Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7773 - 8 Epub 2003 Jun 13. Inducible nitric oxide synthase expression inhibition by adenovirus E1A; Cao W et al.; Nitric oxide (NO) is an antiviral effector of the innate immune system . Viruses that can interfere with NO synthesis may be able to replicate more rapidly than viruses that cannot limit NO synthesis . We show that the adenovirus E1A protein inhibits NO production by decreasing expression of the inducible NO synthase (NOS2) . The amino-terminal portion of E1A decreases transactivation of the NOS2 5'-flanking region, limiting the DNA binding activity of NF-kappaB and inhibiting NOS2 expression . E1A is thus able to deactivate a critical component of the host defense against viral infection . Viral inhibition of NO production is a mechanism that may enable certain viruses to evade the host innate immune system. Mol Cell Biol, 2003 Jul, 23(13), 4449 - 60 Sequential protein association with nascent 60S ribosomal particles; Saveanu C et al.; Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles . These intermediate particles follow a maturation pathway in which important changes in their protein composition occur . The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood . We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis . The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2 . The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins . Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions. Mol Biol Cell, 2003 Jun, 14(6), 2559 - 69 Epub 2003 Feb 21. Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins; Kimata Y et al.; In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2 . It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism . In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation . To test this model, we analyzed five temperature-sensitive alleles of the yeast KAR2 gene . When cells carrying a mutation in the Kar2 substrate-binding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress . Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway . Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate . We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2. Mol Biol Cell, 2003 Jun, 14(6), 2543 - 58 Epub 2003 Feb 21. Nuclear export and plasma membrane recruitment of the Ste5 scaffold are coordinated with oligomerization and association with signal transduction components; Wang Y et al.; The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a Gbetagamma dimer (Ste4/Ste18) at the plasma membrane in a recruitment event that requires prior nuclear shuttling of Ste5 . Genetic evidence suggests that Ste5 must oligomerize to function, but its impact on Ste5 function and localization is unknown . Herein, we show that oligomerization affects Ste5 activity and localization . The majority of Ste5 is monomeric, suggesting that oligomerization is tightly regulated . Increasing the pool of Ste5 oligomers increases association with Ste11 . Remarkably, Ste5 oligomers are also more efficiently exported from the nucleus, retained in the cytoplasm by Ste11 and better recruited to the plasma membrane, resulting in constitutive activation of the mating mitogen-activated protein kinase cascade . Coprecipitation tests show that the RING-H2 domain is the key determinant of oligomerization . Mutational analysis suggests that the leucine-rich domain limits the accessibility of the RING-H2 domain and inhibits export and recruitment in addition to promoting Ste11 association and activation . Our results suggest that the major form of Ste5 is an inactive monomer with an inaccessible RING-H2 domain and Ste11 binding site, whereas the active form is an oligomer that is more efficiently exported and recruited and has a more accessible RING-H2 domain and Ste11 binding site. Mol Biol Cell, 2003 Jun, 14(6), 2357 - 71 Epub 2003 Mar 07. A novel Golgi membrane protein is a partner of the ARF exchange factors Gea1p and Gea2p; Chantalat S et al.; The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking . The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors . However, these interactions remain largely unknown . Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs . Gmh1p is an evolutionarily conserved protein . We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p . This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins . A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain . We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER . Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function . These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins. Mol Biol Cell, 2003 Jun, 14(6), 2342 - 56 Epub 2003 Feb 06. Mgm1p, a dynamin-related GTPase, is essential for fusion of the mitochondrial outer membrane; Sesaki H et al.; In Saccharomyces cerevisiae, mitochondrial fusion requires at least two outer membrane proteins, Fzo1p and Ugo1p . We provide direct evidence that the dynamin-related Mgm1 protein is also required for mitochondrial fusion . Like fzo1 and ugo1 mutants, cells disrupted for the MGM1 gene contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells . Fragmentation of mitochondria in mgm1 mutants is rescued by disrupting DNM1, a gene required for mitochondrial division . In zygotes formed by mating mgm1 mutants, mitochondria do not fuse and mix their contents . Introducing mutations in the GTPase domain of Mgm1p completely block mitochondrial fusion . Furthermore, we show that mgm1 mutants fail to fuse both their mitochondrial outer and inner membranes . Electron microscopy demonstrates that although mgm1 mutants display aberrant mitochondrial inner membrane cristae, mgm1 dnm1 double mutants restore normal inner membrane structures . However, mgm1 dnm1 mutants remain defective in mitochondrial fusion, indicating that mitochondrial fusion requires Mgm1p regardless of the morphology of mitochondria . Finally, we find that Mgm1p, Fzo1p, and Ugo1p physically interact in the mitochondrial outer membrane . Our results raise the possibility that Mgm1p regulates fusion of the mitochondrial outer membrane through its interactions with Fzo1p and Ugo1p. Mol Biol Cell, 2003 Jun, 14(6), 2292 - 302 Epub 2003 Apr 04. MTG1 codes for a conserved protein required for mitochondrial translation; Barrientos A et al.; The MTG1 gene of Saccharomyces cerevisiae, corresponding to ORF YMR097c on chromosome XIII, codes for a mitochondrial protein essential for respiratory competence . A human homologue of Mtg1p capable of partially rescuing the respiratory deficiency of a yeast mtg1 mutant has also been localized in mitochondria . Mtg1p is a member of a family of GTPases with largely unknown functions . The respiratory deficiency of mtg1 mutants stems from a defect in mitochondrial protein synthesis . Mutations in the 21S rRNA locus are able to suppress the translation defect of mtg1 null mutants . This points to the 21S rRNA or the large ribosomal subunit as the most likely target of Mtg1p action . The presence of mature size 15S and 21S mitochondrial rRNAs in mtg1 mutants excludes Mtg1p from being involved in transcription or processing of these RNAs . More likely, Mtg1p functions in assembly of the large ribosomal subunit . This is consistent with the peripheral localization of Mtg1p on the matrix side of the inner membrane and the results of in vivo mitochondrial translation assays in a temperature-sensitive mtg1 mutant. Mol Biol Cell, 2003 Jun, 14(6), 2206 - 15 Epub 2003 Mar 07. Drosophila MCM10 interacts with members of the prereplication complex and is required for proper chromosome condensation; Christensen TW et al.; Mcm10 is required for the initiation of DNA replication in Saccharomyces cerevisiae . We have cloned MCM10 from Drosophila melanogaster and show that it complements a ScMCM10 null mutant . Moreover, Mcm10 interacts with key members of the prereplication complex: Mcm2, Dup (Cdt1), and Orc2 . Interactions were also detected between Mcm10 and itself, Cdc45, and Hp1 . RNAi depletion of Orc2 and Mcm10 in KC cells results in loss of DNA content . Furthermore, depletion of Mcm10, Cdc45, Mcm2, Mcm5, and Orc2, respectively, results in aberrant chromosome condensation . The condensation defects observed resemble previously published reports for Orc2, Orc5, and Mcm4 mutants . Our results strengthen and extend the argument that the processes of chromatin condensation and DNA replication are linked. J Biol Chem, 2003 Sep 5, 278(36), 34373 - 9 Epub 2003 Jun 14. The structure of Aip1p, a WD repeat protein that regulates Cofilin-mediated actin depolymerization; Voegtli WC et al.; Actin-interacting protein 1 (Aip1p) is a 67-kDa WD repeat protein known to regulate the depolymerization of actin filaments by cofilin and is conserved in organisms ranging from yeast to mammals . The crystal structure of Aip1p from Saccharomyces cerevisiae was determined to a 2.3-A resolution and a final crystallographic R-factor of 0.204 . The structure reveals that the overall fold is formed by two connected seven-bladed beta-propellers and has important implications for the structure of Aip1 from other organisms and WD repeat-containing proteins in general . These results were unexpected because a maximum of 10 WD repeats had been reported in the literature for this protein using sequence data . The surfaces of the beta-propellers formed by the D-A and B-C loops are positioned adjacent to one another, giving Aip1p a shape that resembles an open "clamshell." The mapping of conserved residues to the structure of Aip1p reveals dense patches of conserved residues on the surface of one beta-propeller and at the interface of the two beta-propellers . These two patches of conserved residues suggest a potential binding site for F-actin on Aip1p and that the orientation of the beta-propellers with respect to one another plays a role in binding an actin-cofilin complex . In addition, the conserved interface between the domains is mediated by a number of interactions that appear to impart rigidity between the two domains of Aip1p and may make a large substrate-induced conformational change difficult. J Biol Chem, 2003 Sep 5, 278(36), 34347 - 55 Epub 2003 Jun 14. Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated; Vartiainen MK et al.; Twinfilin is a highly conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals . In addition to the previously characterized mammalian twinfilin-1, a second protein with approximately 65% sequence identity to twinfilin-1 exists in mouse and humans . However, previous studies failed to identify any actin binding activity in this protein (Rohwer, A., Kittstein, W., Marks, F., and Gschwendt, M . (1999) Eur . J . Biochem . 263, 518-525) . Here we show that this protein, which we named twinfilin-2, is indeed an actin monomer-binding protein . Similar to twinfilin-1, mouse twinfilin-2 binds ADP-G-actin with a higher affinity (KD = 0.12 microM) than ATP-G-actin (KD = 1.96 microM) and efficiently inhibits actin filament assembly in vitro . Both mouse twinfilins inhibit the nucleotide exchange on actin monomers and directly interact with capping protein . Furthermore, the actin interactions of mouse twinfilin-1 and twinfilin-2 are inhibited by phosphatidylinositol (4,5)-bisphosphate . Although biochemically very similar, our Northern blots and in situ hybridizations show that these two proteins display distinct expression patterns . Twinfilin-1 is the major isoform in embryos and in most adult mouse non-muscle cell-types, whereas twinfilin-2 is the predominant isoform of adult heart and skeletal muscles . Studies with isoform-specific antibodies demonstrated that although the two proteins show similar localizations in unstimulated cells, they are regulated by different mechanisms . The small GTPases Rac1 and Cdc42 induce the redistribution of twinfilin-1 to membrane ruffles and cell-cell contacts, respectively, but do not affect the localization of twinfilin-2 . Taken together, these data show that mammals have two twinfilin isoforms, which are differentially expressed and regulated through distinct cellular signaling pathways. J Biol Chem, 2003 Aug 29, 278(35), 33436 - 44 Epub 2003 Jun 13. ATP uptake in the Golgi and extracellular release require Mcd4 protein and the vacuolar H+-ATPase; Zhong X et al.; Extracellular nucleotides signal via a large group of purinergic receptors . Although much is known about these receptors, the mechanism of nucleotide transport out of the cytoplasm is unknown . We developed a functional screen for ATP release to the extracellular space and identified Mcd4p, a 919-amino acid membrane protein with 14 putative transmembrane domains, as a participant in glucose-dependent ATP release from Saccharomyces cerevisiae . This release occurred through the vesicular trafficking pathway initiated by ATP uptake into the Golgi compartment . Both the compartmental uptake and the extracellular release of ATP were regulated by the activity of the vacuolar H+-ATPase . It is likely that the Mcd4p pathway is generally involved in non-mitochondrial ATP movement across membranes, it is essential for Golgi and endoplasmic reticulum function, and its occurrence led to the appearance of P2 purinergic receptors. Carcinogenesis, 2003 Jul, 24(7), 1247 - 55 Epub 2003 Jun 05. Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (-)benzo{a}pyrene-7R-trans-7,8-dihydrodiol; Mammen JS et al.; Genetic differences that underlie inter-individual variation in the metabolism of common carcinogens are important potential sources of cancer susceptibility . Cytochrome P450 1B1 (CYP1B1), a central enzyme in the activation of the ubiquitous environmental carcinogen benzo{a}pyrene (B{a}P), has several genetic variants . This study investigated six rare mutations and four common polymorphisms for their effects on B{a}P metabolism . Five missense mutations associated with congenital glaucoma (Gly61Glu, Gly365Trp, Asp374Asn, Pro437Leu and Arg469Tryp) dramatically decreased the capacity of CYP1B1 to convert (-)benzo{a}pyrene-7R-trans-7,8-dihyrodiol (B{a}P-7,8-diol) to (+/-)benzo{a}pyrene-r-7,t-8-dihydrodiol-9,10-epoxides . These five mutations resulted in enzymes with 3-12% of normal activity when assayed in vitro using an Saccharomyces cerevisiae microsomal expression system . A 10 bp deletion mutation produced no detectable protein or activity . In contrast, proteins containing all possible combinations of four common single nucleotide polymorphisms (Arg48Gly, Ala199Ser, Val432Leu, Asn453Ser) had modest effects on B{a}P-7,8-diol metabolism . Michaelis-Menten analysis suggested that two alleles, Arg48, Ala119, Val432, Ser453 (RAVS) and Arg48, Ala119, Leu432, Ser453 (RALS), have KM values 2-fold lower than Arg48, Ala119, Val432, Ser453 (RAVN): 1.4+/-0.3 and 1.3+/-0.4 microM, respectively, compared with 2.8+/-0.8 microM (P<0.05) . However, these differences could not be confirmed with direct measurements of rate at low substrate concentration . There were no significant differences for either of two other kinetic parameters, kcat or kcat/KM . Allele frequency analysis in three populations reveals the Ser453 variant is rare among those of Asian (<1%) and African ancestry (<4%), and more common in individuals of European ancestry (16%) . Haplotypes containing the Ser453 variant were uncommon; only RALS was detectable in our small populations . The RALS allele occurred between 0.5% in Asians and 15% in Europeans . Our study demonstrates that rare, disease-associated mutations in CYP1B1 significantly decrease the enzyme's metabolism of B{a}P-7,8-diol; however, our results do not identify any major differences in this metabolism due to four common single amino acid polymorphisms. Toxicol Sci, 2003 Sep, 75(1), 40 - 6 Epub 2003 Jun 12. Antiandrogenic effects of bisphenol A and nonylphenol on the function of androgen receptor; Lee HJ et al.; By using a yeast detection system for androgenic and antiandrogenic effects of chemicals, we identified bisphenol A (BPA) and nonylphenol (NP) as antiandrogens . In this study, we report molecular mechanisms for the antiandrogenic action of BPA and NP . In the ARhLBD-activating signal cointegrator 1 (ASC1) yeast two-hybrid system, which reflects the androgen-dependent interaction between androgen receptor (AR) and its coactivator, ASC1, BPA and NP acted as potent AR antagonists comparable to a known strong antagonist, cyproterone acetate . Ligand competition assays revealed that {3H}5alpha-dihydroxytestosterone (DHT) binding to AR is inhibited a maximum of 30 and 40% at approximately 5 nM of NP and 50 nM of BPA, respectively . In addition, the nuclear translocation of green fluorescent protein (GFP)-AR fusion protein in the presence of testosterone was affected by the addition of BPA and NP, which cause rather dispersed distribution of GFP-AR between the nuclear and the cytoplasmic compartments . Furthermore, in transient transfection assays, BPA and NP inhibited androgen-induced AR transcriptional activity . Taken together, the results suggest that BPA and NP affect multiple steps of the activation and function of AR, thereby inhibiting the binding of native androgens to AR, AR nuclear localization, AR interaction with its coregulator, and its subsequent transactivation . These data may help us better understand the biological alterations induced by these environmental compounds. Plant Physiol, 2003 Jun, 132(2), 757 - 67 Epub 2003 Apr 17. Arabidopsis hot mutants define multiple functions required for acclimation to high temperatures; Hong SW et al.; Plants acquire thermotolerance to lethal high temperatures if first exposed to moderately high temperature or if temperature is increased gradually to an otherwise lethal temperature . We have taken a genetic approach to dissecting acquired thermotolerance by characterizing loss-of-function thermotolerance mutants in Arabidopsis . In previous work, we identified single recessive alleles of four loci required for thermotolerance of hypocotyl elongation, hot1-1, hot2-1, hot3-1, and hot4-1 . Completed screening of M2 progeny from approximately 2500 M1 plants has now identified new alleles of three of these original loci, along with three new loci . The low mutant frequency suggests that a relatively small number of genes make a major contribution to this phenotype or that other thermotolerance genes encode essential or redundant functions . Further analysis of the original four loci was performed to define the nature of their thermotolerance defects . Although the HOT1 locus was shown previously to encode a major heat shock protein (Hsp), Hsp101, chromosomal map positions indicate that HOT2, 3, and 4 do not correspond to major Hsp or heat shock transcription factor genes . Measurement of thermotolerance at different growth stages reveals that the mutants have growth stage-specific heat sensitivity . Analysis of Hsp accumulation shows that hot2 and hot4 produce normal levels of Hsps, whereas hot3 shows reduced accumulation . Thermotolerance of luciferase activity and of ion leakage also varies in the mutants . These data provide the first direct genetic evidence, to our knowledge, that distinct functions, independent of Hsp synthesis, are required for thermotolerance, including protection of membrane integrity and recovery of protein activity/synthesis. Plant Physiol, 2003 Jun, 132(2), 544 - 55 The SAC domain-containing protein gene family in Arabidopsis; Zhong R et al.; The SAC domain was first identified in the yeast (Saccharomyces cerevisiae) Sac1p phosphoinositide phosphatase protein and subsequently found in a number of proteins from yeast and animals . The SAC domain is approximately 400 amino acids in length and is characterized by seven conserved motifs . The SAC domains of several proteins have been recently demonstrated to possess phosphoinositide phosphatase activities . Sac1p has been shown to regulate the levels of various phosphoinositides in the phosphoinositide pool and affect diverse cellular functions such as actin cytoskeleton organization, Golgi function, and maintenance of vacuole morphology . The Arabidopsis genome contains a total of nine genes encoding SAC domain-containing proteins (AtSACs) . The SAC domains of the AtSACs possess the conserved amino acid motifs that are believed to be important for the phosphoinositide phosphatase activities of yeast and animal SAC domain proteins . AtSACs can be divided into three subgroups based on their sequence similarities, hydropathy profiles, and phylogenetic relationship . Gene expression analysis demonstrated that the AtSAC genes exhibited differential expression patterns in different organs and, in particular, the AtSAC6 gene was predominantly expressed in flowers . Moreover, the expression of the AtSAC6 gene was highly induced by salinity . These results provide a foundation for future studies on the elucidation of the cellular functions of SAC domain-containing proteins in Arabidopsis. Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7581 - 6 Epub 2003 Jun 12. The coiled-coil of the human Rad50 DNA repair protein contains specific segments of increased flexibility; van Noort J et al.; Protein structural features are usually determined by defining regularities in a large population of homogeneous molecules . However, irregular features such as structural variation and flexibility are likely to be missed, despite their vital role for their biological function . In this paper, we report the observation of striking irregularities in the flexibility of the coiled-coil region of the human Rad50 DNA repair protein . Existing methods to quantitatively analyze flexibility are applicable to homogeneous polymers only . Because protein coiled-coils cannot be assumed to be homogeneous, we develop a method to quantify the local flexibility from high-resolution atomic force microscopy images . Indeed, in Rad50 coiled-coils, two positions of increased flexibility are observed . We discuss how this dynamic structural feature is integral to Rad50 function. J Biol Chem, 2003 Aug 15, 278(33), 30421 - 4 Epub 2003 Jun 12. Diverse but overlapping functions of the two forkhead-associated (FHA) domains in Rad53 checkpoint kinase activation; Pike BL et al.; Forkhead-associated (FHA) domains are phosphothreonine-binding modules prevalent in proteins with important cell cycle and DNA damage response functions . The yeast checkpoint kinase Rad53 is unique in containing two FHA domains . We have generated novel recessive rad53 alleles with abolished FHA domain functions resulting from Ala substitution of the critical phosphothreonine-binding residues Arg70 and Arg605 . In asynchronous cells, inactivation of the N-terminal FHA1 domain did not impair Rad53 activation and downstream functions, whereas inactivation of the C-terminal FHA2 domain led to reduced Rad53 activation and significantly increased DNA damage sensitivity . Simultaneous inactivation of both FHA domains abolished Rad53 activation and all downstream functions and dramatically increased the sensitivity to DNA damage and replication blocks similar to kinase-defective and rad53 null alleles, but did not compromise the essential viability function of Rad53 . Interestingly, in G2/M synchronized cells, mutation of either FHA domain prevented Rad53 activation and impaired the cell cycle arrest checkpoint . Our data demonstrate that both FHA domains are required for normal Rad53 functions and indicate that the two FHA domains have differential but partially overlapping roles in Rad53 activation and downstream signaling. Circ Res, 2003 Jul 25, 93(2), 155 - 61 Epub 2003 Jun 12. Shear stress-mediated chromatin remodeling provides molecular basis for flow-dependent regulation of gene expression; Illi B et al.; Shear stress (SS), the tangential component of hemodynamic forces, modulates the expression of several genes in endothelial cells . However, no information is available about its effect on chromatin structure, which plays a key role in gene transcription . In this study, a link between SS and chromatin remodeling was established in human umbilical vein endothelial cells (HUVECs) . HUVECs were exposed to SS of 10 dyne/cm2 per second, in the presence or absence of the histone deacetylase inhibitor trichostatin A, and assayed for histone H3 and histone H4 modifications . SS induced histone H3 serine phosphorylation at position 10 (S10) and lysine acetylation at position 14 (K14) but required trichostatin A to induce H3 phosphoacetylation and H4 acetylation . The phosphatidylinositol 3-kinase inhibitor wortmannin and the mitogen-activated protein kinase inhibitor PD98059 decreased SS-dependent histone H3 phosphorylation, without affecting its acetylation; the p38 inhibitor SB203580 reduced both H3 phosphorylation and acetylation, whereas the protein kinase A inhibitor PKI-tide reduced histone H3 acetylation . Remarkably, the abrogation of histone acetylation inhibited SS-dependent c-fos expression . SS also activated ribosomal S6 kinase-2 and mitogen- and stress-activated kinase-1 protein kinases and promoted the formation of a cAMP-responsive element-binding protein (CREB)/CREB-binding protein complex, providing the molecular basis for the increase in histone acetyltransferase activity observed in HUVECs exposed to SS . Finally, the effect of SS on chromatin remodeling was examined . In HUVECs exposed to SS, chromatin within c-fos and c-jun promoters was specifically immunoprecipitated by an antibody against acetylated histone H3 on K14 . These results indicate that SS induces posttransduction modifications of histones; this is an early step toward the flow-dependent regulation of gene expression. EMBO J, 2003 Jun 16, 22(12), 3188 - 97 Multiple roles of Rev3, the catalytic subunit of polzeta in maintaining genome stability in vertebrates; Sonoda E et al.; Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major postreplicational repair (PRR) pathways . The REV3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta, which is involved in mutagenic TLS . To investigate the role of REV3 in vertebrates, we disruped the gene in chicken DT40 cells . REV3(-/-) cells are sensitive to various DNA-damaging agents, including UV, methyl methanesulphonate (MMS), cisplatin and ionizing radiation (IR), consistent with its role in TLS . Interestingly, REV3(-/-) cells showed reduced gene targeting efficiencies and significant increase in the level of chromosomal breaks in the subsequent M phase after IR in the G(2) phase, suggesting the involvement of Rev3 in HR-mediated double-strand break repair . REV3(-/-) cells showed significant increase in sister chromatid exchange events and chromosomal breaks even in the absence of exogenous genotoxic stress . Furthermore, double mutants of REV3 and RAD54, genes involved in HR, are synthetic lethal . In conclusion, Rev3 plays critical roles in PRR, which accounts for survival on naturally occurring endogenous as well as induced damages during replication. EMBO J, 2003 Jun 16, 22(12), 3175 - 87 The nuclear actin-related proteins Arp7 and Arp9: a dimeric module that cooperates with architectural proteins for chromatin remodeling; Szerlong H et al.; Nuclear actin-related proteins (ARPs) are essential components of chromatin remodeling and modifying complexes, but their functions and