NELF-B and NELF-C or NELF-D are integral subunits that bring NELF-A and NELF-E together, and coexpression of these four proteins in insect cells resulted in the reconstitution of functionally active NELF . Detailed analyses using mutated recombinant complexes indicated that the small region of NELF-A with similarity to HDAg is critical for RNAPII binding and for transcriptional pausing . This study defines several important protein-protein interactions and opens the way for understanding the mechanism of DSIF- and NELF-induced transcriptional pausing.

Bioinformatics, 2003 Mar 1, 19(4), 467 - 73
A multivariate approach applied to microarray data for identification of genes with cell cycle-coupled transcription; Johansson D et al.; We have analyzed microarray data using a modeling approach based on the multivariate statistical method partial least squares (PLS) regression to identify genes with periodic fluctuations in expression levels coupled to the cell cycle in the budding yeast, Saccharomyces cerevisiae . PLS has major advantages for analyzing microarray data since it can model data sets with large numbers of variables and with few observations . A response model was derived describing the expression profile over time expected for periodically transcribed genes, and was used to identify budding yeast transcripts with similar profiles . PLS was then used to interpret the importance of the variables (genes) for the model, yielding a ranking list of how well the genes fitted the generated model . Application of an appropriate cutoff value, calculated from randomized data, allows the identification of genes whose expression appears to be synchronized with cell cycling . Our approach also provides information about the stage in the cell cycle where their transcription peaks . Three synchronized yeast cell microarray data sets were analyzed, both separately and combined . Cell cycle-coupled periodicity was suggested for 455 of the 6,178 transcripts monitored in the combined data set, at a significance level of 0.5% . Among the candidates, 85% of the known periodic transcripts were included . Analysis of the three data sets separately yielded similar ranking lists, showing that the method is robust.

Biochem J, 2003 Jun 1, 372(Pt 2), 435 - 41
Carbonic anhydrase-related protein is a novel binding protein for inositol 1,4,5-trisphosphate receptor type 1; Hirota J et al.; The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated Ca(2+) channel that is located on intracellular Ca(2+) stores and modulates Ca(2+) signalling . Using the yeast two-hybrid system, we screened a mouse brain cDNA library with bait constructs for mouse IP(3)R type 1 (IP(3)R1) to identify IP(3)R1-associated proteins . In this way, we found that carbonic anhydrase-related protein (CARP) is a novel IP(3)R1-binding protein . Western blot analysis revealed that CARP is expressed exclusively in Purkinje cells of the cerebellum, in which IP(3)R1 is abundantly expressed . Immunohistochemical analysis showed that the subcellular localization of CARP in Purkinje cells is coincident with that of IP(3)R1 . Biochemical analysis also showed that CARP is co-precipitated with IP(3)R1 . Using deletion mutagenesis, we established that amino acids 45-291 of CARP are essential for its association with IP(3)R1, and that the CARP-binding site is located within the modulatory domain of IP(3)R1 amino acids 1387-1647 . CARP inhibits IP(3) binding to IP(3)R1 by reducing the affinity of the receptor for IP(3) . As reported previously, sensitivity to IP(3) for IP(3)-induced Ca(2+) release in Purkinje cells is low compared with that in other tissues . This could be due to co-expression of CARP with IP(3)R in Purkinje cells and its inhibitory effects on IP(3) binding.

Nat Genet, 2003 Mar, 33 Suppl, 276 - 84
Tailoring the genome: the power of genetic approaches; Nagy A et al.; In the last century, genetics has developed into one of the most powerful tools for addressing basic questions concerning inheritance, development, individual and social operations and death . Here we summarize the current approaches to these questions in four of the most advanced models organisms: Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (worm), Drosophila melanogaster (fly) and Mus musculus (mouse) . The genomes of each of these four models have been sequenced, and all have well developed methods of efficient genetic manipulations.

