EMBO J, 2003 Oct 15, 22(20), 5358 - 69 Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import; Matsuura Y et al.; The yeast nucleoporin Nup2p is associated primarily with the nuclear basket of nuclear pore complexes and is required for efficient importin-alpha:beta-mediated nuclear protein import as well as efficient nuclear export of Kap60p/importin-alpha . Residues 1-51 of Nup2p bind tightly to Kap60p and are required for Nup2p function in vivo . We have determined the 2.6 A resolution crystal structure of a complex between this region of Nup2p and the armadillo repeat domain of Kap60p . Nup2p binds along the inner concave groove of Kap60p, but its interaction interface is different from that employed for nuclear localization signal (NLS) recognition although there is some overlap between them . Nup2p binds Kap60p more strongly than NLSs and accelerates release of NLSs from Kap60p . Nup2p itself is released from Kap60p by Cse1p:RanGTP only in the presence of the importin-beta binding (IBB) domain of Kap60p . These data indicate that Nup2p increases the overall rate of nuclear trafficking by coordinating nuclear import termination and importin recycling as a concerted process. Genes Cells, 2003 Oct, 8(10), 779 - 88 Transcription elongation factor S-II maintains transcriptional fidelity and confers oxidative stress resistance; Koyama H et al.; BACKGROUND: During transcription elongation, RNA polymerase II is arrested on the template when incorrect ribonucleotides are incorporated into the nascent transcripts . Transcription factor S-II enhances the excision of these mis-incorporated nucleotides by RNA polymerase II and stimulates transcription elongation in vitro . This mechanism is considered to be transcriptional proof-reading, but its physiological relevance remains unknown . RESULTS: We report that S-II contributes to the maintenance of transcriptional fidelity in vivo . We employed a genetic reporter assay utilizing a mutated lacZ gene from which active beta-galactosidase protein is expressed when mRNA proof-reading is compromised . In S-II-disrupted mutant yeasts, beta-galactosidase activity was ninefold higher than that in wild-type . The S-II mutant exhibited sensitivity to oxidants, which was suppressed by introduction of the S-II gene . The mutant S-II proteins, which are unable to stimulate transcription by RNA polymerase II in vitro, did not suppress the sensitivity of the mutants to oxidative stress or maintain transcriptional fidelity . CONCLUSION: These results suggest that S-II confers oxidative stress resistance by providing an mRNA proof-reading mechanism during transcription elongation. Mov Disord, 2003 Oct, 18 Suppl 7, S71 - 80 Dopamine transporter: basic science and human variation of a key molecule for dopaminergic function, locomotion, and parkinsonism; Uhl GR; We review the basic science of the dopamine transporter (DAT), a key neurotransmitter for locomotor control and reward systems, including those lost or deranged in Parkinson's disease (PD) . Physiology, pharmaceutical features, expression, cDNA, protein structure/function relationships, and phosphorylation and regulation are discussed . The localization of DAT provides the best marker for the integrity of just the pre-synaptic dopaminergic systems that are most affected in PD . Its function is key for the actions of several toxins that provide some of the best current models for idiopathic parkinsonism, and its variation can clearly alter movement . The wealth of information about this interesting molecule that has been developed over the last 12 years has led to increased interest in DAT among workers interested in both normal and abnormal movement . Nucleic Acids Res, 2003 Oct 15, 31(20), 6053 - 61 Detection of regulatory circuits by integrating the cellular networks of protein-protein interactions and transcription regulation; Yeger-Lotem E et al.; The post-genomic era is marked by huge amounts of data generated by large-scale functional genomic and proteomic experiments . A major challenge is to integrate the various types of genome-scale information in order to reveal the intra- and inter- relationships between genes and proteins that constitute a living cell . Here we present a novel application of classical graph algorithms to integrate the cellular networks of protein-protein interactions and transcription regulation . We demonstrate how integration of these two networks enables the discovery of simple as well as complex regulatory circuits that involve both protein-protein and protein-DNA interactions . These circuits may serve for positive or negative feedback mechanisms . By applying our approach to data from the yeast Saccharomyces cerevisiae, we were able to identify known simple and complex regulatory circuits and to discover many putative circuits, whose biological relevance has been assessed using various types of experimental data . The newly identified relations provide new insight into the processes that take place in the cell, insight that could not be gained by analyzing each type of data independently . The computational scheme that we propose may be used to integrate additional functional genomic and proteomic data and to reveal other types of relations, in yeast as well as in higher organisms. Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12510 - 5 Epub 2003 Oct 06. Topological analysis of a plant vacuolar Na+/H+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity; Yamaguchi T et al.; We conducted an analysis of the topology of AtNHX1, an Arabidopsis thaliana vacuolar Na+/H+ antiporter . Several hydrophilic regions of the antiporter were tagged with a hemagglutinin epitope, and protease protection assays were conducted to determine the membrane topology of the antiporter by using yeast as a heterologous expression system . The overall structure of AtNHX1 is distinct from the human Na+/H+ antiporter NHE1 or any known Na+/H+ antiporter . It is comprised of nine transmembrane domains and a hydrophilic C-terminal domain . Three hydrophobic regions do not appear to span the tonoplast membrane, yet appear to be membrane associated . Our results also indicate that, whereas the N terminus of AtNHX1 is facing the cytosol, almost the entire C-terminal hydrophilic region resides in the vacuolar lumen . Deletion of the hydrophilic C terminus resulted in a dramatic increase in the relative rate of Na+/H+ transport . The ratio of Na+/K+ transport was twice that of the unmodified AtNHX1 . This altered ratio resulted from a relatively small decrease in K+/H+ transport with a large increase in Na+/H+ transport . The vacuolar localization of the C terminus of the AtNHX1, taken together with the regulation of the antiporter selectivity by its C terminus, demonstrates the existence of luminal vacuolar regulatory mechanisms of the antiporter activity. J Biol Chem, 2003 Dec 19, 278(51), 51441 - 7 Epub 2003 Oct 06. Insight into functional aspects of Stt3p, a subunit of the oligosaccharyl transferase . Evidence for interaction of the N-terminal domain of Stt3p with the protein kinase C cascade; Chavan M et al.; Over a decade ago, the gene STT3 was identified in a staurosporine and temperature sensitivity screen of yeast . Subsequently the product of this gene was shown to be a subunit of the endoplasmic reticulum-localized oligosaccharyl transferase (OT) complex . Although stt3 mutants are known to be staurosporine-sensitive, we found that mutants of other OT subunits (except ost4 Delta) are staurosporine-resistant, which indicates that this phenotype of stt3 mutants is not simply a consequence of their defect in glycosylation, as previously speculated . Staurosporine sensitivity was found to be an allele-specific phenotype restricted to cells harboring mutations in highly conserved residues in the N-terminal domain of the STT3 protein . Cells bearing mutations in one of the cytosolic-oriented loops (amino acids 158-168) in the N terminus of Stt3p were found to be specifically susceptible to staurosporine . Staurosporine is a specific inhibitor of Pkc1p, and a genetic link had previously been suggested between PKC1 and STT3 . It is known that overexpression of PKC1 suppresses the staurosporine sensitivity of the stt3 mutants in an allele-specific manner, which is typical of mutants of Pkc1p cascade . It has been shown that the pkc1 null mutant exhibits lowered OT activity . Our results combined with these previous observations indicate that the