relationship to actin remain elusive . The yeast SWI/SNF and RSC complexes contain Arp7 and Arp9, and are shown to form a stable heterodimer with the properties of a functional module . Arp7 and Arp9 rely on their actin-related regions for heterodimerization, and their unique C-termini cooperate for assembly into RSC . We suggest that regulated ARP-ARP (and possibly ARP-beta-actin) heterodimerization might be a conserved feature of chromatin complexes . A RSC complex lacking Arp7/9 was isolated that displays robust nucleosome remodeling activity, suggesting a separate essential role for ARPs in the regulation of chromatin structure . A screen for suppressors of arp mutations yielded the DNA bending architectural transcription factor Nhp6, which interacts with RSC complex physically and functionally and shows facilitated binding to nucleosomes by RSC . We propose that Arp7/9 dimers function with DNA bending proteins to facilitate proper chromatin architecture and complex- complex interactions. EMBO J, 2003 Jun 16, 22(12), 3131 - 41 High-affinity DNA binding by a Mot1p-TBP complex: implications for TAF-independent transcription; Gumbs OH et al.; Yeast Mot1p, an abundant conserved member of the Snf2p-ATPase family of proteins, both dissociates TBP from DNA in vitro using the energy of ATP and represses gene transcription in vivo, yet paradoxically, loss of Mot1p function also leads to decreased transcription of certain genes . We conducted experiments utilizing fluorescently labeled DNA, TBP, fluorescence anisotropy spectroscopy and native gel electrophoresis to study Mot1p action . We have made a number of observations, the most intriguing being that a stable Mot1p-TBP complex has the ability to bind TATA DNA with high affinity, albeit with dramatically altered specificity . We propose that this altered TBP-DNA recognition is integral to Mot1p's ability to regulate transcription, and further postulate that the Mot1p-TBP complex delivers TBP to TAF-independent mRNA encoding genes. EMBO J, 2003 Jun 16, 22(12), 3062 - 72 Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD; Baas AF et al.; The LKB1 gene encodes a serine/threonine kinase mutated in Peutz-Jeghers cancer syndrome . Despite several proposed models for LKB1 function in development and in tumour suppression, the detailed molecular action of LKB1 remains undefined . Here, we report the identification and characterization of an LKB1-specific adaptor protein and substrate, STRAD (STe20 Related ADaptor) . STRAD consists of a STE20- like kinase domain, but lacks several residues that are indispensable for intrinsic catalytic activity . Endogenous LKB1 and STRAD form a complex in which STRAD activates LKB1, resulting in phosphorylation of both partners . STRAD determines the subcellular localization of wild-type, but not mutant LKB1, translocating it from nucleus to cytoplasm . One LKB1 mutation previously identified in a Peutz-Jeghers family that does not compromise its kinase activity is shown here to interfere with LKB1 binding to STRAD, and hence with STRAD-dependent regulation . Removal of endogenous STRAD by siRNA abrogates the LKB1-induced G(1) arrest . Our results imply that STRAD plays a key role in regulating the tumour suppressor activities of LKB1. Mol Biol Cell, 2003 May, 14(5), 1953 - 63 Epub 2003 Feb 06. Constriction and Dnm1p recruitment are distinct processes in mitochondrial fission; Legesse-Miller A et al.; Mitochondria undergo cycles of fusion and fission crucial for organelle homeostasis . Fission is regulated partially by recruitment of the large GTPase Dnm1p to the outer mitochondrial membrane . Using three-dimensional time-lapse fluorescence imaging of Saccharomyces cerevisiae cells, we found that Dnm1p-EGFP appears and disappears at "hot spots" along mitochondrial tubes . It forms patches that convert rapidly into different shapes regardless of whether mitochondrial fission ensues or not . Moreover, the thickness of the mitochondrial matrix displays frequent temporal fluctuations apparently unrelated to fission or to recruitment of Dnm1p-EGFP . These results suggest that mitochondrial fission requires coordination of at least two distinct processes. Mol Biol Cell, 2003 May, 14(5), 1852 - 67 Epub 2003 Feb 06. Dual prenylation is required for Rab protein localization and function; Calero M et al.; The majority of Rab proteins are posttranslationally modified with two geranylgeranyl lipid moieties that enable their stable association with membranes . In this study, we present evidence to demonstrate that there is a specific lipid requirement for Rab protein localization and function . Substitution of different prenyl anchors on Rab GTPases does not lead to correct function . In the case of YPT1 and SEC4, two essential Rab genes in Saccharomyces cerevisiae, alternative lipid tails cannot support life when present as the sole source of YPT1 and SEC4 . Furthermore, our data suggest that double geranyl-geranyl groups are required for Rab proteins to correctly localize to their characteristic organelle membrane . We have identified a factor, Yip1p that specifically binds the di-geranylgeranylated Rab and does not interact with mono-prenylated Rab proteins . This is the first demonstration that the double prenylation modification of Rab proteins is an important feature in the function of this small GTPase family and adds specific prenylation to the already known determinants of Rab localization. Mol Biol Cell, 2003 May, 14(5), 1818 - 34 Epub 2003 Feb 06. Centrin4p, a novel mammalian centrin specifically expressed in ciliated cells; Gavet O et al.; Centriole assembly plays an important role in centrosome duplication during the cell cycle and is a prerequisite for cilia formation during the differentiation of ciliated cells . In spite of numerous investigations, the molecular machinery that governs centriole/basal body formation remains enigmatic . Recent reports suggest that the ubiquitously expressed mammalian centrins, centrin2p and centrin3p, could be involved in the centriole duplication process . To better understand the specific functions of these proteins, we performed a systematic search for novel mammalian centrins . We isolated a cDNA and the corresponding gene coding for a novel murine centrin, centrin4p, which is more closely related to centrin2p . Like centrin2p, centrin4p accumulates to centrioles and procentrioles when ectopically expressed in HeLa cells . However, centrin4p possesses two splice variants that do not localize to centrioles, suggesting a posttranscriptional regulation mechanism . We also observed that centrin4p does not share the same centriolar targeting properties with centrin2p and 3p, indicating that these proteins could recognize different centriolar partners . Centrin4 mRNA possesses a restricted expression profile and is only detected in brain, kidney, lung, and ovary . In brain, centrin4p is exclusively expressed in ependymal and choroidal ciliated cells where it is localized to basal bodies . Together, our present data suggest that centrin4p could be more specifically involved in basal bodies assembly or in a subsequent step of ciliogenesis. Bioinformatics, 2003 Jun 12, 19(9), 1110 - 5 Novel clustering algorithm for microarray expression data in a truncated SVD space; Horn D et al.; MOTIVATION: This paper introduces the application of a novel clustering method to microarray expression data . Its first stage involves compression of dimensions that can be achieved by applying SVD to the gene-sample matrix in microarray problems . Thus the data (samples or genes) can be represented by vectors in a truncated space of low dimensionality, 4 and 5 in the examples studied here . We find it preferable to project all vectors onto the unit sphere before applying a clustering algorithm . The clustering algorithm used here is the quantum clustering method that has one free scale parameter . Although the method is not hierarchical, it can be modified to allow hierarchy in terms of this scale parameter . RESULTS: We apply our method to three data sets . The results are very promising . On cancer cell data we obtain a dendrogram that reflects correct groupings of cells . In an AML/ALL data set we obtain very good clustering of samples into four classes of the data . Finally, in clustering of genes in yeast cell cycle data we obtain four groups in a problem that is estimated to contain five families . AVAILABILITY: Software is available as Matlab programs at http://neuron.tau.ac.il/~horn/QC.htm. Trends Genet, 2003 Jun, 19(6), 321 - 9 The diverse functions of histone acetyltransferase complexes; Carrozza MJ et al.; Although histone acetylation has historically been linked to transcription activation, recent studies indicate that this modification and the enzymes that catalyze it have much broader and diverse functions . Histone acetyltransferase complexes are involved in such diverse processes as transcription activation, gene silencing, DNA repair and cell-cycle progression . The high conservation of the acetyltransferase complexes and their functions illustrates their central role in cell growth and development. Trends Genet, 2003 Jun, 19(6), 295 - 8 Splicing goes global; Barrass JD et al.; Transcriptomics, the analysis of the complement of mRNAs transcribed from a cell's genome, currently focuses mainly on mature, processed mRNAs . However, posttranscriptional processing of primary transcripts can significantly affect both the quantity and the structure of the mature mRNAs and therefore of the protein products . Recently, the development of an intron-specific microarray has permitted a preliminary analysis of the splicing of all intron-containing transcripts in Saccharomyces cerevisiae . Here, we discuss the findings and what might be learned from this kind of approach. Gene, 2003 May 22, 310, 161 - 8 Identification of a larger form of the histone acetyl transferase Tip60; Legube G et al.; The histone acetyl transferase Tat interactive protein 60kD (Tip60) plays major roles in the cellular response to extra- or intra-cellular signalling . Tip60 activity appears to be tightly regulated in cells through post-translational modifications or subcellular localisation of the protein . In addition, several alternatively spliced forms of Tip60 have been described . We found here that in a significant proportion of cytoplasmic poly adenylated Tip60 mRNAs, the intron 1 was not excised . Translation of this mRNA would predictably lead to the production of a longer Tip60 protein, containing a 33 amino acids insertion four amino acids after the ATG . By transient transfection experiments, we could demonstrate that this protein was significantly produced . Thus, taken together, our results indicate the existence of a longer Tip60 protein . Whether this protein could be differentially regulated and could play different roles than the classical Tip60 protein is an intriguing possibility. Gene, 2003 May 22, 310, 113 - 21 Characterization of a SNF1 homologue from the phytopathogenic fungus Sclerotinia sclerotiorum; Vacher S et al.; In yeast, the SNF1 gene product is essential for the release of catabolic repression . We report the isolation and characterization of an SNF1 homologue from the necrotrophic pathogen Sclerotinia sclerotiorum . Ss snf1 encodes a 765-amino-acid protein in which the catalytic domain has an overall identity with the yeast proteins varying from 55 to 76% while the C-terminal half of Ss SNF1 has a weak homology of about 20% with the yeast sequences . Reverse transcription-polymerase chain reaction showed that its transcripts were weakly and constitutively expressed in planta and in vitro regardless of the nature of the carbon sources and of the presence or absence of glucose . Expression of Ss snf1 in yeast cells allowed the snf1 mutant cells to utilize sucrose, raffinose or glycerol for growth while expression of the Ss snf1 catalytic domain did not restore growth on raffinose or glycerol . Ss SNF1 is structurally homologous to Snf1p, suggesting that the interactions between the kinase and the accessory subunits to activate the enzymatic complex are conserved in fungi. World J Gastroenterol, 2003 Jun, 9(6), 1165 - 9 DNA polymerase zeta: new insight into eukaryotic mutagenesis and mammalian embryonic development; Zhu F et al.; Information about the mechanisms that generate mutations in eukaryotes is likely to be useful for understanding human health concerns, such as genotoxicity and cancer . Eukaryotic mutagenesis is largely the outcome of attacks by endogenous and environmental agents . Except for DNA repair, cell cycle checkpoints and DNA damage avoidance, cells have also evolved DNA damage tolerance mechanism, by which lesion-targeted mutation might occur in the genome during replication by specific DNA polymerases to bypass the lesions (translesion DNA synthesis, TLS), or mutation on undamaged DNA templates (untargeted mutation) might be induced . DNA polymerase zeta (pol zeta), which was found firstly in budding yeast Saccharomyces cerevisiae and consists of catalytic subunit scRev3 and stimulating subunit scRev7, has received more attention in recent years . Pol zeta is a member of DNA polymerase eta subfamily, which belongs to DNA polymerase B family, and exists in almost all eukaryotes . Human homolog of the scRev3 gene is located in chromosome region 6q21, and the mouse equivalent maps to chromosome 10, distal to the c-myb gene and close to the Macs gene . Alternative splicing, upstream out-of frame ATG can be found in yeast scRev3, mouse and human homologs . Furthermore, the sequence from 253-323 immediate upstream of the