Science, 2003 Feb 28, 299(5611), 1351 - 4
Genetics and the specificity of the aging process; Hekimi S et al.; The identification and study of long-lived mutant animals has provided valuable insights into the mechanisms that limit the life-span of organisms . Findings with the gene SIR2 suggest that the rate of aging can be regulated under certain conditions . Indeed, increased expression of SIR2 lengthens life-span by acting on biological processes that promote survival under conditions of scarcity . In addition, studies of mutant strains of Caenorhabditis elegans, in particular daf-2, clk-1, and isp-1 mutants, suggest that the biology of reactive oxygen species in the mitochondria and elsewhere might be the main determinant of life-span in this organism . Thus, the aging process may be more specific than previously anticipated on evolutionary grounds.

Science, 2003 Mar 14, 299(5613), 1751 - 3 Epub 2003 Feb 27.
Asymmetric inheritance of oxidatively damaged proteins during cytokinesis; Aguilaniu H et al.; Carbonylated proteins were visualized in single cells of the budding yeast Saccharomyces cerevisiae, revealing that they accumulate with replicative age . Furthermore, carbonylated proteins were not inherited by daughter cells during cytokinesis . Mother cells of a yeast strain lacking the sir2 gene, a life-span determinant, failed to retain oxidatively damaged proteins during cytokinesis . These findings suggest that a genetically determined, Sir2p-dependent asymmetric inheritance of oxidatively damaged proteins may contribute to free-radical defense and the fitness of newborn cells.

J Virol, 2003 Mar, 77(6), 3734 - 48
A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin alpha/beta pathway; Lischka P et al.; The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86 . Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function . To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins . Strong interactions were found between pUL84 and four members of the importin alpha protein family . These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells . Using in vitro transport assays we showed that the pUL84 nuclear import required importin alpha, importin beta, and Ran, thus following the classical importin-mediated import pathway . Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin alpha proteins . Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins . Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity . Thus, a complex structure appears to be essential for importin alpha binding and import activity.

J Biol Chem, 2003 May 9, 278(19), 16675 - 82 Epub 2003 Feb 27.
Involvement of histone acetylation in ovarian steroid-induced decidualization of human endometrial stromal cells; Sakai N et al.; Histone acetyltransferases and histone deacetylases (HDACs) determine the acetylation status of histones, regulating gene transcription . Decidualization is the progestin-induced differentiation of estrogen-primed endometrial stromal cells (ESCs), which is crucial for implantation and maintenance of pregnancy . We here show that trichostatin A (TSA), a specific HDAC inhibitor, enhances the up-regulation of decidualization markers such as insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin in a dose-dependent manner that is directed by 17beta-estradiol (E(2)) plus progesterone (P(4)) in cultured ESCs, but not glandular cells, both isolated from human endometrium . Morphological changes resembling decidual transformation were also augmented by co-addition of TSA . Acid urea triton gel analysis and immunoblot using acetylated histone type-specific antibodies demonstrated that treatment with E(2) plus P(4) significantly increased the levels of acetylated H3 and H4 whose increment was augmented by co-treatment with TSA . Chromatin immunoprecipitation assay revealed that treatment with E(2) plus P(4) increased the amount of proximal progesterone-responsive region of IGFBP-1 promoter associated with acetylated H4, which was dramatically enhanced by co-addition of TSA . Taken together, our results suggest that histone acetylation is deeply involved in differentiation of human ESCs and that TSA has a potential as an enhancer of decidualization through promotion of progesterone action.

J Biol Chem, 2003 May 2, 278(18), 15905 - 10 Epub 2003 Feb 27.
TIP120A associates with cullins and modulates ubiquitin ligase activity; Min KW et al.; The cullin-containing ubiquitin-protein isopeptide ligases (E3s) play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling . They have multisubunit modular structures in which substrate recognition and the catalysis of ubiquitination are carried out by distinct polypeptides . In a search for proteins involved in regulation of cullin-containing E3 ubiquitin ligases we immunopurified CUL4B-containing complex from HeLa cells and identified TIP120A as an associated protein by mass spectrometry . Immunoprecipitation of cullins revealed that all cullins tested specifically interacted with TIP120A . Reciprocal immunoaffinity purification of TIP120A confirmed the stable interaction of TIP120A with cullin family proteins . TIP120A formed a complex with CUL1 and Rbx1, but interfered with the binding of Skp1 and F-box proteins to CUL1 . TIP120A greatly reduced the ubiquitination of phosphorylated IkappaBalpha by SCF(beta-TrCP) ubiquitin ligase . These results suggest that TIP120A functions as a negative regulator of SCF E3 ubiquitin ligases and may modulate other cullin ligases in a similar fashion.