N-terminal domain of Stt3p may interact with members of the Pkc1p cascade and consequently mutations in this domain result in staurosporine sensitivity . We further speculate that the Pkc1p regulates OT activity through the N-terminal domain of Stt3p, the C-terminal domain of which possesses the recognition and/or catalytic site of the OT complex. J Biol Chem, 2003 Dec 19, 278(51), 51841 - 50 Epub 2003 Oct 06. A single protease, Apg4B, is specific for the autophagy-related ubiquitin-like proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L; Hemelaar J et al.; Apg8 is a ubiquitin-like protein involved in autophagy in yeast . Apg8 is covalently but transiently attached to membrane lipids through the actions of activating, conjugating, and processing/deconjugating enzymes . The mammalian Apg8 homologues GATE-16, GARARAP, and MAP1-LC3 have been implicated in intra-Golgi transport, receptor sorting, and autophagy, respectively . All are served by a single set of activating and conjugating enzymes . Here we identify a novel mammalian Apg8 homologue, which we name Apg8L, and describe the synthesis of electrophilic probes based on the GATE-16, GARARAP, MAP1-LC3, and Apg8L proteins . These probes not only form specific adducts in crude cell lysates, but also allow identification of the cellular proteases specific for the C termini of these Apg8 homologues . We find a single protease, Apg4B/autophagin-1, capable of acting on GATE-16, GABARAP, MAP1-LC3, and Apg8L . The Apg4B/autophagin-1 protease thus serves as a processing/deconjugating enzyme for these four highly divergent mammalian Apg8 homologues. J Mol Biol, 2003 Oct 17, 333(2), 229 - 35 Interaction of the MAGUK family member Acvrinp1 and the cytoplasmic domain of the Notch ligand Delta1; Pfister S et al.; The evolutionarily conserved Notch signal transduction pathway regulates cell fate and cellular differentiation in various tissues and has essential functions in embryonic patterning and tumorigenesis . Cell-cell signaling by the Notch pathway is mediated by the interaction of the transmembrane receptor Notch with its ligands Delta or Jagged presented on adjacent cells . Whereas signal transduction to Notch expressing cells has been described, it is unclear whether Delta-dependent signaling may exist within the Delta-expressing cell . Here, we report on the identification of Acvrinp1, a MAGUK family member, interacting with the intracellular domain of Delta1 (Dll1) . We confirmed the interaction between Dll1 and Acvrinp1 by pull-down experiments in vitro and in a mammalian two-hybrid system in vivo . We delimited the fourth PDZ domain of Acvrinp1 and the PDZ-binding domain of Dll1 as major interacting domains . In situ expression analyses in mouse embryos revealed that Dll1 and Acvrinp1 show partly overlapping but distinct expression patterns, for example, in the central nervous system and the vibrissae buds . Further, we found that expression of Acvrinp1 is altered in Dll1 loss-of-function mouse embryos. Mol Biol Cell, 2004 Jan, 15(1), 24 - 36 Epub 2003 Oct 03. Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase; Rudge SA et al.; In the budding yeast Saccharomyces cerevisiae, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is synthesized by a single phosphatidylinositol 3-phosphate 5-kinase, Fab1 . Cells deficient in PtdIns(3,5)P2 synthesis exhibit a grossly enlarged vacuole morphology, whereas increased levels of PtdIns(3,5)P2 provokes the formation of multiple small vacuoles, suggesting a specific role for PtdIns(3,5)P2 in vacuole size control . Genetic studies have indicated that Fab1 kinase is positively regulated by Vac7 and Vac14; deletion of either gene results in ablation of PtdIns(3,5)P2 synthesis and the formation of a grossly enlarged vacuole . More recently, a suppressor of vac7Delta mutants was identified and shown to encode a putative phosphoinositide phosphatase, Fig4 . We demonstrate that Fig4 is a magnesium-activated PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro . Analysis of a Fig4-GFP fusion protein revealed that the Fig4 phosphatase is localized to the limiting membrane of the vacuole . Surprisingly, in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole . However, Fig4-GFP remains localized to the grossly enlarged vacuoles of vac7 deletion mutants . Consistent with these observations, we found that Fig4 physically associates with Vac14 in a common membrane-associated complex . Our studies indicate that Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of the Fig4 PtdIns(3,5)P2 phosphatase. J Biol Chem, 2003 Dec 26, 278(52), 52689 - 99 Epub 2003 Oct 03. The human phosphatidylinositol phosphatase SAC1 interacts with the coatomer I complex; Rohde HM et al.; The Saccharomyces cerevisiae SAC1 gene encodes an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus . Yeast SAC1 mutants display a wide array of phenotypes including inositol auxotrophy, cold sensitivity, secretory defects, disturbed ATP transport into the ER, or suppression of actin gene mutations . At present, it is not clear how these phenotypes relate to the finding that SAC1 displays polyphosphoinositide phosphatase activity . Moreover, it is still an open question whether SAC1 functions similarly in mammalian cells, since some phenotypes are yeast-specific . Potential protein interaction partners and, connected to that, possible regulatory circuits have not been described . Therefore, we have cloned human SAC1 (hSAC1), show that it behaves similar to ySac1p in terms of substrate specificity, demonstrate that the endogenous protein localizes to the ER and Golgi, and identify for the first time members of the coatomer I (COPI) complex as interaction partners of hSAC1 . Mutation of a putative COPI interaction motif (KXKXX) at its C terminus abolishes interaction with COPI and causes accumulation of hSAC1 in the Golgi . In addition, we generated a catalytically inactive mutant, demonstrate that its lipid binding capacity is unaltered, and show that it accumulates in the Golgi, incapable of interacting with the COPI complex despite the presence of the KXKXX motif . These results open the possibility that the enzymatic function of hSAC1 provides a switch for accessibility of the COPI interaction motif. Gene, 2003 Sep 18, 314, 1 - 13 A POLYCOMB group gene of rice (Oryza sativa L . subspecies indica), OsiEZ1, codes for a nuclear-localized protein expressed preferentially in young seedlings and during reproductive development; Thakur JK et al.; The SET domains are conserved amino acid sequences present in chromosomal proteins that contribute to the epigenetic control of gene expression by altering regional organization of the chromatin structure . The SET domain proteins are divided into four subgroups as categorized by their Drosophila members; enhancer of zeste (E(Z)), trithorax (TRX), absent small or homeotic 1 (ASH1) and supressor of variegation (SU(VAR)3-9) . Homologs of all four classes have been characterized in yeast, mammals and plants . We report here the isolation and characterization of rice (Oryza sativa L . subspecies indica) cDNA, OsiEZ1, as a monocot member of this family . The OsiEZ1 cDNA is 3133 bp long with an ORF of 2799 bp, and the predicted amino acid sequence (895 residues) corresponds to a protein of ca . 