AUG initiator codon has the potential to form a stem-loop hairpin secondary structure in REV3 mRNA, suggesting that human REV3 protein may be expressed at low levels in human cells under normal growth conditions . The functional domain analysis showed that yeast Rev3-980 tyrosine in conserved region II is at the polymerase active site . Human REV3 amino acid residues 1 776-2 195 provide a REV7 binding domain, and REV7 amino acid residues 1-211 provide a bind domain for REV1, REV3 and REV7 itself . More interestingly, REV7 interacts with hMAD2 and therefore might function in the cell cycle control by affecting the activation of APC (anaphase promoting complex) . Currently it has been known that pol zeta is involved in most spontaneous mutation, lesion-targeted mutation via TLS, chemical carcinogen induced untargeted mutation and somatic hypermutation of antibody genes in mammalian . In TLS pathway, pol zeta acts as a "mismatch extender" with combination of other DNA polymerases, such as pol iota . Unlike in yeast, it was found that pol zeta also functioned in mouse embryonic development more recently . It was hypothesized that the roles of pol zeta in TLS and cell cycle control might contribute to mouse embryonic lethality. Int J Cancer, 2003 Aug 20, 106(2), 244 - 51 Novel tumor antigens identified by autologous antibody screening of childhood medulloblastoma cDNA libraries; Behrends U et al.; Medulloblastoma is an embryonal childhood malignancy with poor prognosis . By screening 4 medulloblastoma cDNA expression libraries (SEREX) with autologous sera, 15 different antigens were identified . These antigens were encoded by 3 novel genes, genes of unknown function (KIAA0445, KIAA1853, KIAA0665, FLJ13942, HSPC213), a proto-oncogene (rab18), candidate tumor suppressor genes (BAP1, PRDM13) and genes encoding a motor protein (kinesin-2), a histone (H2A1.2), the ankyrin residue-rich nasopharyngeal cancer susceptibility protein (NZ16) and the transcription factor TZP, which is homologous to the tumor-associated antigens HCA58 and GLEA2 . In a consecutive analysis of serum antibody titers and tumor load, a more than 10-fold increase in serum antibodies against PRDM13 preceded the clinical diagnosis of recurrent tumor growth in a patient with aggressive large cell medulloblastoma . When sera of pediatric patients with cancer (n = 40) and healthy controls (n = 40) were tested for humoral responses against the SEREX-defined antigens, 5 antigens were exclusively recognized by sera from cancer patients . These antigens included a novel rab18 gene product translated from mRNA sequences formerly described as 3' untranslated region . Humoral responses against 2 of the remaining 10 antigens were found preferentially in cancer patients . Antibodies against these antigens were detected in 8/40 and 12/40 cancer patients, respectively, but in only 1 healthy control . The 2 antigens were characterized by a tumor-specific deletion and a tumor-specific mutation, respectively . These findings indicate that the humoral immune response against medulloblastoma is directed against diverse antigens that may be useful as diagnostic markers or targets for immunotherapy . J Biosci, 2003 Jun, 28(4), 423 - 36 The transcriptional activator GAL4-VP16 regulates the intra-molecular interactions of the TATA-binding protein; Mishra AK et al.; Binding characteristics of yeast TATA-binding protein (yTBP) over five oligomers having different TATA variants and lacking a UASGAL, showed that TATA-binding protein (TBP)-TATA complex gets stabilized in the presence of the acidic activator GAL4-VP16 . Activator also greatly suppressed the non-specific TBP-DNA complex formation . The effects were more pronounced over weaker TATA boxes . Activator also reduced the TBP dimer levels both in vitro and in vivo, suggesting the dimer may be a direct target of transcriptional activators . The transcriptional activator facilitated the dimer to monomer transition and activated monomers further to help TBP bind even the weaker TATA boxes stably . The overall stimulatory effect of the GAL4-VP16 on the TBP-TATA complex formation resembles the known effects of removal of the N-terminus of TBP on its activity, suggesting that the activator directly targets the N-terminus of TBP and facilitates its binding to the TATA box. Nucleic Acids Res, 2003 Jun 15, 31(12), 3157 - 65 The Upf-dependent decay of wild-type PPR1 mRNA depends on its 5'-UTR and first 92 ORF nucleotides; Kebaara B et al.; mRNAs containing premature translation termination codons (nonsense mRNAs) are targeted for deadenylation-independent degradation in a mechanism that depends on Upf1p, Upf2p and Upf3p . This decay pathway is often called nonsense- mediated mRNA decay (NMD) . Nonsense mRNAs are decapped by Dcp1p and then degraded 5' to 3' by Xrn1p . In the yeast Saccharomyces cerevisiae, a significant number of wild-type mRNAs accumulate in upf mutants . Wild-type PPR1 mRNA is one of these mRNAs . Here we show that PPR1 mRNA degradation depends on the Upf proteins, Dcp1p, Xrn1p and Hrp1p . We have mapped an Upf1p-dependent destabilizing element to a region located within the 5'-UTR and the first 92 bases of the PPR1 ORF . This element targets PPR1 mRNA for Upf-dependent decay by a novel mechanism. Nucleic Acids Res, 2003 Jun 15, 31(12), 3114 - 22 The SV40 T antigen modulates CBP histone acetyltransferase activity; Valls E et al.; Histone acetyltransferases (HATs) play a key role in transcription control, cell proliferation and differentiation by modulating chromatin structure; however, little is known about their own regulation . Here we show that expression of the viral oncoprotein SV40 T antigen increases histone acetylation and global cellular HAT activities . In addition, it enhances CREB-binding protein HAT activity and modulates its transcriptional activity . Finally, we show that inhibition of cellular histone deacetylases by trichostatin A increases the SV40 infectivity rate . These findings highlight the importance of histone acetylation in the regulation of the cell cycle by oncoviral proteins. Nucleic Acids Res, 2003 Jun 15, 31(12), 3006 - 15 Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro; Bae KH et al.; We have previously shown that replication- protein A (RPA), the heterotrimeric single-stranded DNA binding protein of eukaryotes, plays a role in Okazaki fragment processing by acting as a molecular switch between the two endonucleases, Dna2 and Fen1, to ensure the complete removal of primer RNAs in Saccharomyces cerevisiae . The stimulation of Dna2 endonuclease activity by RPA requires direct protein-protein interaction . In this report we have analyzed genetically and biochemically the interaction of Dna2 with RPA . RFA1, the gene encoding the large subunit of RPA, displayed allele-specific interactions with DNA2 that included synthetic lethality and intergenic complementation . In addition, we identified physical and functional interactions between these proteins and found that RPA binds Dna2 predominantly through its large subunit, Rpa1 . Consistent with the mapping of synthetic lethal mutations, robust interaction localizes to the C-termini of these proteins . Moreover, the N-terminal domains of Dna2 and Rpa1 appear to be important for a functional interaction because the N-terminal domain of RPA1 was required to maximally stimulate Dna2 endonuclease activity . We propose that a bimodal interaction of Dna2 with Rpa1 is important for Dna2 function both in vivo and in vitro . The relevance of each interaction with respect to the function of the Dna2 endonuclease activity is discussed. J Cell Sci, 2003 Aug 1, 116(Pt 15), 3069 - 77 Epub 2003 Jun 10. Relationship of DNA double-strand breaks to synapsis in Drosophila; Jang JK et al.; The relationship between synaptonemal complex formation (synapsis) and double-strand break formation (recombination initiation) differs between organisms . Although double-strand break creation is required for normal synapsis in Saccharomyces cerevisiae and the mouse, it is not necessary for synapsis in Drosophila and Caenorhabditis elegans . To investigate the timing of and requirements for double-strand break formation during Drosophila meiosis, we used an antibody that recognizes a histone modification at double-strand break sites, phosphorylation of HIS2AV (gamma-HIS2AV) . Our results support the hypothesis that double-strand break formation occurs after synapsis . Interestingly, we detected a low (10-25% of wildtype) number of gamma-HIS2AV foci in c(3)G mutants, which fail to assemble synaptonemal complex, suggesting that there may be both synaptonemal complex-dependent and synaptonemal complex-independent mechanisms for generating double-strand breaks . Furthermore, mutations in Drosophila Rad54 (okr) and Rad51 (spnB) homologs cause delayed and prolonged gamma-HIS2AV staining, suggesting that double-strand break repair is delayed but not eliminated in these mutants . There may also be an interaction between the recruitment of repair proteins and phosphorylation. Genome Res, 2003 Jun, 13(6A), 1231 - 43 Kinase pathway database: an integrated protein-kinase and NLP-based protein-interaction resource; Koike A et al.; Protein kinases play a crucial role in the regulation of cellular functions . Various kinds of information about these molecules are important for understanding signaling pathways and organism characteristics . We have developed the Kinase Pathway Database, an integrated database involving major completely sequenced eukaryotes . It contains the classification of protein kinases and their functional conservation, ortholog tables among species, protein-protein, protein-gene, and protein-compound interaction data, domain information, and structural information . It also provides an automatic pathway graphic image interface . The protein, gene, and compound interactions are automatically extracted from abstracts for all genes and proteins by natural-language processing (NLP).The method of automatic extraction uses phrase patterns and the GENA protein, gene, and compound name dictionary, which was developed by our group . With this database, pathways are easily compared among species using data with more than 47,000 protein interactions and protein kinase ortholog tables . The database is available for querying and browsing at http://kinasedb.ontology.ims.u-tokyo.ac.jp/. Genome Res, 2003 Jun, 13(6A), 1056 - 66 New evidence for genome-wide duplications at the origin of vertebrates using an amphioxus gene set and completed animal genomes; Panopoulou G et al.; The 2R hypothesis predicting two genome duplications at the origin of vertebrates is highly controversial . Studies published so far include limited sequence data from organisms close to the hypothesized genome duplications . Through the comparison of a gene catalog from amphioxus, the closest living invertebrate relative of vertebrates, to 3453 single-copy genes orthologous between Caenorhabditis elegans (C), Drosophila melanogaster (D), and Saccharomyces cerevisiae (Y), and to Ciona intestinalis ESTs, mouse, and human genes, we show with a large number of genes that the gene duplication activity is significantly higher after the separation of amphioxus and the vertebrate lineages, which we estimate at 650 million years (Myr) . The majority of human orthologs of 195 CDY groups that could be dated by the molecular clock appear to be duplicated between 300 and 680 Myr with a mean at 488 million years ago (Mya) . We detected 485 duplicated chromosomal segments in the human genome containing CDY orthologs, 331 of which are found duplicated in the mouse genome and within regions syntenic between human and mouse, indicating that these were generated earlier than the human-mouse split . Model based calculations of the codon substitution rate of the human genes included in these segments agree with the molecular clock duplication time-scale prediction . Our results favor at least one large duplication event at the origin of vertebrates, followed by smaller scale duplication closer to the bird-mammalian split. J Mol Biol, 2003 Jun 20, 329(5), 931 - 9 Mechanism of DNA binding by the ADR1 zinc finger transcription factor as determined by SPR; Schaufler LE et al.; The ADR1 protein recognizes a six base-pair consensus DNA sequence using two zinc fingers and an adjacent accessory motif . Kinetic measurements were performed on the DNA-binding domain of ADR1 using surface plasmon resonance . Binding by ADR1 was characterized to two known native binding sequences from the ADH2 and CTA1 promoter regions, which differ in two of the six consensus positions . In addition, non-specific binding by ADR1 to a random DNA sequence was measured . ADR1 binds the native sites with nanomolar affinities . Remarkably, ADR1 binds non-specific DNA with affinities only approximately tenfold lower than the native sequences . The specific and non-specific binding affinities are conferred mainly by differences in the association phase of DNA binding . The association rate for the complex is strongly influenced by the proximal accessory region, while the dissociation reaction and specificity of binding are controlled by the two zinc fingers . Binding kinetics of two ADR1 mutants was also examined . ADR1 containing an R91K mutation in the accessory region bound with similar affinity to wild-type, but with slightly less sequence specificity . The R91K mutation was observed to increase binding affinity to a suboptimal sequence by decreasing the complex dissociation rate . L146H, a change-of-specificity mutation at the +3 position of the second zinc finger, bound its preferred sequence with a slightly higher affinity than wild-type . The L146H mutant indicates that beneficial protein-DNA contacts provide similar levels of stabilization to the complex, whether they are hydrogen-bonding or van der Waals interactions. J Mol Graph Model, 2003 Sep, 22(1), 31 - 40 Analysis and optimization of structure-based virtual screening protocols . 