Biophys J, 2003 Mar, 84(3), 1867 - 75
Thermodynamic characterization of the folding coupled DNA binding by the monomeric transcription activator GCN4 peptide; Wang X et al.; Dimerization is a widely believed critical requirement for the yeast transcriptional activator GCN4 specifically recognizing its DNA target sites . Nonetheless, the binding of the monomeric GCN4 to DNA target sites AP-1 and ATF/CREB was recently detected by kinetic studies . Here, for the first time, we present a detailed description of the thermodynamics of a monomeric peptide GCN4-br, the basic region (226-252) of GCN4, binding to AP-1, and ATF/CREB . GCN4 specifically binds to AP-1 and ATF/CREB in the monomeric form as shown by our circular dichroism thermal unfolding measurements . Isothermal titration calorimetry experiments indicate that the binding process of GCN4-br with DNA is enthalpically driven, accompanied by an unfavorable entropy change . The temperature dependence of DeltaH(0) reveals negative changes in heat capacity DeltaC(p): DeltaC(p) = -0.92 kJ . mol(-1) K(-1) and DeltaC(p) = -0.95 kJ . mol(-1) K(-1) for GCN4-br binding to AP-1 and ATF/CREB, respectively, which is a striking manifestation of GCN4-br specifically recognizing DNA target sites . These thermodynamic characteristics may give new insight into the mechanism by which GCN4 protein binds to DNA target sites for its transcriptional regulation.

EMBO J, 2003 Mar 3, 22(5), 1158 - 67
The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo; Riggs DL et al.; Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5 . The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function . FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51 . The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52.

J Biol Chem, 2003 May 2, 278(18), 15917 - 21 Epub 2003 Feb 26.
The mRNA transcription/processing factor Ssu72 is a potential tyrosine phosphatase; Meinhart A et al.; Ssu72 is an essential and highly conserved protein involved in mRNA transcription and 3'-end processing . The biochemical function of Ssu72 was so far unknown . We report here evidence that Ssu72 is a phosphatase that resembles protein tyrosine phosphatases (PTPases) . First, recombinant Ssu72 cleaves the phosphotyrosine analogue p-nitrophenylphosphate, and this catalytic activity is impaired by PTPase-inhibiting agents . Second, the Ssu72 sequence contains the CX(5)R signature motif of PTPases; mutation of the catalytic cysteine in this motif abolishes Ssu72 activity in vitro and has been shown to confer lethality in vivo . Third, secondary structure prediction and site-directed mutagenesis predict that Ssu72 adopts the fold of PTPases of the low molecular weight family . Distinguishing features, such as a short "aspartate loop" at the active site, suggest however that Ssu72 is the founding member of a new phosphatase subfamily . The novel Ssu72 activity may regulate coupling events during mRNA biogenesis.