98 kDa . All the characteristic domains known to be conserved in E(Z) homologs (subgroup I) of SET domain containing proteins are present in OsiEZ1 . In the rice genome, a 7499 bp long OsiEZ1 sequence is split into 17 exons interrupted by 16 introns . Southern analysis indicates that OsiEZ1 is represented as single copy in the rice genome . Expression studies revealed that the OsiEZ1 transcript level was highest in rice flowers, almost undetectable in developing seeds of 1-2 days post-fertilization but increased significantly in young seeds of 3-5 days post-fertilization . The OsiEZ1 transcript was barely detectable in mature zygotic embryos, but its levels were significantly higher in callus derived from rice scutellum, somatic embryos and young seedlings . The OsiEZ1/GUS recombinant protein was confined to the nucleus in living cells of particle-bombarded onion peels . The expression of OsiEZ1 complemented a set1Delta Saccharomyces cerevisiae mutant that is impaired in telomeric silencing . We suggest that the nuclear-localized OsiEZ1 has a role in regulating various aspects of plant development, and this control is most likely brought about by repressing the activity of downstream regulatory genes. FEBS Lett, 2003 Sep 25, 552(2-3), 155 - 9 Adhesion forces measured between a calcium blocker drug and its receptor in living cells using atomic force microscope; de Souza Pereira R et al.; The adhesion force between the tip of an atomic force microscope cantilever derivatized with nimodipine (a calcium blocker, from the dihydropyridine class, currently used in clinical medicine for hypertension) and living cells of Saccharomyces cerevisiae (unicellular eukaryotes which portray ultrastructural features characteristic of higher eukaryotic cells) was measured . This methodology allowed us to locate (and visualize) pores on the cell surface which may be responsible for calcium transportation in the living cells . The interaction of the cantilever derivatized with the calcium blocker and a pore, which can be a calcium channel, is more intense than a non-derivatized cantilever and the pore . Outside the pore (on the rest of cell surface), a derivatized or a non-derivatized cantilever has the same pattern of adhesion force . The information obtained with this method is very important for the design of new, more potent and less toxic drugs for pharmacological use. Mol Cell, 2003 Sep, 12(3), 783 - 90 BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases; Geyer R et al.; Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases . Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members . We show here that S . pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome . The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction . Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner . Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1 . We propose that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes. Mol Cell, 2003 Sep, 12(3), 699 - 709 A minimal RNA polymerase III transcription system from human cells reveals positive and negative regulatory roles for CK2; Hu P et al.; In higher eukaryotes, RNA polymerase (pol) III is known to use different transcription factors to recognize three basic types of promoters, but in no case have these transcription factors been completely defined . We show that a highly purified pol III complex combined with the recombinant transcription factors SNAP(c), TBP, Brf2, and Bdp1 directs multiple rounds of transcription initiation and termination from the human U6 promoter . The pol III complex contains traces of CK2, and CK2 associates with the U6 promoter region in vivo . Transcription requires CK2 phosphorylation of the pol III complex . In contrast, CK2 phosphorylation of TBP, Brf2, and Bdp1 combined is inhibitory . The results define a minimum core machinery, the ultimate target of regulatory mechanisms, capable of directing all steps of the transcription process-initiation, elongation, and termination-by a metazoan RNA polymerase, and suggest positive and negative regulatory roles for CK2 in transcription by pol III. Mol Cell, 2003 Sep, 12(3), 663 - 73 Cytoskeletal activation of a checkpoint kinase; Hanrahan J et al.; The assembly of cytoskeletal structures is coupled to other cellular processes . We have studied the molecular mechanism by which assembly of the yeast septin cytoskeleton is monitored and coordinated with cell cycle progression by analyzing a key regulatory protein kinase, Hsl1, that becomes activated only when the septin cytoskeleton is properly assembled . We first identified a regulatory region of Hsl1 that physically associates with the kinase domain and found that it performs an autoinhibitory function both in vivo and in vitro . Several septin binding domains lie near and overlap the inhibitory domain; these are important for Hsl1 function, and binding of two septins, Cdc11 and Cdc12, relieves the autoinhibition imposed by the kinase inhibitory domain in vitro . Our results suggest that binding to multiple septins activates Hsl1 kinase activity, thereby promoting cell cycle progression . The high conservation of Hsl1 indicates that similar mechanisms may monitor cytoskeletal organization in other eukaryotes. Mol Cell, 2003 Sep, 12(3), 615 - 25 Conserved structural motifs in intracellular trafficking pathways: structure of the gammaCOP appendage domain; Hoffman GR et al.; The formation of coated vesicles is a fundamental step in many intracellular trafficking pathways . COPI and clathrin represent two important and distinct sets of vesicle coating machinery, involved primarily in mediating intra-Golgi and endocytic transport, respectively . Here we identify an important functional region at the carboxyl terminus of the gamma subunit of the COPI complex (gammaCOP) and describe the X-ray crystal structure of this domain at 2.3 A resolution . This domain of gammaCOP exhibits unexpected structural similarity to the carboxyl-terminal appendage domains of the alpha and beta subunits of the AP2 adaptor proteins, integral components of clathrin-coated vesicles . The remarkable structural conservation exhibited by the gammaCOP appendage domain, coupled with functional data and primary sequence analysis, supports a model of COPI function with significant structural and mechanistic parallels to vesicular transport by the clathrin/AP2 system. Plant Physiol, 2003 Oct, 133(2), 630 - 41 Epub 2003 Oct 02. Plasma membrane aquaporins are involved in winter embolism recovery in walnut tree; Sakr S et al.; In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity . Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels . Low water potential leads to water movement from the parenchyma cells into the xylem vessels . The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity . We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia) . First, we established that xylem parenchyma starch is converted to sucrose in the winter months . Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins . The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes . The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period . In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose . Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels . Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels. J Biol Chem, 2003 Dec 12, 278(50), 50158 - 62 Epub 2003 Oct 02. Tup1-Ssn6 interacts with multiple class I histone deacetylases in vivo; Davie JK et al.; The Tup1-Ssn6 corepressor complex in Saccharomyces cerevisiae represses the transcription of a diverse set of genes . Chromatin is an important component of Tup1-Ssn6-mediated repression . Tup1 binds to underacetylated histone tails and requires multiple histone deacetylases (HDACs) for its repressive functions . Here, we describe physical interactions of the corepressor complex with the class I HDACs Rpd3, Hos2, and Hos1 . In contrast, no in vivo interaction was observed between Tup-Ssn6 and Hda1, a class II HDAC . We demonstrate that Rpd3 interacts with both Tup1 and Ssn6 . Rpd3 and Hos2 interact with Ssn6 independently of Tup1 via distinct tetratricopeptide domains within Ssn6, suggesting that these two HDACs may contact the corepressor at the same time. Genome Res, 2003 Oct, 13(10), 2242 - 51 Two distinct modes of microsatellite mutation processes: evidence from the complete genomic sequences of nine species; Dieringer D et al.; We surveyed microsatellite distribution in 10 completely sequenced genomes . Using a permutation-based statistic, we assessed for all 10 genomes whether the microsatellite distribution significantly differed from expectations . Consistent with previous reports, we observed a highly significant excess of long microsatellites . Focusing on short microsatellites containing only a few repeat units, we demonstrate that this repeat class is significantly underrepresented in most genomes . This pattern was observed across different repeat types . Computer simulations indicated that neither base substitutions nor a combination of length-dependent slippage and base substitutions could explain the observed pattern of microsatellite distribution . When we introduced one additional mutation process, a length-independent slippage (indel slippage) operating at repeats with few repetitions, our computer simulations captured the observed pattern of microsatellite distribution. Genome Res, 2003 Oct, 13(10), 2229 - 35 Gene loss, protein sequence divergence, gene dispensability, expression level, and interactivity are correlated in eukaryotic evolution; Krylov DM et al.; Lineage-specific gene loss, to a large extent, accounts