2 . Examination of docked ligand orientation sampling methodology: mapping a pharmacophore for success; Good AC et al.; An important element of any structure-based virtual screening (SVS) technique is the method used to orient the ligands in the target active site . This has been a somewhat overlooked issue in recent SVS validation studies, with the assumption being made that the performance of an algorithm for a given set of orientation sampling settings will be representative for the general behavior of said technique . Here, we analyze five different SVS targets using a variety of sampling paradigms within the DOCK, GOLD and PROMETHEUS programs over a data set of approximately 10,000 noise compounds, combined with data sets containing multiple active compounds . These sets have been broken down by chemotype, with chemotype hit rate used to provide a measure of enrichment with a potentially improved relevance to real world SVS experiments . The variability in enrichment results produced by different sampling paradigms is illustrated, as is the utility of using pharmacophores to constrain sampling to regions that reflect known structural biology . The difference in results when comparing chemotype with compound hit rates is also highlighted. Bioorg Med Chem Lett, 2003 Jul 7, 13(13), 2235 - 7 Borrelidin induces the transcription of amino acid biosynthetic enzymes via a GCN4-dependent pathway; Eastwood EL et al.; Global cellular profiling of messenger RNA levels has been used to provide insight into the effects of the angiogenesis inhibitor borrelidin on the eukaryotic model organism Saccharomyces cerevisiae . The most notable result of treatment with borrelidin is the induction of amino acid biosynthetic enzymes in a time-dependent fashion . We have ascertained that induction of this pathway involves the GCN4 transcription factor . This conclusion was determined by treating a yeast strain lacking this gene and observing the absence of increased gene transcription under Gcn4p control. Biochem J, 2003 Sep 1, 374(Pt 2), 349 - 58 Differential expression, localization and activity of two alternatively spliced isoforms of human APC regulator CDH1; Zhou Y et al.; The timely destruction of key regulators through ubiquitin-mediated proteolysis ensures the orderly progression of the cell cycle . The APC (anaphase-promoting complex) is a major component of this degradation machinery and its activation is required for the execution of critical events . Recent studies have just begun to reveal the complex control of the APC through a regulatory network involving WD40 repeat proteins CDC20 and CDH1 . In the present paper, we report on the identification and characterization of human CDH1beta, a novel alternatively spliced isoform of CDH1 . Both CDH1alpha and CDH1beta can bind to the APC and stimulate the degradation of cyclin B1, but they are differentially expressed in human tissues and cells . CDH1alpha contains a nuclear localization signal which is absent in CDH1beta . Intracellularly, CDH1alpha appears in the nucleus whereas CDH1beta is a predominantly cytoplasmic protein . The forced overexpression of CDH1alpha in cultured cells correlates with the reduction of nuclear cyclin A, but the steady-state amount of cyclin A does not change noticeably in CDH1beta-overexpressed cells . In Xenopus embryos, ectopic overexpression of human CDH1alpha, but not of CDH1beta, induces cell-cycle arrest during the first G(1) phase at the mid-blastula transition . Taken together, our findings document the differential expression, subcellular localization and cell-cycle-regulatory activity of human CDH1 isoforms. Nat Genet, 2003 Jul, 34(3), 326 - 9 Cadmium is a mutagen that acts by inhibiting mismatch repair; Jin YH et al.; Most errors that arise during DNA replication can be corrected by DNA polymerase proofreading or by post-replication mismatch repair (MMR) . Inactivation of both mutation-avoidance systems results in extremely high mutability that can lead to error catastrophe . High mutability and the likelihood of cancer can be caused by mutations and epigenetic changes that reduce MMR . Hypermutability can also be caused by external factors that directly inhibit MMR . Identifying such factors has important implications for understanding the role of the environment in genome stability . We found that chronic exposure of yeast to environmentally relevant concentrations of cadmium, a known human carcinogen, can result in extreme hypermutability . The mutation specificity along with responses in proofreading-deficient and MMR-deficient mutants indicate that cadmium reduces the capacity for MMR of small misalignments and base-base mismatches . In extracts of human cells, cadmium inhibited at least one step leading to mismatch removal . Together, our data show that a high level of genetic instability can result from environmental impediment of a mutation-avoidance system. Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7551 - 6 Epub 2003 Jun 09. Spreading of Sir3 protein in cells with severe histone H3 hypoacetylation; Kristjuhan A et al.; Heterochromatin formation in yeast involves deacetylation of histones, but the precise relationship between acetylation and the association of proteins such as Sir3, Sir4, and the histone deacetylase Sir2 with chromatin is still unclear . Here we show that Sir3 protein spreads to subtelomeric DNA in cells lacking the transcription-related histone acetyltransferases GCN5 and ELP3 . Spreading correlates with hypoacetylation of lysines in the histone H3 tail and results in deacetylation of lysine 16 in histone H4 . De-repression of genes situated very close to the ends of the chromosomes in gcn5 elp3 suggests that Sir3 spreads into subtelomeric DNA from the tip of the telomere . Interestingly, growth defects caused by gcn5 elp3 mutation can be suppressed by SIR deletion, suggesting that Sir proteins become detrimental for growth when chromatin is severely hypoacetylated. Plant J, 2003 Jun, 34(6), 788 - 801 Sodium transport and HKT transporters: the rice model; Garciadeblas B et al.; Na+ uptake in the roots of K+-starved seedlings of barley, rice, and wheat was found to exhibit fast rate, low Km, and high sensitivity to K+ . Sunflower plants responded in a similar manner but the uptake was not K+ sensitive . Ba2+ inhibited Na+ uptake, but not K+ uptake in rice roots . This demonstrated that Na+ and K+ uptake are mediated by different transporters, and that K+ blocked but was not transported by the Na+ transporter . The genome of rice cv . Nipponbare contains seven HKT genes, which may encode Na+ transporters, plus two HKT pseudogenes . Yeast expressions of OsHKT1 and OsHKT4 proved that they are Na+ transporters of high and low affinity, respectively, which are sensitive to K+ and Ba2+ . Parallel experiments of K+ and Na+ uptake in yeast expressing the wheat or rice HKT1 transporters proved that they were very different; TaHKT1 transported K+ and Na+, and OsHKT1 only Na+ . Transcript expressions in shoots of the OsHKT genes were fairly constant and insensitive to changes in the K+ and Na+ concentrations of the nutrient solution . In roots, the expressions were much lower than in shoots, except for OsHKT4 and OsHKT1 in K+-starved plants . We propose that OsHKT transporters are involved in Na+ movements in rice, and that OsHKT1 specifically mediates Na+ uptake in rice roots when the plants are K+ deficient . The incidence of HKT ESTs in several plant species suggests that the rice model with many HKT genes applies to other plants. Traffic, 2003 Jul, 4(7), 479 - 90 PtdIns(3,5)P2 is required for delivery of endocytic cargo into the multivesicular body; Shaw JD et al.; The endocytic pathway transports cargo from the plasma membrane to early endosomes, where certain cargoes are sorted to the late endosome/multivesicular body . Biosynthetic cargo destined for the lysosome is also trafficked through the multivesicular body . Once delivered to the multivesicular body, cargo destined for the interior of the lysosome is selectively sorted into vesicles that bud into the lumen of the multivesicular body . These vesicles are released into the lumen of the lysosome upon the fusion of the multivesicular body and lysosomal limiting membranes . The yeast protein Fab1, which catalyzes the production of phosphatidylinositol (3,5) bisphosphate {PtdIns(3,5)P2}, is necessary for proper sorting of biosynthetic cargo in the multivesicular body . Utilizing an endocytosis screen, we isolated a novel allele of FAB1 that contains a point mutation in the lipid kinase domain . Characterization of this allele revealed reduced PtdIns(3,5)P2 production, altered vacuole morphology, and biosynthetic protein sorting defects . We also found that endocytosis of the plasma membrane protein Ste3 is partially blocked downstream of the internalization step, and that delivery of the dye FM4-64 to the vacuole is delayed in fab1 mutants . Additionally, Ste3 is not efficiently sorted into multivesicular body vesicles in fab1 mutants and instead localizes to the vacuolar limiting membrane . These data show that PtdIns(3,5)P2 is necessary for proper trafficking and sorting of endocytic cargo through the late endosome/multivesicular body. Biochemistry, 2003 Jun 17, 42(23), 7068 - 76 Transient protein interactions studied by NMR spectroscopy: the case of cytochrome C and adrenodoxin; Worrall JA et al.; The interaction between yeast iso-1-cytochrome c (C102T) and two forms of bovine adrenodoxin, the wild type and a truncated form comprising residues 4-108, has been investigated using a combination of one- and two-dimensional heteronuclear NMR spectroscopy . Chemical shift perturbations and line broadening of amide resonances in the {(15)N,(1)H}HSQC spectrum for both (15)N-labeled cytochrome c and adrenodoxin in the presence of the unlabeled partner protein indicate the formation of a transient complex, with a K(a) of (4 +/- 1) x 10(4) M(-)(1) and a lifetime of <3 ms . The perturbed residues map over a large surface area for both proteins . For cytochrome c, the dominating effects are located around the exposed heme edge but with other areas also affected upon formation of the complex . In the case of adrenodoxin, effects are seen in both the recognition and core domains, with the largest perturbations in the recognition domain . These results indicate that the complex has a dynamic nature, with delocalized binding of cytochrome c on adrenodoxin . A comparison with other transient complexes of redox proteins places this complex between well-defined complexes such as the cytochrome c-cytochrome c peroxidase complex and entirely dynamic complexes such as the cytochrome b(5)-myoglobin complex. Biochemistry, 2003 Jun 17, 42(23), 7013 - 22 Substrate-induced changes in the ammonia channel for imidazole glycerol phosphate synthase; Myers RS et al.; IGP synthase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the unusual nucleotide, N(1)-{(5'-phosphoribulosyl)-formimino}-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to form 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR) and imidazole glycerol phosphate (IGP) . A common feature of all glutamine amidotransferases is the upregulation of glutamine hydrolysis in the presence of an acceptor substrate . A refined assay system was developed to establish that Saccharomyces cerevisae IGP synthase shows a 4900-fold stimulation of glutaminase in the presence of the substrate acceptor PRFAR . The structure and function of IGP synthase acceptor substrate binding site were probed with competitive inhibitors that are nucleotide substrate and product analogues . In addition, these analogues were also used to establish that the normal steady-state turnover cycle involves a random sequential mechanism . Upregulation of the glutaminase active site occurs when these competitive inhibitors bind in the nucleotide site over 30 A away . One of the key structural features of IGP synthase is that the transfer of ammonia from the glutaminase site occurs through the (beta/alpha)(8) core of the protein . Upon the basis of the recent substrate-occupied structure for yeast IGP synthase (1), kinetic investigations of site-directed mutants revealed that a conserved K258 residue is key to productive binding and the overall stoichiometry of the reaction . The binding of the ribulosyl phosphate portion of the substrate PRFAR appears to be transduced through reorientation of K258 resulting in a conformational switch at the base of the (beta/alpha)(8) core that enables the passage of ammonia through the core of the protein . The overall analysis also leads to further discussion of how the residues that cover the opening of the (beta/alpha)(8) in the closed state may assist the channeling of ammonia at the interface of the two functional domains in the open state. J Biotechnol, 2003 May 8, 102(3), 269 - 79 Cell volume changes during rapid temperature shifts; Gervais P et al.; The effect of a rapid temperature increase on the volume of different types of cells was investigated . Experiments were carried out using continuous microscopic image analysis . Volume variation of yeast cells, yeast spheroplasts and human leukaemia cells was measured during the transient phase after a