Biol Reprod, 2003 Jun, 68(6), 2241 - 8 Epub 2003 Jan 22.
A-kinase anchoring protein 4 binding proteins in the fibrous sheath of the sperm flagellum; Brown PR et al.; The fibrous sheath is a unique cytoskeletal structure located in the principal piece of the sperm flagellum and is constructed of two longitudinal columns connected by closely spaced circumferential ribs . Cyclic AMP-dependent protein kinases are secured within specific cytoplasmic domains by A-kinase anchoring proteins (AKAPs), and the most abundant protein in the fibrous sheath is AKAP4 . Several other fibrous sheath proteins have been identified, but how the fibrous sheath assembles is not understood . Yeast two-hybrid assays and deletion mutagenesis were used to identify AKAP4-binding proteins and to map the binding regions on AKAP4 and on the proteins identified . We found that AKAP4 binds AKAP3 and two novel spermatogenic cell-specific proteins, Fibrous Sheath Interacting Proteins 1 and 2 (FSIP1, FSIP2) . Transcription of Akap4, Akap3, and Fsip1 begins in early spermatid development, whereas transcription of Fsip2 begins in late spermatocyte development . AKAP3 is synthesized in round spermatids and incorporated into the fibrous sheath concurrently with formation of the rib precursors . However, AKAP4 is synthesized and incorporated into the nascent fibrous sheath late in spermatid development . The AKAP4 precursor is processed in the flagellum and only the mature form of AKAP4 appears to bind AKAP3 . These results suggest that AKAP3 is involved in organizing the basic structure of the fibrous sheath, whereas AKAP4 has a major role in completing fibrous sheath assembly.

Am J Respir Cell Mol Biol, 2003 Aug, 29(2), 232 - 8 Epub 2003 Feb 26.
Characterization of a lanosterol 14 alpha-demethylase from Pneumocystis carinii; Morales IJ et al.; Pneumocystis carinii (PC) causes severe pneumonia in immunocompromised patients . PC is intrinsically resistant to treatment with azole antifungal medications . The enzyme lanosterol 14 alpha-demethylase (Erg11) is the target for azole antifungals . We cloned PCERG11 and compared its sequence to Erg11 proteins present in azole-resistant organisms, and performed chromosomal and Northern blot analysis for PCERG11 . Of 13 potential sites which could confer resistance to azoles, two were identical to azole-resistant Candida . By site-directed mutagenesis we changed these two sites in PCERG11 to those present in azole-sensitive Candida to generate PCERG11-SDM (E113D, T125K) . We tested the susceptibility of ERG11 deletion strains of Saccharomyces cerevisiae (SC) expressing PCERG11, PCERG11-SDM, and wild-type SCERG11 to three azole antifungals: fluconazole, itraconazole, and voriconazole . PCERG11 required a 2.2-fold higher dose of voriconazole and 3.5-fold higher dose of fluconazole than SCERG11 for a 50% reduction in growth . No difference was observed in the sensitivity to itraconazole . PCERG11-SDM has increased sensitivity to fluconazole and voriconazole, but not itraconazole . We believe that the molecular structure of the lanosterol 14 alpha-demethylase encoded by PCERG11 confers inherent resistance to azole antifungals and plays an integral part in the overall resistance of this PC to azole therapy.

FEBS Lett, 2003 Feb 27, 537(1-3), 210 - 4
Perturbation of protein kinase CK2 uncouples executive part of phosphate maintenance pathway from cyclin-CDK control; Barz T et al.; The budding yeast Saccharomyces cerevisiae encounters phosphate starvation by the transcription-regulated PHO pathway . We find that genetic perturbation of protein kinase CK2, a conserved tetrameric Ser/Thr phosphotransferase with links to cell cycle and transcription, affects expression of PHO pathway genes in a subunit- and isoform-specific manner . Remarkably, the genes encoding phosphate supplying phosphatases and transporters are significantly repressed, while the genes encoding components of the central pathway regulator complex, a cyclin-dependent kinase (CDK), a cyclin, and a CDK inhibitor, remain unaltered . Thus, perturbation of CK2 uncouples the executive part of the PHO pathway from its cyclin-CDK control complex.