for the differences in gene repertoires between genomes, particularly among eukaryotes . We derived a parsimonious scenario of gene losses for eukaryotic orthologous groups (KOGs) from seven complete eukaryotic genomes . The scenario involves substantial gene loss in fungi, nematodes, and insects . Based on this evolutionary scenario and estimates of the divergence times between major eukaryotic phyla, we introduce a numerical measure, the propensity for gene loss (PGL) . We explore the connection among the propensity of a gene to be lost in evolution (PGL value), protein sequence divergence, the effect of gene knockout on fitness, the number of protein-protein interactions, and expression level for the genes in KOGs . Significant correlations between PGL and each of these variables were detected . Genes that have a lower propensity to be lost in eukaryotic evolution accumulate fewer substitutions in their protein sequences and tend to be essential for the organism viability, tend to be highly expressed, and have many interaction partners . The dependence between PGL and gene dispensability and interactivity is much stronger than that for sequence evolution rate . Thus, propensity of a gene to be lost during evolution seems to be a direct reflection of its biological importance. BMC Evol Biol . 2003 Oct 02;3(1):21. Apparent dependence of protein evolutionary rate on number of interactions is linked to biases in protein-protein interactions data sets; Bloom JD et al.; BACKGROUND: Several studies have suggested that proteins that interact with more partners evolve more slowly . The strength and validity of this association has been called into question . Here we investigate how biases in high-throughput protein-protein interaction studies could lead to a spurious correlation . RESULTS: We examined the correlation between evolutionary rate and the number of protein-protein interactions for sets of interactions determined by seven different high-throughput methods in Saccharomyces cerevisiae . Some methods have been shown to be biased towards counting more interactions for abundant proteins, a fact that could be important since abundant proteins are known to evolve more slowly . We show that the apparent tendency for interactive proteins to evolve more slowly varies directly with the bias towards counting more interactions for abundant proteins . Interactions studies with no bias show no correlation between evolutionary rate and the number of interactions, and the one study biased towards counting fewer interactions for abundant proteins actually suggests that interactive proteins evolve more rapidly . In all cases, controlling for protein abundance significantly decreases the observed correlation between interactions and evolutionary rate . Finally, we disprove the hypothesis that small data set size accounts for the failure of some interactions studies to show a correlation between evolutionary rate and the number of interactions . CONCLUSIONS: The only correlation supported by a careful analysis of the data is between evolutionary rate and protein abundance . The reported correlation between evolutionary rate and protein-protein interactions cannot be separated from the biases of some protein-protein interactions studies to count more interactions for abundant proteins. Phys Rev Lett . 2003 Sep 26;91(13):138701 . Epub 2003 Sep 26. Preferential attachment in the protein network evolution; Eisenberg E et al.; The Saccharomyces cerevisiae protein-protein interaction map, as well as many natural and man-made networks, shares the scale-free topology . The preferential attachment model was suggested as a generic network evolution model that yields this universal topology . However, it is not clear that the model assumptions hold for the protein interaction network . Using a cross-genome comparison, we show that (a) the older a protein, the better connected it is, and (b) the number of interactions a protein gains during its evolution is proportional to its connectivity . Therefore, preferential attachment governs the protein network evolution . Evolutionary mechanisms leading to such preference and some implications are discussed. Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12081 - 6 Epub 2003 Oct 01. DcpS can act in the 5'-3' mRNA decay pathway in addition to the 3'-5' pathway; van Dijk E et al.; Eukaryotic mRNA degradation proceeds through two main pathways, both involving mRNA cap breakdown . In the 3'-5' mRNA decay pathway, mRNA body degradation generates free m7GpppN that is hydrolyzed by DcpS generating m7GMP . In the 5'-3' pathway, the recently identified human Dcp2 decapping enzyme cleaves the cap of deadenylated mRNAs to produce m7GDP and 5'-phosphorylated mRNA . We investigated mRNA decay in human cell extracts by using a new assay for decapping . We observed that 5'-phosphorylated intermediates resulting from decapping appear after incubation of a substrate RNA in human cell extracts, indicating the presence of an active 5'-3' mRNA decay pathway . Surprisingly, however, the cognate m7GDP product was not detected, whereas abundant amounts of m7GMP were generated . Additional experiments revealed that m7GDP is, unexpectedly, efficiently converted to m7GMP in extracts from various organisms . The factor necessary and sufficient for this reaction was identified as DcpS in both yeast and human . m7GMP is thus a general, pathway-independent, by-product of eukaryotic mRNA decay . m7GDP breakdown should prevent misincorporation of methylated nucleotides in nucleic acids and could generate a unique indicator allowing the cell to monitor mRNA decay. Sci Aging Knowledge Environ . 2003 Oct 01;2003(39):PE27. Ceramide, stress, and a "LAG" in aging; Obeid LM et al.; Recent studies have implicated the longevity assurance gene LAG1 in ceramide synthesis . In light of a role for ceramide in yeast and mammalian stress responses and mammalian cellular senescence, important connections are emerging between ceramide and organismal aging . In this Perspective, we examine the evidence for these connections in yeast, Drosophila, and mammals, and speculate on their implications. J Biol Chem, 2003 Dec 12, 278(50), 50732 - 43 Epub 2003 Sep 30. Permease recycling and ubiquitination status reveal a particular role for Bro1 in the multivesicular body pathway; Nikko E et al.; Ubiquitination of the yeast Gap1 permease at the plasma membrane triggers its endocytosis followed by targeting to the vacuolar lumen for degradation . We previously identified Bro1 as a protein essential to this down-regulation . In this study, we show that Bro1 is essential neither to ubiquitination nor to the early steps of Gap1 endocytosis . Bro1 rather intervenes at a late step of the multivesicular body (MVB) pathway, after the core components of the endosome-associated ESCRT-III protein complex and before or in conjunction with Doa4, the ubiquitin hydrolase mediating protein deubiquitination prior to their incorporation into MVB vesicles . Bro1 markedly differs from other class E vacuolar protein sorting factors involved in MVB sorting as lack of Bro1 leads to recycling of the internalized permease back to the plasma membrane by passing through the Golgi . This recycling seems to be accompanied by deubiquitination of the permease and unexpectedly requires a normal endosome-to-vacuole transport function. J Biol Chem, 2003 Dec 19, 278(51), 50985 - 98 Epub 2003 Sep 30. Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases; Jackson MD et al.; Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine epsilon-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose . Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span . Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established . Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2'-ribose hydroxyl of ADP-ribose . Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD+ (transglycosidation) . The cellular implications are discussed . A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation . A Bronsted plot of the data yielded a slope of +0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity . NAD+ analogues including beta-2'-deoxy-2'-fluororibo-NAD+ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer . We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2'-OH ribose . The observed enzyme-catalyzed formation of a labile 1'-acetylated-ADP-fluororibose intermediate using beta-2'-deoxy-2'-fluororibo-NAD+ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1' ribose to form an initial iminium adduct. Genes Dev, 2003 Oct 1, 17(19), 2421 - 35 KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C . elegans; Desai A et al.; Segregation of the replicated genome during cell division requires kinetochores, mechanochemical organelles that assemble on mitotic chromosomes to connect them to spindle microtubules . CENP-A, a histone H3 variant, and CENP-C, a conserved structural protein, form the DNA-proximal foundation for kinetochore assembly . Using RNA interference-based genomics in Caenorhabditis elegans, we identified KNL-1, a novel kinetochore protein whose depletion, like that of CeCENP-A or CeCENP-C, leads to a "kinetochore-null" phenotype . KNL-1 is downstream of CeCENP-A and CeCENP-C in a linear assembly hierarchy . In embryonic extracts, KNL-1 exhibits substoichiometric interactions with CeCENP-C and forms a near-stoichiometric complex with CeNDC-80 and HIM-10, the C . elegans homologs of Ndc80p/HEC1p and Nuf2p-two widely conserved outer kinetochore components . However, CeNDC-80 and HIM-10 are not functionally equivalent to KNL-1 because their inhibition, although preventing formation of a mechanically stable kinetochore-microtubule interface and causing chromosome missegregation, does not result in a kinetochore-null phenotype . The greater functional importance of KNL-1 may be due to its requirement for targeting multiple components of the outer kinetochore, including CeNDC-80 and HIM-10 . Thus, KNL-1 plays a central role in translating the initiation of kinetochore assembly by CeCENP-A and CeCENP-C into the formation of a functional microtubule-binding interface. Genes Dev, 2003 Oct 1, 17(19), 2396 - 405 mSin3-associated protein, mSds3, is essential for pericentric heterochromatin formation and chromosome segregation in mammalian cells; David G et al.; The histone code guides many aspects of chromosome biology including the equal distribution of chromosomes during cell division . In the chromatin domains surrounding the centromere, known as pericentric heterochromatin, histone modifications, particularly deacetylation and methylation, appear to be essential for proper chromosome segregation . However, the specific factors and their precise roles in this highly orchestrated process remain under active investigation . Here, we report that germ-line or somatic deletion of mSds3, an essential component of the functional mSin3/HDAC corepressor complex, generates a cell-lethal condition associated with rampant aneuploidy, defective karyokinesis, and consequently, a failure of cytokinesis . mSds3-deficient cells fail to deacetylate and methylate pericentric heterochromatin histones and to recruit essential heterochromatin-associated proteins, resulting in aberrant associations among heterologous chromosomes via centromeric regions and consequent failure to properly segregate chromosomes . Mutant mSds3 molecules that are defective in mSin3 binding fail to rescue the mSds3 null phenotypes . On the basis of these findings, we propose that mSds3 and its associated mSin3/HDAC components play a central role in initiating the cascade of pericentric heterochromatin-specific modifications necessary for the proper distribution of chromosomes during cell division in mammalian cells. Arch Biochem Biophys, 2003 Oct 15, 418(2), 151 - 60 5alpha-reduced C21 steroids are substrates for human cytochrome P450c17; Gupta MK et al.; The 5alpha-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5alpha-reduced C(19) steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate . We studied the metabolism of 5alpha-reduced C(21) steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C(21) steroids to C(19) steroids via its 17alpha-hydroxylase and 17,20-lyase activities . hCYP17 17alpha-hydroxylates 5alpha-pregnan-3,20-dione, but little androstanedione is formed by 17,20-lyase activity . hCYP17 also 17alpha-hydroxylates 5alpha-pregnan-3alpha-ol-20-one and the 5alpha-pregnan-3alpha,17alpha-diol-20-one intermediate is rapidly converted to androsterone by 17,20-lyase activity . Furthermore, 5alpha-pregnan-3alpha,17alpha-diol-20-one is a better substrate for the 17,20-lyase reaction than the preferred substrate 17alpha-hydroxypregnenolone and cytochrome b(5) stimulates androsterone formation only 3-fold . Both 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3alpha,17alpha-diol-20-one bind to hCYP17 with higher affinity than does progesterone . We conclude that 5alpha-reduced, 3alpha-hydroxy-C(21) steroids are excellent, high-affinity substrates for hCYP17 . The brisk metabolism of 5alpha-pregnan-3alpha,17alpha-diol-20-one to androsterone by CYP17 explains how, when 5alpha-reductases are present, the testis can produce C(19) steroids androsterone and androstanediol from 17alpha-hydroxyprogesterone without the intermediacy of androstenedione and testosterone. Biochem Biophys Res Commun, 2003 Oct 17, 310(2), 312 - 7 Expression of histone acetyltransferases was down-regulated in poly(ADP-ribose) polymerase-1-deficient murine cells; Ota K et al.; NF-kappaB-dependent, as well as human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR)-dependent, reporter gene expression was significantly impaired in cells derived from poly(ADP-ribose) polymerase-1 (PARP-1)-knockout (PARP-1 -/-) mice . In addition, the level of protein acetylation was markedly lower in PARP-1 -/- cells than control (PARP-1 +/+) cells . Surprisingly, the expression levels of histone acetyltransferases (HATs), p300, cAMP response element-binding protein-binding protein (CBP), and p300/CBP-associated factor (PCAF), were significantly reduced in PARP-1 -/- cells, as compared with PARP-1 +/+ cells . These results suggest that PARP-1 is required for the proper expression of particular HATs . Since p300 and CBP are coactivators of NF-kappaB, we propose here that PARP-1 participates in NF-kappaB-dependent transcription by means of maintaining the expression of HATs. Curr Biol, 2003 Sep 30, 13(19), 1740 - 5 Cell cycle-regulated transcription through the FHA domain of Fkh2p and the coactivator Ndd1p; Darieva Z et al.; Recent studies in Saccharomyces cerevisiae by using global approaches have significantly enhanced our knowledge of the components involved in the transcriptional regulation of the cell cycle . The Mcm1p-Fkh2p complex, in combination with the coactivator Ndd1p, plays an important role in the cell cycle-dependent expression of the CLB2 gene cluster during the G2 and M phases ({4-7}; see {8-10}for reviews) . Fkh2p is phosphorylated in a cell cycle-dependent manner, and peak phosphorylation occurs coincidentally with maximal expression of Mcm1p-Fkh2p-dependent gene expression . However, the mechanism by which this complex is activated in a cell cycle-dependent manner is unknown . Here, we demonstrate that the forkhead-associated (FHA) domain of Fkh2p directs cell cycle-regulated transcription and that the activity of this domain is dependent on the coactivator Ndd1p . Ndd1p was found to be phosphorylated in a cell cycle-dependent manner by Cdc28p-Clb2p, and, importantly, this phosphorylation event promotes interactions between Ndd1p and the FHA domain of Fkh2p . Furthermore, mutation of the FHA domain blocks these phosphorylation-dependent interactions and abolishes transcriptional activity . Our data therefore link the transcriptional activity of the FHA domain with cell cycle-dependent phosphorylation of the coactivator Ndd1p and reveal a mechanism that permits precise temporal activation of the Mcm1p-Fkh2p complex. Curr Biol, 2003 Sep 30, 13(19), 1669 - 78 Mathematical modeling suggests cooperative interactions between a disordered polyvalent ligand and a single receptor site; Klein P et al.; BACKGROUND: The CDK inhibitor Sic1 must be phosphorylated on at least six sites in order to allow its recognition by the SCF ubiquitin ligase subunit Cdc4 . However, because Cdc4 appears to have only a single phospho-epitope binding site, the apparent cooperative dependence on the number of phosphorylation sites in Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity . RESULTS: We develop a general kinetic model, which predicts an unexpected multiplicative increase in affinity as a function of ligand sites . This effect, termed allovalency, derives from a high local concentration of interaction sites moving independently of each other . Modeling of this interaction by a first exit time approach indicates that the probability