thermal shift . The thermal shift was found to induce rapid increase in cell volume for cells lacking a cell wall (yeast spheroplasts and human leukaemia cells) . This increase in cell volume is assumed to be a main cause of the heat shock-induced cell death . A theoretical mechanistic model that explains the behaviour of these cells is finally proposed. J Biol Chem, 2003 Aug 22, 278(34), 32227 - 35 Epub 2003 Jun 06. Phosphorylation by glycogen synthase kinase-3 beta down-regulates Notch activity, a link for Notch and Wnt pathways; Espinosa L et al.; Phosphorylation of Notch proteins has been indirectly correlated with Notch activation and nuclear translocation as well as cellular transformation . There is evidence that the Wnt signaling pathway, which results in glycogen synthase kinase-3 beta (GSK-3 beta) inhibition, cross-talks with the Notch pathway . In this study, we show that GSK-3 beta is able to bind and phosphorylate Notch2 in vitro and in vivo . We identify three specific phosphorylation sites in the Notch2 serine/threonine-rich domain that are dependent on GSK-3 beta activity . Phosphorylation of the serine/threonine-rich domain has been shown previously to be crucial in regulating cytokine-specific cell differentiation . Coimmunoprecipitation experiments show that full-length Notch2 binds more efficiently than intracellular Notch2 to GSK-3 beta . Nevertheless, only the processed Notch2 is a substrate for the kinase, thus suggesting that GSK-3 beta-dependent phosphorylation may be specifically regulating the activated Notch molecule . Consistent with this, GSK-3 beta inhibits the transcriptional activation of Notch target genes both in vitro and in vivo, whereas lithium chloride treatment or Wnt-1 overexpression that results in GSK-3 beta inhibition leads to the up-regulation of the Hes-1 promoter . Together, our results suggest that cross-talk between Notch and Wnt pathways may be partially mediated by specific regulation of GSK-3 beta-dependent Notch phosphorylation. J Biol Chem, 2003 Aug 22, 278(34), 32091 - 9 Epub 2003 Jun 06. The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans; Artal-Sanz M et al.; Prohibitins in eukaryotes consist of two subunits (PHB1 and PHB2) that together form a high molecular weight complex in the mitochondrial inner membrane . The evolutionary conservation and the ubiquitous expression in mammalian tissues of the prohibitin complex suggest an important function among eukaryotes . The PHB complex has been shown to play a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes in the yeast Saccharomyces cerevisiae . We have used Caenorhabditis elegans as model system to study the role of the PHB complex during development of a multicellular organism . We demonstrate that prohibitins in C . elegans form a high molecular weight complex in the mitochondrial inner membrane similar to that of yeast and humans . By using RNA-mediated gene inactivation, we show that PHB proteins are essential during embryonic development and are required for somatic and germline differentiation in the larval gonad . We further demonstrate that a deficiency in PHB proteins results in altered mitochondrial biogenesis in body wall muscle cells . This paper reports a strong loss of function phenotype for prohibitin gene inactivation in a multicellular organism and shows for the first time that prohibitins serve an essential role in mitochondrial function during organismal development. Science, 2003 Jun 6, 300(5625), 1542 - 8 Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes; Zou L et al.; The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage . Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells . In vitro, RPA stimulates the binding of ATRIP to ssDNA . The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA . Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner . A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro . Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling. J Biol Chem, 2003 Aug 8, 278(32), 29792 - 8 Epub 2003 Jun 05. The multiple site binding of carboxypeptidase Y inhibitor (IC) to the cognate proteinase . Implications for the biological roles of the phosphatidylethanolamine-binding protein; Mima J et al.; The serine carboxypeptidase inhibitor in the cytoplasm of Saccharomyces cerevisiae, IC, specifically inhibits vacuolar carboxypeptidase Y (CPY) and belongs to a functionally unknown family of phosphatidylethanolamine-binding proteins (PEBPs) . In the presence of 1 M guanidine hydrochloride, a CPY-IC complex is formed and is almost fully activated . The reactivities of phenylmethylsulfonyl fluoride, p-chloromercuribenzoic acid, and diisopropyl fluorophosphate toward the complex are considerably increased in 1 M guanidine hydrochloride, indicating that IC contains a binding site other than its inhibitory reactive site . IC is able to form the complex with diisopropyl fluorophosphate-modified CPY . Tryptic digestion of the complex indicates that two fragments from IC are involved in complex formation with CPY . These findings demonstrate the multiple site binding of IC with CPY . Considering the fact that mouse PEBP has recently been identified as a novel thrombin inhibitor, the binding that characterizes the CPY-IC complex could be a common feature of PEBPs. J Biol Chem, 2003 Aug 15, 278(33), 31312 - 8 Epub 2003 Jun 05. Molecular basis for atovaquone binding to the cytochrome bc1 complex; Kessl JJ et al.; Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis . It is thought to act on these organisms by inhibiting the cytochrome bc1 complex . We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus . Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center . These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein . A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone . When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone . These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes. Eur J Pharm Sci, 2003 Jun, 19(2-3), 151 - 64 Novel high energy intermediate analogues with triazasterol-related structures as inhibitors of ergosterol biosynthesis . III . Synthesis and antifungal activity of N4-alkyl-1,6,7,11b-tetrahydro-2H-pyrimido{4,3-a}isoquinolin-4-amine salts; Gossnitzer E et al.; A series of N4-alkyl-1,6,7,11b-tetrahydro-2H-pyrimido{4,3-a}isoquinolinamine hydroiodides with triazasterol-related structures was designed and synthesized to mimic, as stable analogues, native high energy intermediates (HEI) of ergosterol biosynthesis . The title compounds can be regarded as 8,13,15-triaza-13,17-secosteroids with aromatic ring A bearing the positive charge in the guanidinium moiety . Hence, these compounds present structural similarities with corresponding carbocationic intermediates occurring during the enzyme catalyzed transformation of squalene into ergosterol . The N4-alkylaminopyrimidoisoquinolinium salts were prepared by reaction of respective S-methylthiotetrahydropyrimidoisoquinoline hydroiodides with octylamine, and appropriately methyl-branched alkyl- and alkenylamines . In order to prepare (3R)-6-isopropyl-3-methyl-6-hepten-1-amine several synthetic routes were investigated . The structures of all reported compounds were proved and completely assigned on the basis of homo- and heteronuclear correlated 1D and 2D NMR spectroscopy . The in vitro antifungal susceptibility tests of the title compounds with a standard panel of eight pathogenic fungi revealed especially against the used dermatophytes and yeasts with MICs in the range of 1-32 microg/ml moderate to good antimycotic effects . Depending on the nature of the N4-alkyl substituents structure-activity relationships were found with a maximum of antifungal efficacy of the N4-3,7-dimethyloctylaminopyrimidoisoquinolinium iodide. Dev Cell, 2003 Jun, 4(6), 769 - 70 A mitochondrial rhomboid protease; van der Bliek AM et al.; Rhomboid proteases are integral membrane proteins, typically associated with cleavage of peptide hormones along the secretory pathway . Recent publications demonstrate that yeast mitochondria contain a rhomboid protease required for the cleavage of two mitochondrial intermembrane space proteins, suggesting that rhomboid proteases play a regulatory role in mitochondria. Structure (Camb), 2003 Jun, 11(6), 637 - 49 Structure of the coiled-coil dimerization motif of Sir4 and its interaction with Sir3; Chang JF et al.; The yeast silent information regulators Sir2, Sir3, and Sir4 physically interact with one another to establish a transcriptionally silent state by forming repressive chromatin structures . The Sir4 protein contains binding sites for both Sir2 and Sir3, and these protein-protein interactions are required for gene silencing . Here, we report the X-ray structure of the coiled-coil dimerization motif within the C-terminus of Sir4 and show that it forms a stable 1:1 complex with a dimeric fragment of Sir3 (residues 464-978) . We have identified a cluster of residues on the surface of the Sir4 coiled coil required for specific interactions with Sir3 . The histone deacetylase Sir2 can also bind to this complex, forming a ternary complex with the truncated Sir3 and Sir4 proteins . The dual interactions of Sir4 with Sir3 and Sir2 suggest a physical basis for recruiting Sir3 to chromatin by virtue of its interactions with Sir4 and with deacetylated histones in chromatin. Mol Microbiol, 2003 Jun, 48(6), 1693 - 709 Different roles of the Mre11 complex in the DNA damage response in Aspergillus nidulans; Semighini CP et al.; The Mre11-Rad50-Nbs1 protein complex has emerged as a central player in the cellular DNA damage response . Mutations in scaANBS1, which encodes the apparent homologue of human Nbs1 in Aspergillus nidulans, inhibit growth in the presence of the anti-topoisomerase I drug camptothecin . We have used the scaANBS1 cDNA as a bait in a yeast two-hybrid screening and report the identification of the A . nidulans Mre11 homologue (mreA) . The inactivated mreA strain was more sensitive to several DNA damaging and oxidative stress agents . Septation in A . nidulans is dependent not only on the uvsBATR gene, but also on the mre11 complex . scaANBS1 and mreA genes are both involved in the DNA replication checkpoint whereas mreA is specifically involved in the intra-S-phase checkpoint . ScaANBS1 also participates in G2-M checkpoint control upon DNA damage caused by MMS . In addition, the scaANBS1 gene is also important for ascospore viability, whereas mreA is required for successful meiosis in A . nidulans . Consistent with this view, the Mre11 complex and the uvsCRAD51 gene are highly expressed at the mRNA level during the sexual development. J Biol Chem, 2003 Aug 29, 278(35), 33501 - 18 Epub 2003 Jun 04. Mice lacking phosphatidylinositol transfer protein-alpha exhibit spinocerebellar degeneration, intestinal and hepatic steatosis, and hypoglycemia; Alb JG Jr et al.; Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and cellular functions . We now report that ablation of PITP alpha function leads to aponecrotic spinocerebellar disease, hypoglycemia, and intestinal and hepatic steatosis in mice . The data indicate that hypoglycemia is in part associated with reduced proglucagon gene expression and glycogenolysis that result from pancreatic islet cell defects . The intestinal and hepatic steatosis results from the intracellular accumulation of neutral lipid and free fatty acid mass in these organs and suggests defective trafficking of triglycerides and diacylglycerols from the endoplasmic reticulum . We propose that deranged intestinal and hepatic lipid metabolism and defective proglucagon gene expression contribute to hypoglycemia in PITP alpha-/- mice, and that hypoglycemia is a significant contributing factor in the onset of spinocerebellar disease . Taken together, the data suggest an unanticipated role for PITP alpha in with glucose homeostasis and in mammalian endoplasmic reticulum functions that interface with transport of specific luminal lipid cargoes. J Biol Chem, 2003 Aug 15, 278(33), 30875 - 80 Epub 2003 Jun 04. Mutagenesis reveals a specific role for Cox17p in copper transport to cytochrome oxidase; Punter FA et al.; The provision of copper to cytochrome oxidase is one of the requisite steps in the assembly of the holoenzyme . Several proteins are involved in this process including Cox17p, Sco1p, and Cox11p . Cox17p, an 8-kDa protein, is the only molecule thought to be involved in shuttling copper from the cytoplasm into mitochondria . Given the small size of Cox17p, we have taken a random and site-directed mutagenesis approach to studying structure-function relationships in Cox17p . Mutations have been generated in 70% of the Cox17p amino acid residues, with only a small subset leading to a detectable respiration-deficient phenotype . We have characterized the respiration-deficient cox17 mutants and found in addition to the expected cytochrome oxidase deficiency, a specific lack of Cox2p and the presence of a misassembled cytochrome oxidase in a subset of mutants . These results suggest that Cox17p is involved upstream of Sco1p in delivering copper specifically to subunit 2 of cytochrome oxidase and predict the existence of a subunit 1-specific copper chaperone. Curr Opin Genet Dev, 2003 Jun, 13(3), 262 - 70 Nuclear genes in mitochondrial disorders; Zeviani M et al.; Nuclear genes encode hundreds of proteins involved in mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) . Nevertheless, the identification of nuclear genes responsible for OXPHOS-related disorders has proceeded at a much slower pace, compared with the discovery and characterization of mtDNA mutations . Reasons for such a gap include rarity of syndromes, genetic heterogeneity, and ignorance on this nuclear gene repertoire in humans . This scenario is changing rapidly, thanks to the discovery of several OXPHOS-related human genes, and to the identification in some of them of disease-associated mutations . In addition, new strategies - based on transcriptome and proteome analysis, and functional complementation assays - have been applied successfully to mitochondrial medicine. Curr Opin Cell Biol, 2003 Jun, 15(3), 275 - 80 Telomerase: what are the Est proteins doing? Taggart AK, Zakian VA. Saccharomyces cerevisiae has proven to be a useful model organism for the study of telomerase, a specialized cellular reverse transcriptase that helps maintain genomic stability by adding telomeric DNA repeats to the ends of chromosomes . Yeast telomerase is thought to be a holoenzyme containing Est2p and TLC1 RNA, the catalytic subunit and its intrinsic template, respectively, as well as the TLC1-RNA-associated factors Est1p and Est3p . Cdc13p, a sequence-specific telomere-DNA-binding protein, is also required for action in vivo . A current model for telomerase regulation is that telomere-associated Cdc13p binds Est1p, thereby recruiting telomerase . However, recent chromatin immunoprecipitation experiments suggest an alternate role for Est1p in activating Est2p-TLC1-RNA that is already bound to the telomere . Three models for Est1p activation are presented. Cell, 2003 May 30, 113(5), 621 - 30 Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding; Kang RS et al.; Monoubiquitination serves as a regulatory signal in a variety of cellular processes . Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs . Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination . The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces, including the Leu8-Ile44-Val70 patch on ubiquitin . The contact surface extends beyond this patch and encompasses Lys48, a site of polyubiquitin chain formation . This suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling . The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch . Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin . The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins. Cell, 2003 May 30, 113(5), 609 - 20 Mechanism of ubiquitin recognition by the CUE domain of Vps9p; Prag G et al.; Coupling of ubiquitin conjugation to ER degradation (CUE) domains are approximately 50 amino acid monoubiquitin binding motifs found in proteins of trafficking and ubiquitination pathways . The 2.3 A structure of the Vps9p-CUE domain is a dimeric domain-swapped variant of the ubiquitin binding UBA domain . The 1.7 A structure of the CUE:ubiquitin complex shows that one CUE dimer binds one ubiquitin molecule . The bound CUE dimer is kinked relative to the unbound CUE dimer and wraps around ubiquitin . The CUE monomer contains two ubiquitin binding surfaces on opposite faces of the molecule that cannot bind simultaneously to a single ubiquitin molecule . Dimerization of the CUE domain allows both surfaces to contact a single ubiquitin molecule, providing a mechanism for high-affinity binding to monoubiquitin. Cell, 2003 May 30, 113(5), 554 - 6 CUE'd up for Monoubiquitin; Lima CD; The first structures have been obtained for complexes between CUE domains and monoubiquitin, one by NMR (Kang et al., this issue of Cell) and one by X-ray crystallography (Prag et al., this issue of Cell), thus providing insights into ubiquitin recognition by CUE domains . Structural comparisons suggest that different CUE surfaces can interact with ubiquitin, indicating that not all CUE domains are created equal. Genes Cells, 2003 Jun, 8(6), 559 - 71 Terminal deoxynucleotidyltransferase forms a ternary complex with a novel chromatin remodeling protein with 82 kDa and core histone; Fujita K et al.; BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances the Ig and TcR gene diversity in the N region at the junctions of variable (V), diversity (D) and joining (J) segments in B- and T-cells . TdT synthesizes the N region in concert with many proteins including DNA-PKcs, Ku70 and Ku86 . To elucidate the molecular mechanism of the N region synthesis, we first attempted to isolate the genes with products that directly interact with TdT . RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT . This protein was designated as TdT interacting factor 2 (TdIF2) . The confined region of the C-terminal in TdIF2 is involved in specific interaction with the entire C-terminal in TdT . TdIF2 contains an acidic region comprised of 42 residues . TdIF2 was shown to bind specifically to a core histone by pull down assay using specific antibodies against TdIF2 . When a TdT/TdIF2 complex was applied on to a DNA-cellulose column, only TdT bound to the column while TdIF2 passed through . TdIF2 reduces the TdT activity to 46% of its maximum value in vitro assay system using activated DNA as primer . CONCLUSIONS: TdIF2 binds directly to TdT and core histone . Furthermore, TdT, TdIF2 and core histone form a ternary complex . TdIF2 liberates H2A/H2B from a core histone in correlation with PCNA . The enzymatic consequence of the TdIF2/TdT complex is the reduction of TdT activity in vitro . TdIF2 would function as a chromatin remodeling protein at the N region synthesis. Genes Cells, 2003 Jun, 8(6), 525 - 35 Transmembrane topology of sphingoid long-chain base-1-phosphate phosphatase, Lcb3p; Kihara A et al.; BACKGROUND: Sphingoid long-chain base-1-phosphates (LCBPs) are thought to act as intracellular signalling molecules in yeast . Lcb3p is a member of the LCBPs-specific phosphatase family (SPP family) . Other yeast phosphatases, Lpp1p and Dpp1p, are members of a different lipid phosphatase family (LPP family) known to exhibit broader substrate specificities . Until now, only the membrane topology of mammalian LPP family members has been reported, whereas that of the SPP family has remained unclear . RESULTS: In our in vitro system, Lcb3p displayed major phosphatase activity against dihydrosphingosine-1-phosphate, while Dpp1p and Lpp1p also exhibited activities . Here, we determined that Lpp1p and Dpp1p exhibit the topology common to the LPP family . Moreover, we examined the transmembrane topology of Lcb3p using a C-terminal reporter approach . From our results we deduced a structural model illustrating that Lcb3p has eight membrane-spanning domains with its highly conserved phosphatase motifs positioned within the endoplasmic reticulum (ER) lumen . Consistent with this result, Lcb3p collected in low speed pellet fractions was highly resistant to exogenous proteinase K unless the membrane was disrupted . CONCLUSION: Our results suggest that the active site of Lcb3p is located in the ER lumen and, thus, the phosphate group of the LCBP is hydrolysed on the lumenal side. Genes Dev, 2003 Jun 15, 17(12), 1524 - 39 Epub 2003 Jun 03. Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2; Benjamin KR et al.; Meiosis is thought to require the protein kinase Ime2 early for DNA replication and the cyclin-dependent kinase Cdc28 late for chromosome segregation . To elucidate the roles of these kinases, we inhibited their activities early and late using conditional mutants that are sensitive to chemical inhibitors . Our studies reveal that both Cdc28 and Ime2 have critical roles in meiotic S phase and M phase . Early inhibition of analog-sensitive cdc28-as1 blocked DNA replication, revealing a previously undetected role for Cdc28 . Yet Cdc28 was dispensable for one of its functions in the mitotic cell cycle, degradation of Sic1 . Late addition of inhibitor to ime2-as1 revealed unexpected roles of Ime2 in the initiation and execution of chromosome segregation . The requirement of Ime2 for M phase is partially explained by its stimulation of the key meiotic transcription factor Ndt80, which is needed in turn for high Cdc28 activity . In accordance with a late role for Ime2, we observed an increase in its activity during M phase that depended on Cdc28 and Ndt80 . We speculate that several unique features of the meiotic cell division reflect a division of labor and regulatory coordination between Ime2 and Cdc28. Hum Mol Genet, 2003 Jun 15, 12(12), 1427 - 37 The p38 subunit of the aminoacyl-tRNA synthetase complex is a Parkin substrate: linking protein biosynthesis and neurodegeneration; Corti O et al.; Parkinson's disease (PD) is a severe neurological disorder, characterized by the progressive degeneration of the dopaminergic nigrostriatal pathway and the presence of Lewy bodies (LBs) . The discovery of genes responsible for familial forms of the disease has provided insights into its pathogenesis . Mutations in the parkin gene, which encodes an E3 ubiquitin-protein ligase involved in the ubiquitylation and proteasomal degradation of specific protein substrates, have been found in nearly 50% of patients with autosomal-recessive early-onset parkinsonism . The abnormal accumulation of substrates due to loss of Parkin function may be the cause of neurodegeneration in parkin-related parkinsonism . Here, we demonstrate that Parkin interacts with, ubiquitylates and promotes the degradation of p38, a key structural component of the mammalian aminoacyl-tRNA synthetase complex . We found that the ubiquitylation of p38 is abrogated by truncated variants of Parkin lacking essential functional domains, but not by the pathogenic Lys161Asn point mutant . Expression of p38 in COS7 cells resulted in the formation of aggresome-like inclusions in which Parkin was systematically sequestered . In the human dopaminergic neuroblastoma-derived SH-SY5Y cell line, Parkin promoted the formation of ubiquitylated p38-positive inclusions . Moreover, the overexpression of p38 in SH-SY5Y cells caused significant cell death against which Parkin provided protection . Analysis of p38 expression in the human adult midbrain revealed strong immunoreactivity in normal dopaminergic neurons and the labeling of LBs in idiopathic PD . This suggests that p38 plays a role in the pathogenesis of PD, opening the way for a detailed examination of its potential non-canonical role in neurodegeneration. Hum Mol Genet, 2003 Jun 15, 12(12), 1415 - 25 IL1 receptor accessory protein like, a protein involved in X-linked mental retardation, interacts with Neuronal Calcium Sensor-1 and regulates exocytosis; Bahi N et al.; Previously, human genetics-based approaches allowed us to show that mutations in the IL-1 receptor accessory protein-like gene (IL1RAPL) are responsible for a non-specific form of X-linked mental retardation . This gene encodes a predicted protein of 696 amino acids that belongs to a novel class of the IL-1/Toll receptor family . In addition to the extracellular portion consisting of three Ig-like domains and the intracellular TIR domain characteristic of the IL-1/Toll receptor family, IL1RAPL contains a specific 150 amino acid carboxy terminus that has no significant homology with any protein of known function . In order to begin to elucidate the function of this IL-1/Toll receptor-like protein, we have assessed the effect of recombinant IL1RAPL on the binding affinity of type I IL-1R for its ligands IL-1alpha and beta and searched for proteins interacting with the specific carboxy terminus domain of IL1RAPL . Our results show that IL1RAPL is not a protein receptor for IL-1 . In addition we present here the identification of Neuronal Calcium Sensor-1 (NCS-1) as an IL1RAPL interactor . Remarkably, although NCS-1 and its non-mammalian homologue, frequenin, are members of a highly conserved EF-hand Ca(2+) binding protein family, our data show that IL1RAPL interacts only with NCS-1 through its specific C-terminal domain . The functional relevance of IL1RAPL activity was further supported by the inhibitory effect on exocytosis in PC12 cells overexpressing IL1RAPL . Taken together, our data suggest that IL1RAPL may regulate calcium-dependent exocytosis and provide insight into the understanding of physiopathological mechanisms underlying cognitive impairment resulting from IL1RAPL dysfunction. Plant Cell, 2003 Jun, 15(6), 1375 - 85 PmSUC3: characterization of a SUT2/SUC3-type sucrose transporter from Plantago major; Barth I et al.; Higher plants possess medium-sized gene families that encode plasma membrane-localized sucrose transporters . For several plant species, it has been shown that at least one of these genes (e.g., AtSUC3 in Arabidopsis and LeSUT2 in tomato) differs from all other family members in several features, such as the length of the open reading frame, the number of introns, and the codon usage bias . For these reasons, and because two of these proteins did not rescue a yeast mutant defective in sucrose utilization, it had been speculated that this subgroup of transporters might have sensor functions . Here, we describe the detailed functional characterization and cellular localization of PmSUC3, the orthologous transporter from the Plantago major transporter family . The PmSUC3 protein is localized in the sieve elements of the Plantago phloem and mediates the energy-dependent transport of sucrose and maltose . In contrast to the situation in solanaceous plants, PmSUC3 is not colocalized with PmSUC2, the source-specific, phloem-loading sucrose transporter of Plantago . Moreover, PmSUC3 also was identified in sieve elements of sink leaves and in several nonphloem cells and tissues . Arguments for and against a potential sensor function for this type of sucrose transporter are presented, and the role of this type of transporter in the regulation of sucrose fluxes is discussed. Plant Cell, 