Eur J Biochem, 2003 Jan, 270(2), 293 - 306
Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2alpha (eIF2alpha) kinase; Pomar N et al.; Four distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases phosphorylate eIF2alpha at S51 and regulate protein synthesis in response to various environmental stresses . These are the hemin-regulated inhibitor (HRI), the interferon-inducible dsRNA-dependent kinase (PKR), the endoplasmic reticulum (ER)-resident kinase (PERK) and the GCN2 protein kinase . Whereas HRI and PKR appear to be restricted to mammalian cells, GCN2 and PERK seem to be widely distributed in eukaryotes . In this study, we have characterized the second eIF2alpha kinase found in Drosophila, a PERK homologue (DPERK) . Expression of DPERK is developmentally regulated . During embryogenesis, DPERK expression becomes concentrated in the endodermal cells of the gut and in the germ line precursor cells . Recombinant wild-type DPERK, but not the inactive DPERK-K671R mutant, exhibited an autokinase activity, specifically phosphorylated Drosophila eIF2alpha at S50, and functionally replaced the endogenous Saccharomyces cerevisiae GCN2 . The full length protein, when expressed in 293T cells, located in the ER-enriched fraction, and its subcellular localization changed with deletion of different N-terminal fragments . Kinase activity assays with these DPERK deletion mutants suggested that DPERK localization facilitates its in vivo function . Similar to mammalian PERK, DPERK forms oligomers in vivo and DPERK activity appears to be regulated by ER stress . Furthermore, the stable complexes between wild-type DPERK and DPERK-K671R mutant were mediated through the N terminus of the proteins and exhibited an in vitro eIF2alpha kinase activity.

Proc Natl Acad Sci U S A, 2003 Mar 4, 100(5), 2462 - 7 Epub 2003 Feb 25.
Nonhomologous end joining and V(D)J recombination require an additional factor; Dai Y et al.; DNA nonhomologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks in mammalian cells . It also functions to carry out rearrangements at the specialized breaks introduced during V(D)J recombination . Here, we describe a patient with T(-)B(-) severe combined immunodeficiency, whose cells have defects closely resembling those of NHEJ-defective rodent cells . Cells derived from this patient show dramatic radiosensitivity, decreased double-strand break rejoining, and reduced fidelity in signal and coding joint formation during V(D)J recombination . Detailed examination indicates that the patient is defective neither in the known factors involved in NHEJ in mammals (Ku70, Ku80, DNA-dependent protein kinase catalytic subunit, Xrcc4, DNA ligase IV, or Artemis) nor in the Mre11/Rad50/Nbs1 complex, whose homologue in Saccharomyces cerevisiae functions in NHEJ . These results provide strong evidence that additional activities are crucial for NHEJ and V(D)J recombination in mammals.

J Biol Chem, 2003 May 16, 278(20), 18346 - 52 Epub 2003 Feb 25.
Dynamic regulation of histone H3 methylated at lysine 79 within a tissue-specific chromatin domain; Im H et al.; Post-translational modifications of individual lysine residues of core histones can exert unique functional consequences . For example, methylation of histone H3 at lysine 79 (H3-meK79) has been implicated recently in gene silencing in Saccharomyces cerevisiae . However, the distribution and function of H3-meK79 in mammalian chromatin are not known . We found that H3-meK79 has a variable distribution within the murine beta-globin locus in adult erythroid cells, being preferentially enriched at the active betamajor gene . By contrast, acetylated H3 and H4 and H3 methylated at lysine 4 were enriched both at betamajor and at the upstream locus control region . H3-meK79 was also enriched at the active cad gene, whereas the transcriptionally inactive loci necdin and MyoD1 contained very little H3-meK79 . As the pattern of H3-meK79 at the beta-globin locus differed between adult and embryonic erythroid cells, establishment and/or maintenance of H3-meK79 was developmentally dynamic . Genetic complementation analysis in null cells lacking the erythroid and megakaryocyte-specific transcription factor p45/NF-E2 showed that p45/NF-E2 preferentially establishes H3-meK79 at the betamajor promoter . These results support a model in which H3-meK79 is strongly enriched in mammalian chromatin at active genes but not uniformly throughout active chromatin domains . As H3-meK79 is highly regulated at the beta-globin locus, we propose that the murine ortholog of Disruptor of Telomeric Silencing-1-like (mDOT1L) methyltransferase, which synthesizes H3-meK79, regulates beta-globin transcription.