of ligand rebinding increases exponentially with the number of sites . This type of interaction is relatively immune to loss of any one site and may be easily tuned to any given threshold by adjusting the properties of individual sites . CONCLUSIONS: The allovalency model suggests that a previously undescribed mechanism may underlie certain cooperative interactions . The widespread occurrence of flexible polyvalent ligands in biological systems suggests that this principle may be broadly applicable. J Med Chem, 2003 Oct 9, 46(21), 4609 - 24 N-hydroxy-3-phenyl-2-propenamides as novel inhibitors of human histone deacetylase with in vivo antitumor activity: discovery of (2E)-N-hydroxy-3-{4-{{(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl}amino}methyl}phenyl}-2-propenamide (NVP-LAQ824); Remiszewski SW et al.; A series of N-hydroxy-3-phenyl-2-propenamides were prepared as novel inhibitors of human histone deacetylase (HDAC) . These compounds were potent enzyme inhibitors, having IC(50)s < 400 nM in a partially purified enzyme assay . However, potency in cell growth inhibition assays ranged over 2 orders of magnitude in two human carcinoma cell lines . Selected compounds having cellular IC(50) < 750 nM were tested for maximum tolerated dose (MTD) and for efficacy in the HCT116 human colon tumor xenograft assay . Four compounds having an MTD > or = 100 mg/kg were selected for dose-response studies in the HCT116 xenograft model . One compound, 9 (NVP-LAQ824), had significant dose-related activity in the HCT116 colon and A549 lung tumor models, high MTD, and low gross toxicity . On the basis, in part, of these properties, 9 has entered human clinical trials in 2002. Life Sci, 2003 Oct 24, 73(23), 2991 - 3004 Direct and biochemical interaction between dopamine D3 receptor and elongation factor-1Bbetagamma; Cho DI et al.; Novel signaling components of dopamine D3 receptor (D3R) were searched using yeast two-hybrid system, and the gamma subunit of elongation Factor-1B (eEF1Bgamma) was found to interact with D3R . This interaction was observed specifically between eEF1Bgamma and D3R but not with D2R or D4R . Immunocytochemical studies showed that D3R and eEF1Bgamma form clusters on the plasma membrane and their co-localization was evident in these clusters . The beta subunit of eEF1B (eEF1Bbeta), which forms a tight complex with eEF1Bgamma, was phosphorylated on serine residues in response to the stimulation of D3R . Phosphorylation of eEF1Bbeta was insensitive to pertussis toxin or wortmannin, however, stimulation of cellular protein kinase C (PKC) directly phosphorylated eEF1Bbeta and depletion of PKC abolished D3R-mediated phosphorylation of eEF1Bbeta . These results suggest the involvement of PKC, but not Gi/o proteins or phosphatidylinositol 3-kinase, in D3R-mediated phosphorylation of eEF1Bbeta . Stimulation of D3R did not activate PKC, but the activation of PKC resulted in the phosphorylation of D3R . These results show that PKC has a permissive role for the D3R-mediated phosphorylation of eEF1Bbeta, and suggest that PKC could modulate the mutual interaction between two protein by phosphorylating both D3R and eEF1Bbeta . Therefore, the cellular PKC level would be important for the D3R-mediated modulation of eEF1B, and for their cellular regulations such as protein synthesis or cellular proliferation. Curr Opin Cell Biol, 2003 Oct, 15(5), 590 - 7 Roles of Rho-family GTPases in cell polarisation and directional migration; Fukata M et al.; Polarised cell migration is a tightly regulated process that occurs in tissue development, chemotaxis and wound healing . Rho-family GTPases, including Cdc42, Rac1 and RhoA, play a central role in establishing cell polarisation, which requires asymmetric and ordered distribution of the signalling molecules and the cytoskeleton . Recent advances reveal that Rho GTPases, together with phosphatidylinositol 3-kinase, contribute to asymmetric phosphatidylinositol 3,4,5-trisphosphate distribution via a positive-feedback loop . Phosphatidylinositol 3,4,5-trisphosphate thereby activates the signalling cascades to the cytoskeleton as a second messenger . Rho GTPases also capture and stabilise microtubules through their effectors (e.g . IQGAP1, mDia and Par6) near the cell cortex, leading to polarised cell morphology and directional cell migration . Thus, elucidation of the signal transduction cascades from receptors to Rho GTPases and, subsequently, from Rho GTPases to microtubules has begun. Mol Cancer Res, 2003 Sep, 1(11), 836 - 47 Decreased frequency and highly aberrant spectrum of ultraviolet-induced mutations in the hprt gene of mouse fibroblasts expressing antisense RNA to DNA polymerase zeta; Diaz M et al.; In the budding yeast Saccharomyces cerevisiae, DNA polymerase zeta (pol zeta) is responsible for the great majority of mutations generated during error-prone translesion replication of DNA that contains UV-induced lesions . The catalytic subunit of pol zeta is encoded by the Rev3 gene . The orthologue of Rev3 has been cloned from higher eukaryotic cells, including human, but its role in mutagenesis and carcinogenesis remains obscure . Investigation into the cellular function of pol zeta has been hindered by the fact that Rev3 knockout mice do not survive beyond midgestation, and embryonic stem cells used to derive these mice are not genetically stable . We have generated a transgenic mouse that expresses antisense RNA transcripts to mRev3 endogeneous RNA . These mice are viable, have greatly reduced levels of Rev3 transcript, and have reduced levels of B cells and impaired development of high-affinity memory B cells . Here, we report that exposure of fibroblasts derived from these mice to UV resulted in a 4-5-fold reduction in mutant frequency at the hprt locus at every dose examined, and the mutation spectrum was highly aberrant compared with the control cells . In the control cells, 80% of the mutations were transitions and approximately 75% of these arose from photoproducts in the putative leading strand template . Strikingly, in transgenic cells, most of the mutations were transversions and there was a complete loss of strand bias . This mutation spectrum is highly aberrant and is similar to that induced by UV in human xeroderma pigmentosum variant cells, which lack polymerase eta . These data indicate that most UV-induced mutations are dependent on DNA pol zeta, a function that has been conserved from yeast to higher eukaryotic cells . However, in mammalian cells, other DNA polymerase(s) may accomplish error-prone translesion replication and are responsible for residual UV mutagenesis observed in the absence of pol zeta . Further, these data support a central role for DNA polymerase eta in the error-free bypass of UV photoproducts . The antisense Rev3 mice should be a useful model to study mutagenic lesion bypass by pol zeta in mammalian cells and to investigate the role this polymerase plays in carcinogenesis. Mol Biol Cell, 2003 Oct, 14(10), 4126 - 39 Epub 2003 Jul 11. The WD-repeats of Net2p interact with Dnm1p and Fis1p to regulate division of mitochondria; Cerveny KL et al.; The Net2, Fis1, and Dnm1 proteins are required for the division of mitochondria in the yeast Saccharomyces cerevisiae . Net2p has an amino-terminal region that contains predicted coiled-coil motifs and a carboxyl-terminal domain composed of WD-40 repeats . We found that the amino-terminal part of Net2p interacts with Fis1p, whereas the carboxyl-terminal region interacts with both Dnm1p and Fis1p . Overproduction of either domain of Net2p in yeast cells poisons mitochondrial fission, and the dominant-negative effect caused by the WD-repeats of Net2p is suppressed by increased levels of Dnm1p . Point mutations in the WD-region of Net2p or in the GTPase region of Dnm1p disrupt the normal Net2p-Dnm1p interaction, causing Net2p to lose its normal punctate distribution . Our results suggest that Dnm1p interacts with the WD-repeats of Net2p and in a GTP-dependent manner recruits Net2p to sites of mitochondrial division . Furthermore, our results indicate that Net2p is required for proper assembly of the mitochondrial fission components to regulate organelle division. Mol Biol Cell, 2003 Oct, 14(10), 4015 - 27 Epub 2003 Jul 11. The role of mVps18p in clustering, fusion, and intracellular localization of late endocytic organelles; Poupon V et al.; Delivery of endocytosed macromolecules to mammalian cell lysosomes occurs by direct