2003 Jun, 15(6), 1347 - 59 Small ubiquitin-like modifier modulates abscisic acid signaling in Arabidopsis; Lois LM et al.; Post-translational modification of proteins by small polypeptides, such as ubiquitin, has emerged as a common and important mechanism for regulating protein function . Small ubiquitin-like modifier (SUMO) is a small protein that is structurally related to but functionally different from ubiquitin . We report the identification and functional analysis of AtSUMO1, AtSUMO2, and AtSCE1a as components of the SUMO conjugation (sumoylation) pathway in Arabidopsis . In yeast-two hybrid assays, AtSUMO1/2 interacts specifically with a SUMO-conjugating enzyme but not with a ubiquitin-conjugating enzyme . AtSCE1a, the Arabidopsis SUMO-conjugating enzyme ortholog, conjugates SUMO to RanGAP in vitro . AtSUMO1/2 and AtSCE1a colocalize at the nucleus, and AtSUMO1/2 are conjugated to endogenous SUMO targets in vivo . Analysis of transgenic plants showed that overexpression of AtSUMO1/2 does not have any obvious effect in general plant development, but increased sumoylation levels attenuate abscisic acid (ABA)-mediated growth inhibition and amplify the induction of ABA- and stress-responsive genes such as RD29A . Reduction of AtSCE1a expression levels accentuates ABA-mediated growth inhibition . Our results suggest a role for SUMO in the modulation of the ABA signal transduction pathway. Biochim Biophys Acta, 2003 Jun 10, 1632(1-3), 1 - 15 Plant sphingolipids: structural diversity, biosynthesis, first genes and functions; Sperling P et al.; In mammals and Saccharomyces cerevisiae, sphingolipids have been a subject of intensive research triggered by the interest in their structural diversity and in mammalian pathophysiology as well as in the availability of yeast mutants and suppressor strains . More recently, sphingolipids have attracted additional interest, because they are emerging as an important class of messenger molecules linked to many different cellular functions . In plants, sphingolipids show structural features differing from those found in animals and fungi, and much less is known about their biosynthesis and function . This review focuses on the sphingolipid modifications found in plants and on recent advances in the functional characterization of genes gaining new insight into plant sphingolipid biosynthesis . Recent studies indicate that plant sphingolipids may be also involved in signal transduction, membrane stability, host-pathogen interactions and stress responses. Genomics, 2003 Jun, 81(6), 570 - 8 Genomic organization and expression of mouse Tpt1 gene; Fiucci G et al.; The translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF), is encoded by a gene (Tpt1) that is highly conserved throughout phylogeny . TCTP is implicated in cell growth, acute allergic response, and apoptosis . In the present study, seven putative Tpt1 genes with different chromosomal localizations were identified in the mouse genome . In six of them, analysis of the 5' and 3' untranslated regions revealed the presence of flanking direct repeats and residual poly(A) tails typical of pseudogenes . Only three of the seven genes can produce a protein of the expected molecular weight . We isolated the genomic DNA of these three genes to analyze their sequence, genomic organization, and in vitro promoter activity . We found that mouse Tpt1 is localized on chromosome 14 with a canonical intron-exon organization, a functional promoter, and only one transcript that is ubiquitously expressed in all tissues. Curr Biol, 2003 May 27, 13(11), R449 - 60 Captivating capture: how microtubules attach to kinetochores; Biggins S et al.; Accurate chromosome segregation is essential to ensure genomic stability because the aneuploidy that results from segregation errors leads to birth defects and contributes to the development of cancer . Chromosome segregation is directed by the kinetochore, the chromosomal site of attachment to dynamic polymers called microtubules (MTs) . Although the fidelity of chromosome segregation depends on precise interactions between kinetochores and MTs, it is still unclear how this interaction is mediated and regulated . Here we discuss current progress in determining how kinetochores assemble and attach to MTs during mitosis as well as how they correct errors. Curr Biol, 2003 May 27, 13(11), R439 - 41 Telomere replication: an Est fest; Lundblad V; The search for subunits of the telomerase enzyme has uncovered orthologs of the budding years Est1 protein in several species, including humans . Thus, positive regulation of telomerase by Est1 appears to be a widely utilized mechanism for maintaining telomere length homeostasis. Curr Biol, 2003 May 27, 13(11), R430 - 2 Microtubule cytoskeleton: navigating the intracellular landscape; Bloom K; Recent studies have significantly advanced our understanding of how a dividing cell asymmetrically positions the mitotic spindle--a key process in metazoan development--while maintaining a dynamic spindle state that can respond and reorient when necessary. Biochemistry, 2003 Jun 10, 42(22), 6709 - 18 2.4 A resolution crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor; Holyoak T et al.; This paper reports the first structure of a member of the Kex2/furin family of eukaryotic pro-protein processing proteases, which cleave sites consisting of pairs or clusters of basic residues . Reported is the 2.4 A resolution crystal structure of the two-domain protein ssKex2 in complex with an Ac-Ala-Lys-boroArg inhibitor (R = 20.9%, R(free) = 24.5%) . The Kex2 proteolytic domain is similar in its global fold to the subtilisin-like superfamily of degradative proteases . Analysis of the complex provides a structural basis for the extreme selectivity of this enzyme family that has evolved from a nonspecific subtilisin-like ancestor . The P-domain of ssKex2 has a novel jelly roll like fold consisting of nine beta strands and may potentially be involved, along with the buried Ca(2+) ion, in creating the highly determined binding site for P(1) arginine. Nat Cell Biol, 2003 Jul, 5(7), 661 - 7 Ubp3 requires a cofactor, Bre5, to specifically de-ubiquitinate the COPII protein, Sec23; Cohen M et al.; Ubiquitination is important for a broad array of cellular functions . Although reversal of this process, de-ubiquitination, most probably represents an important regulatory step contributing to cellular homeostasis, the specificity and properties of de-ubiquitination enzymes remain poorly understood . Here, we show that the Saccharomyces cerevisiae ubiquitin protease Ubp3 requires an additional protein, Bre5, to form an active de-ubiquitination complex that cleaves ubiquitin from specific substrates . In particular, this complex rescues Sec23p, a COPII subunit essential for the transport between the endoplasmic reticulum and the Golgi apparatus, from degradation by the proteasome . This probably contributes to maintaining and adapting a Sec23 expression level that is compatible with an efficient secretion pathway, and consequently with cell growth and viability. Biochem J, 2003 Aug 1, 373(Pt 3), 733 - 8 X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase; Erskine PT et al.; The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 A) resolution . The cyclic intermediate is bound covalently to Lys(263) with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion . The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen . The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C-C bond linking both substrates in the intermediate is formed before the C-N bond. Plant Mol Biol, 2003 Apr, 51(6), 851 - 7 Pectate lyase gene expression and enzyme activity in ripening banana fruit; Marin-Rodriguez MC et al.; Two distinct cDNA clones showing sequence homology to higher-plant pectate lyase (Pel) genes were isolated from ripening banana fruits . The transcripts were detected only in fruit tissue and both were strongly ripening-related . Yeast transformation with the most highly expressed Pel clone produced a recombinant protein with pectate lyase activity, demonstrating that this sequence was likely to encode a pectate lyase protein in planta . An assay developed for measuring the action of the endogenous enzyme from banana pulp tissue revealed a significant increase in calcium-dependent pectate lyase activity during ripening . The enhanced levels of enzyme activity corresponded with an increase in soluble polyuronides from banana pulp. Plant Mol Biol, 2003 Apr, 51(6), 817 - 29 Tobacco bZIP factor TGA10 is a novel member of the TGA family of transcription factors; Schiermeyer A et al.; TGA factors constitute a family of conserved plant bZ1P transcription factors that regulate transcription from as-1-like elements in response to plant signalling molecules salicylic acid (SA), methyl jasmonate (MJ) and auxin . Based on sequence similarities, two subclasses of TGA factors have been identified before in tobacco: class I factors (TGA1a and PG13) are preferentially expressed in root tip meristems, whereas class II factors (TGA2.1 and TGA2.2) are found in leaves and in roots . Here we describe a novel member of the tobacco TGA family (TGA10), which defines a distinct subclass of its own . TGA10 mRNA and TGA10 protein were found in roots but not in leaves of mature tobacco plants . TGA10 binds specifically to the as-1 element, interacts with TGA2.2, and activates transcription in yeast . When ectopically expressed in leaves, TGA10 enhanced SA-, auxin- and MJ-inducibility of target gene Nt103, which responds in the same manner to enhanced levels of TGA2.2 . This indicates that TGA10, albeit normally not present in leaves, can interact with the leaf regulatory network controlling transcription from as-1-containing promoters . However, Nt103 expression was not affected in roots of TGA10-over-expressing plants, implying the existence of root-specific mechanisms which do not allow a positive effect of increased TGA10 levels on target gene expression. J Cell Sci, 2003 Jul 1, 116(Pt 13), 2603 - 11 Formins: signaling effectors for assembly and polarization of actin filaments; Evangelista M et al.; Eukaryotic cells require filamentous actin to maintain their shape and for movement, growth and replication . New actin filaments are formed by the cutting of existing filaments or de novo through the action of specialized nucleators . The most highly characterized nucleator is the Arp2/3 complex, which nucleates the branched actin networks in the lamellae of migrating cells . Recently, Bni1p, which is a member of the formin family of proteins, has been shown to nucleate actin filaments in vitro . Formins are implicated in the formation of actin cables in yeast, stress fibers in tissue culture cells and cytokinesis in many cell types . Formins contain two highly conserved formin-homology domains, FH1 and FH2 . The Bni1p FH2 domain is sufficient to mediate nucleation . The Bni1p FH1 domain binds profilin, an actin-monomer-binding protein that delivers actin to the growing barbed end of filaments . The Bni1p FH1-profilin interaction enhances nucleation . Formins participate in a number of signaling pathways that control the assembly of specific actin structures and bind the barbed end of actin filaments, thereby providing a cytoskeletal basis for the establishment of cell polarity. Nature, 2003 May 29, 423(6939), 537 - 41 Mitochondrial membrane remodelling regulated by a conserved rhomboid protease; McQuibban GA et al.; Rhomboid proteins are intramembrane serine proteases that activate epidermal growth factor receptor (EGFR) signalling in Drosophila . Rhomboids are conserved throughout evolution, and even in eukaryotes their existence in species with no EGFRs implies that they must have additional roles . Here we report that Saccharomyces cerevisiae has two rhomboids, which we have named Rbd1p and Rbd2p . RBD1 deletion results in a respiratory defect; consistent with this, Rbd1p is localized in the inner mitochondrial membrane and mutant cells have disrupted mitochondria . We have identified two substrates of Rbd1p: cytochrome c peroxidase (Ccp1p); and a dynamin-like GTPase (Mgm1p), which is involved in mitochondrial membrane fusion . Rbd1p mutants are indistinguishable from Mgm1p mutants, indicating that Mgm1p is a key substrate of Rbd1p and explaining the rbd1Delta mitochondrial phenotype . Our data indicate that mitochondrial membrane remodelling is regulated by cleavage of Mgm1p and show that intramembrane proteolysis by rhomboids controls cellular processes other than signalling . In addition, mitochondrial rhomboids are conserved throughout eukaryotes and the mammalian homologue, PARL, rescues the yeast mutant, suggesting that these proteins represent a functionally conserved subclass of rhomboid proteases. Mol Cell Biol, 2003 Jun, 23(12), 4344 - 55 The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively; Kallstrom G et al.; We characterized two essential putative GTPases, Nog1p and Lsg1p, that are found associated with free 60S ribosomal subunits affinity purified with the nuclear export adapter Nmd3p . Nog1p and Lsg1p are nucleolar and cytoplasmic, respectively, and are not simultaneously on the same particle, reflecting the path of Nmd3p shuttling in and out of the nucleus . Conditional mutants of both NOG1 and LSG1 are defective in 60S subunit biogenesis and display diminished levels of 60S subunits at restrictive temperature . Mutants of both genes also accumulate the 60S ribosomal reporter Rpl25-eGFP in the nucleolus, suggesting that both proteins are needed for subunit export from the nucleolus . Since Lsg1p is cytoplasmic,