Pac Symp Biocomput . 2003;:391-402.
Evaluation of the vector space representation in text-based gene clustering; Glenisson P et al.; Thanks to its increasing availability, electronic literature can now be a major source of information when developing complex statistical models where data is scarce or contains much noise . This raises the question of how to deeply integrate information from domain literature with experimental data . Evaluating what kind of statistical text representations can integrate literature knowledge in clustering still remains an unsufficiently explored topic . In this work we discuss how the bag-of-words representation can be used successfully to represent genetic annotation and free-text information coming from different databases . We demonstrate the effect of various weighting schemes and information sources in a functional clustering setup . As a quantitative evaluation, we contrast for different parameter settings the functional groupings obtained from text with those obtained from expert assessments and link each of the results to a biological discussion.

Pac Symp Biocomput . 2003;:89-100.
Decomposing gene expression into cellular processes; Segal E et al.; We propose a probabilistic model for cellular processes, and an algorithm for discovering them from gene expression data . A process is associated with a set of genes that participate in it; unlike clustering techniques, our model allows genes to participate in multiple processes . Each process may be active to a different degree in each experiment . The expression measurement for gene g in array a is a sum, over all processes in which g participates, of the activity levels of these processes in array a . We describe an iterative procedure, based on the EM algorithm, for decomposing the expression matrix into a given number of processes . We present results on Yeast gene expression data, which indicate that our approach identifies real biological processes.

Plant Mol Biol, 2003 Jan, 51(1), 99 - 108
Molecular and cellular characterisation of LjAMT2;1, an ammonium transporter from the model legume Lotus japonicus; Simon-Rosin U et al.; Two related families of ammonium transporters have been identified and partially characterised in plants in the past; the AMT1 and AMT2 families . Most attention has focused on the larger of the two families, the AMT1 family, which contains members that are likely to fulfil different, possibly overlapping physiological roles in plants, including uptake of ammonium from the soil . The possible physiological functions of AMT2 proteins are less clear . Lack of data on cellular and tissue location of gene expression, and the intracellular location of proteins limit our understanding of the physiological role of all AMT proteins . We have cloned the first AMT2 family member from a legume, LjAMT2;1 of Lotus japonicus, and demonstrated that it functions as an ammonium transporter by complementing a yeast mutant defective in ammonium uptake . However, like AtAMT2 from Arabidopsis, and unlike AMT1 transporters from several plant species, LjAMT2;1 was unable to transport methylammonium . The LjAMT2;1 gene was found to be expressed constitutively throughout Lotus plants . In situ RNA hybridisation revealed LjAMT2;1 expression in all major tissues of nodules . Transient expression of LjAMT2;1-GFP fusion protein in plant cells indicated that the transporter is located on the plasma membrane . In view of the fact that nodules derive ammonium internally, rather than from the soil, the results implicate LjAMT2;1 in the recovery of ammonium lost from nodule cells by efflux . A similar role may be fulfilled in other organs, especially leaves, which liberate ammonium during normal metabolism.

Plant Mol Biol, 2003 Feb, 51(3), 373 - 84
An interaction between an Arabidopsis poly(A) polymerase and a homologue of the 100 kDa subunit of CPSF; Elliott BJ et al.; The Arabidopsis genome possesses a number of sequences that are predicted to encode proteins that are similar to mammalian and yeast polyadenylation factor subunits . One of these resides on chromosome V and has the potential to encode a polypeptide related to the 100 kDa subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) . This gene encodes a ca . 2400 nucleotide mRNA that in turn can be translated to yield a polypeptide that is 39% identical to the mammalian CPSF100 protein . Antibodies raised against the Arabidopsis protein recognized distinctive polypeptides in nuclear extracts prepared from pea and wheat germ, consistent with the hypothesis that the Arabidopsis protein is resident in a nuclear polyadenylation complex . Interestingly, the Arabidopsis CPSF100 was found to interact with a portion of a nuclear poly(A) polymerase . This interaction was attributable to a 60 amino acid domain in the CPSF100 polypeptide and the N-terminal 220 amino acids of the poly(A) polymerase . An analogous interaction has yet to be described in other eukaryotes . The interaction with PAP thus indicates that the plant CPSF100 polypeptide is likely part of the 3'-end processing machinery, but suggests that this complex may function differently in plants than it does in mammals and yeast.