fusion of late endosomes with lysosomes, resulting in the formation of hybrid organelles from which lysosomes are reformed . The molecular mechanisms of this fusion are analogous to those of homotypic vacuole fusion in Saccharomyces cerevisiae . We report herein the major roles of the mammalian homolog of yeast Vps18p (mVps18p), a member of the homotypic fusion and vacuole protein sorting complex . When overexpressed, mVps18p caused the clustering of late endosomes/lysosomes and the recruitment of other mammalian homologs of the homotypic fusion and vacuole protein sorting complex, plus Rab7-interacting lysosomal protein . The clusters were surrounded by components of the actin cytoskeleton, including actin, ezrin, and specific unconventional myosins . Overexpression of mVps18p also overcame the effect of wortmannin treatment, which inhibits membrane traffic out of late endocytic organelles and causes their swelling . Reduction of mVps18p by RNA interference caused lysosomes to disperse away from their juxtanuclear location . Thus, mVps18p plays a critical role in endosome/lysosome tethering, fusion, intracellular localization and in the reformation of lysosomes from hybrid organelles. Mol Cell Biol, 2003 Oct, 23(20), 7339 - 49 Rds3p is required for stable U2 snRNP recruitment to the splicing apparatus; Wang Q et al.; Rds3p is a well-conserved 12-kDa protein with five CxxC zinc fingers that has been implicated in the activation of certain drug transport genes and in the pre-mRNA splicing pathway . Here we show that Rds3p resides in the yeast spliceosome and is essential for splicing in vitro . Rds3p purified from yeast stably associates with at least five U2 snRNP proteins, Cus1p, Hsh49p, Hsh155p, Rse1p, and Ist3p/Snu17p, and with the Yra1p RNA export factor . A mutation upstream of the first Rds3p zinc finger causes the conditional release of the putative branchpoint nucleotide binding protein, Ist3p/Snu17p, and weakens Rse1p interaction with the Rds3p complex . The resultant U2 snRNP particle migrates exceptionally slowly in polyacrylamide gels, suggestive of a disorganized structure . U2 snRNPs depleted of Rds3p fail to form stable prespliceosomes, although U2 snRNA stability is not affected . Metabolic depletion of Yra1p blocks cell growth but not splicing, suggesting that Yra1p association with Rds3p relates to Yra1p's role in RNA trafficking . Together these data establish Rds3p as an essential component of the U2 snRNP SF3b complex and suggest a new link between the nuclear processes of pre-mRNA splicing and RNA export. EMBO J, 2003 Oct 1, 22(19), 5251 - 9 The mechanisms of {URE3} prion elimination demonstrate that large aggregates of Ure2p are dead-end products; Ripaud L et al.; The yeast prion {URE3} is a self-propagating inactive form (the propagon) of the Ure2 protein . Ure2p is composed of two domains: residues 1-93--the prion-forming domain (PFD)--and the remaining C-terminal part of the protein, which forms the functional domain involved in nitrogen catabolite repression . Guanidine hydrochloride, and the overproduction of Ure2p 1-65 or Ure2-GFP have been shown to induce the elimination of {URE3} . We demonstrate here, two different curing mechanisms: the inhibition of {URE3} replication by guanidine hydrochloride and its destruction by Ure2p aggregation . Such aggregation is observed if PFD or Ure2-GFP are overproduced and in heterozygous URE2/URE2-GFP, {URE3} diploids . We found that the GFP foci associated with the presence of the prion were dead-end products, the propagons remaining soluble . Surprisingly, {URE3} propagated via the Ure2-GFP fusion protein alone is resistant to these two curing mechanisms and cannot promote the formation of foci . The relationship between aggregation, prion and Hsp104 gives rise to a model in which the propagon is in equilibrium with larger aggregates and functional protein. EMBO J, 2003 Oct 1, 22(19), 5230 - 40 TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes; Siegers K et al.; The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood . Here, we show that a group of WD40 beta-propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state . TRiC cooperates in the folding of these proteins with the ribosome-associated heat shock protein (Hsp)70 chaperones Ssb1/2p . In contrast, newly synthesized actin and tubulins, the major known client proteins of TRiC, are independent of Ssb1/2p and instead use the co-chaperone GimC/prefoldin for efficient transfer to the chaperonin . GimC can replace Ssb1/2p in the folding of WD40 substrates such as Cdc55p, but combined deletion of SSB and GIM genes results in loss of viability . These findings expand the substrate range of the eukaryotic chaperonin by a structurally defined class of proteins and demonstrate an essential role for upstream chaperones in TRiC-assisted folding. EMBO J, 2003 Oct 1, 22(19), 5163 - 74 Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo; Green CM et al.; DNA damage and its repair can cause both local and global rearrangements of chromatin structure . In each case, the epigenetic information contained within this structure must be maintained . Using the recently developed method for the localized UV irradiation of cells, we analysed responses that occur locally to damage sites and global events triggered by local damage recognition . We thus demonstrate that, within a single cell, the recruitment of chromatin assembly factor 1 (CAF-1) to UV-induced DNA damage is a strictly local phenomenon, restricted to damage sites . Concomitantly, proliferating cell nuclear antigen (PCNA) locates to the same sites . This localized recruitment suggests that CAF-1 participates directly in chromatin structural rearrangements that occur in the vicinity of the damage . Use of nucleotide excision repair (NER)-deficient cells shows that the NER pathway--specifically dual incision--is required for recruitment of CAF-1 and PCNA . This in vivo demonstration of the local role of CAF-1, depending directly on NER, supports the hypothesis that CAF-1 ensures the maintenance of epigenetic information by acting locally at repair sites. EMBO J, 2003 Oct 1, 22(19), 5147 - 53 Modulation of transcription factor function by an amino acid: activation of Put3p by proline; Sellick CA et al.; Saccharomyces cerevisiae are able to convert proline to glutamate so that it may be used as a source of nitrogen . Here, we show that the activator of the proline utilization genes, Put3p, is transcriptionally inert in the absence of proline but transcriptionally active in its presence . The activation of Put3p requires no additional yeast proteins and can occur in the presence of certain proline analogues: an unmodified pyrrolidine ring is able to activate Put3p as efficiently as proline itself . In addition, we show that a direct interaction occurs between Put3p and proline . These data, which represent direct control of transcriptional activator function by a metabolite, are discussed in terms of the regulation of proline-specific genes in yeast and as a general mechanism of the control of transcription. EMBO J, 2003 Oct 1, 22(19), 5137 - 46 Zinc fingers can act as Zn2+ sensors to regulate transcriptional activation domain function; Bird AJ et al.; The yeast Zap1 transcription factor controls the expression of genes involved in zinc accumulation and storage . Zap1 is active in zinc-limited cells and repressed in replete cells . Zap1 has two activation domains, AD1 and AD2, which are both regulated by zinc . AD2 function was mapped to a region containing two Cys2His2 zinc fingers, ZF1 and ZF2, that are not involved in DNA binding . More detailed mapping placed AD2 almost precisely within the endpoints of ZF2, suggesting a role for these fingers in regulating activation domain function . Consistent with this hypothesis, ZF1 and ZF2 bound zinc in vitro but less stably than did zinc fingers involved in DNA binding . Furthermore, mutations predicted to disrupt zinc binding to ZF1 and/or ZF2 rendered AD2 constitutively active . Our results also indicate that the repressed form of AD2 requires an intramolecular interaction between ZF1 and ZF2 . These studies suggest that these zinc fingers play an unprecedented role as zinc sensors to control activation domain function. EMBO J, 2003 Oct 1, 22(19), 5115 - 24 A common site on TBP for transcription by RNA polymerases II and III; Schroder O et al.; The TATA-binding protein (TBP) is involved in all nuclear transcription . We