Genes Dev, 2003 Feb 15, 17(4), 502 - 15
SWI/SNF-dependent chromatin remodeling of RNR3 requires TAF(II)s and the general transcription machinery; Sharma VM et al.; Gene expression requires the recruitment of chromatin remodeling activities and general transcription factors (GTFs) to promoters . Whereas the role of activators in recruiting chromatin remodeling activities has been clearly demonstrated, the contributions of the transcription machinery have not been firmly established . Here we demonstrate that the remodeling of the RNR3 promoter requires a number of GTFs, mediator and RNA polymerase II . We also show that remodeling is dependent upon the SWI/SNF complex, and that TFIID and RNA polymerase II are required for its recruitment to the promoter . In contrast, Gcn5p-dependent histone acetylation occurs independently of TFIID and RNA polymerase II function, and we provide evidence that acetylation increases the extent of nucleosome remodeling, but is not required for SWI/SNF recruitment . Thus, the general transcription machinery can contribute to nucleosome remodeling by mediating the association of SWI/SNF with promoters, thereby revealing a novel pathway for the recruitment of chromatin remodeling activities.

J Colloid Interface Sci, 2003 Feb 1, 258(1), 75 - 81
Study of the immobilization of alcohol dehydrogenase on Au-colloid modified gold electrode by piezoelectric quartz crystal sensor, cyclic voltammetry, and electrochemical impedance techniques; Liu Y et al.; The immobilization of alcohol dehydrogenase (ADH) on Au-colloid modified gold electrodes has been investigated . Colloidal Au was first self-assembled onto gold electrodes through the thiol groups of an 1,6-hexanedithiol monolayer . Piezoelectric quartz crystal sensor, cyclic voltammetry, and electrochemical impedance techniques were used to investigate the immobilization of ADH on Au colloids . The cyclic voltammogram tends to be more irreversible with increased ADH concentration . In the impedance spectroscopic study, an obvious difference of the electron transfer resistance between the Au-colloid modified electrode and the bare gold electrode was observed . Using the piezoelectric quartz crystal sensor, the Michaelis constant, K(m), and the maximum initial rate, V(max), of the immobilized ADH were estimated as 6.03 x 10(-4) M and 0.63 Hzs (-1), respectively . The binding constant of ADH with nicotinamide adenine dinucleotide (NAD) was also determined as 1.87 x 10(4) M(-1) . Experimental results showed that colloidal Au can be used as a biocompatible matrix for enzyme immobilization.

Cell, 2003 Feb 21, 112(4), 561 - 74
Asymmetric loading of Kar9 onto spindle poles and microtubules ensures proper spindle alignment; Liakopoulos D et al.; Spindle alignment is the process in which the two spindle poles are directed toward preselected and opposite cell ends . In budding yeast, the APC-related molecule Kar9 is required for proper alignment of the spindle with the mother-bud axis . We find that Kar9 localizes to the prospective daughter cell spindle pole . Kar9 is transferred from the pole to cytoplasmic microtubules, which are then guided in a myosin-dependent manner to the bud . Clb4/Cdc28 kinase phosphorylates Kar9 and accumulates on the pole destined to the mother cell . Mutations that block phosphorylation at Cdc28 consensus sites result in localization of Kar9 to both poles and target them both to the bud . Thus, Clb4/Cdc28 prevents Kar9 loading on the mother bound pole . In turn, asymmetric distribution of Kar9 ensures that only one pole orients toward the bud . Our results indicate that Cdk1-dependent spindle asymmetry ensures proper alignment of the mitotic spindle with the cell division axis.

Cell, 2003 Feb 21, 112(4), 423 - 40
Un ménage à quatre: the molecular biology of chromosome segregation in meiosis; Petronczki M et al.; Sexually reproducing organisms rely on the precise reduction of chromosome number during a specialized cell division called meiosis . Whereas mitosis produces diploid daughter cells from diploid cells, meiosis generates haploid gametes from diploid precursors . The molecular mechanisms controlling chromosome transmission during both divisions have started to be delineated . This review focuses on the four fundamental differences between mitotic and meiotic chromosome segregation that allow the ordered reduction of chromosome number in meiosis: (1) reciprocal recombination and formation of chiasmata between homologous chromosomes, (2) suppression of sister kinetochore biorientation, (3) protection of centromeric cohesion, and (4) inhibition of DNA replication between the two meiotic divisions.

Biochem J, 2003 Jun 1, 372(Pt 2), 453 - 63
Direct binding of a fragment of the Wiskott-Aldrich syndrome protein to the C-terminal end of the anaphylatoxin C5a receptor; Tardif M et al.; Migration of myeloid cells towards a source of chemoattractant, such as the C5a anaphylatoxin, is triggered by the activation of a G-protein-coupled receptor . In the present study, we have used a yeast two-hybrid approach to find unknown partners of the C5a receptor (C5aR) . Using the cytosolic C-terminal region of C5aR as bait to screen a human leucocyte cDNA library, we identified the Wiskott-Aldrich syndrome protein (WASP) as a potential partner of C5aR . WASP is known to have an essential function in regulating actin dynamics at the cell leading edge . The interaction was detected with both the fragment of WASP containing amino acids 1-321 (WASP.321) and WASP with its actin-nucleation-promoting domain {verprolin-like, central and acidic (VCA) domain} deleted . The interaction between C5aR and the WASP.321 was supported further by an in vitro binding assay between a radiolabelled WASP.321 fragment and a receptor C-terminus glutathione S-transferase (GST) fusion protein, as well as by GST pull-down, co-immunoprecipitation and immunofluorescence experiments . In the yeast two-hybrid assay, full-length WASP showed no ability to interact with the C-terminal domain of C5aR . This is most probably due to an auto-inhibited conformation imposed by the VCA domain . In HEK-293T cells co-transfected with full-length WASP and C5aR, only a small amount of WASP was co-precipitated with the receptor . However, in the presence of the active form of the GTPase Cdc42 (Cdc42V12), which is thought to switch WASP to an active 'open conformation', the amount of WASP associated with the receptor was markedly increased . We hypothesize that a transient interaction between C5aR and WASP occurs following the stimulation of C5aR and Cdc42 activation . This might be one mechanism by which WASP is targeted to the plasma membrane and by which actin assembly is spatially controlled in cells moving in a gradient of C5a.

Radiat Res, 2003 Mar, 159(3), 420 - 5
Caffeine could not efficiently sensitize homologous recombination repair-deficient cells to ionizing radiation-induced killing; Wang H et al.; Caffeine inhibits ATM and ATR, two important checkpoint regulators, abolishes ionizing radiation-induced checkpoint response, and radiosensitizes cells . Radiation-induced DNA double-strand breaks (DSBs) are repaired by two major processes, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) . It remains unclear which repair process, HRR or NHEJ, is affected when the checkpoint responses are abolished by caffeine . In this study we observed the effect of caffeine on gene-targeted DT40 chicken lymphoblast cells . We show that caffeine efficiently abolishes S- and G(2)-phase checkpoint responses after irradiation in all cell lines tested and greatly radiosensitizes wild-type and ATM(-/-) cells, the partially checkpoint-deficient cells . However, caffeine has a much smaller radiosensitizing effect on RAD54(-/-) cells and has no effect on RAD51-deficient cells . RAD51 and RAD54 are the important factors for HRR . Our results indicate that the checkpoint responses abolished by caffeine (S and G(2)) mainly affect HRR, which results in cell radiosensitization . Copyright 2003 by Radiation Research Society

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