show that a common site on TBP is used for transcription initiation complex formation by RNA polymerases (pols) II and III . TBP, the transcription factor IIB (TFIIB)-related factor Brf1 and the pol III-specific factor Bdp1 constitute TFIIIB . A photochemical cross-linking approach was used to survey a collection of human TBP surface residue mutants for their ability to form TFIIIB-DNA complexes reliant on only the TFIIB-related part of Brf1 . Mutations impairing complex formation and transcription were identified and mapped on the surface of TBP . The most severe effects were observed for mutations in the C-terminal stirrup of TBP, which is the principal site of interaction between TBP and TFIIB . Structural modeling of the Brf1-TBP complex and comparison with its TFIIB-TBP analog further rationalizes the close resemblance of the TBP interaction with the N-proximal part of Brf1 and TFIIB, and establishes the conserved usage of a TBP surface in pol II and pol III transcription for a conserved function in the initiation of transcription. EMBO J, 2003 Oct 1, 22(19), 4945 - 56 Tim14, a novel key component of the import motor of the TIM23 protein translocase of mitochondria; Mokranjac D et al.; The TIM23 translocase mediates the deltaPsi- and ATP-dependent import of proteins into mitochondria . We identified Tim14 as a novel component of the TIM23 translocase . Tim14 is an integral protein of the inner membrane with a typical J-domain exposed to the matrix space . TIM14 genes are present in the genomes of virtually all eukaryotes . In yeast, Tim14 is essential for viability . Mitochondria from cells depleted of Tim14 are deficient in the import of proteins mediated by the TIM23 complex . In particular, import of proteins that require the action of mtHsp70 is affected . Tim14 interacts with Tim44 and mtHsp70 in an ATP-dependent manner . A mutation in the HPD motif of the J-domain of Tim14 is lethal . Thus, Tim14 is a constituent of the mitochondrial import motor . We propose a model in which Tim14 is required for the activation of mtHsp70 and enables this chaperone to act in a rapid and regulated manner in the Tim44-mediated trapping of unfolded preproteins entering the matrix. EMBO J, 2003 Oct 1, 22(19), 4888 - 97 The PB1 domain and the PC motif-containing region are structurally similar protein binding modules; Yoshinaga S et al.; The PC motif is evolutionarily conserved together with the PB1 domain, a binding partner of the PC motif-containing protein . For interaction with the PB1 domain, the PC motif-containing region (PCCR) comprising the PC motif and its flanking regions is required . Because the PB1 domain and the PCCR are novel binding modules found in a variety of signaling proteins, their structural and functional characterization is crucial . Bem1p and Cdc24p interact through the PB1-PCCR interaction and regulate cell polarization in budding yeast . Here, we determined a tertiary structure of the PCCR of Cdc24p by NMR . The tertiary structure of the PCCR is similar to that of the PB1 domain of Bem1p, which is classified into a ubiquitin fold . The PC motif portion takes a compact betabetaalpha-fold, presented on the ubiquitin scaffold . Mutational studies indicate that the PB1-PCCR interaction is mainly electrostatic . Based on the structural information, we group the PB1 domains and the PCCRs into a novel family, named the PB1 family . Thus, the PB1 family proteins form a specific dimer with each other. J Biol Chem, 2003 Dec 12, 278(50), 50428 - 34 Epub 2003 Sep 29. Functional and structural basis of carnitine palmitoyltransferase 1A deficiency; Gobin S et al.; Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid beta-oxidation . Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia . We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E . Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations . To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase . This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant) . Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class . They are located more than 20 A away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex . By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category . They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms . This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders. Virology, 2003 Sep 15, 314(1), 460 - 7 Roles of host cell factors in circularization of retroviral dna; Kilzer JM et al.; Early during retroviral infection, a fraction of the linear reverse-transcribed viral DNA genomes become circularized by cellular enzymes, thereby inactivating the genomes for further replication . Prominent circular DNA forms include 2-long-terminal repeat (LTR) circles, made by DNA end joining, and 1-LTR circles, produced in part by homologous recombination . These reactions provide a convenient paradigm for analyzing the cellular machinery involved in DNA end joining in vertebrate cells . In previous studies, we found that inactivating components of the nonhomologous DNA end-joining (NHEJ) pathway--specifically Ku, ligase 4, or XRCC4--blocked formation of 2-LTR circles . Here we report that inactivating another NHEJ component, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), had at most modest effects on 2-LTR circle formation, providing informative parallels with other end-joining reactions . We also analyzed cells mutant in components of the RAD50/MRE11/NBS1 nuclease and found a decrease in the relative amount of 1-LTR circles, opposite to the effects of NHEJ mutants . In MRE11-mutant cells, a MRE11 gene mutant in the nuclease catalytic site failed to restore 1-LTR circle formation, supporting a model for the role of MRE11 in 1-LTR circle formation . None of the cellular mutations showed a strong effect on normal integration, consistent with the idea that the cellular pathways leading to circularization are not involved in productive integration. J Mol Biol, 2003 Oct 10, 333(1), 201 - 9 Glucose and type 2A protein phosphatase regulate the interaction between catalytic and regulatory subunits of AMP-activated protein kinase; Gimeno-Alcaniz JV et al.; We have expressed in yeast the different subunits of AMP-activated protein kinase (AMPK) and, by using the two-hybrid system, we have found a glucose-regulated interaction between alpha 2 catalytic and gamma 1 regulatory subunits . This regulation was not affected by known regulators of the corresponding yeast orthologue, the SNF1 complex, such as Reg1 or Hxk2, but it was affected by deletion of regulatory subunits of yeast type 2A protein phosphatase (PP2A) complex . We have also found that Tpd3 and PR65 alpha, the corresponding yeast and mammalian A subunits of PP2A, interacted with AMPK alpha 2 both in yeast and mammals, respectively . This interaction occurred only through the regulatory domain of this subunit . These results suggested a direct involvement of PP2A complex in regulating the interaction between AMPK alpha 2 and gamma 1 in a glucose-dependent manner. J Mol Biol, 2003 Oct 10, 333(1), 187 - 99 Tuning the heterogeneous early folding dynamics of phosphoglycerate kinase; Osvath S et al.; We recently reported stretched kinetics during the formation of a collapsed, long-lived intermediate state of the large two-domain enzyme phosphoglycerate kinase (PGK) . It was postulated that intrinsic roughness of the energy landscape on the way downhill to the intermediate causes the lack of a single time-scale . Here, we investigate several alternative explanations for stretched refolding dynamics in more detail: tyrosine fluorescence, multiple tryptophan probes, and rate differences between independently folding domains . To this end, we systematically simplify PGK in several steps from the full protein with two tryptophan residues and all tyrosine residues probed, to a single domain with only one tryptophan residue and no tyrosine residue probed . The kinetics in the 10 micros to 10 ms range are revealed by laser-induced temperature-jump relaxation experiments . The isolated N-terminal domain forms an intermediate by nearly single-exponential kinetics, but the isolated C-terminal domain shows strongly non-exponential kinetics . Thus, domain interaction and a cis-proline residue between the two domains are ruled out as the sole contributors to heterogeneity during the earliest folding dynamics of the C-terminal domain . We apply two limiting models for the roughness of the energy landscape . A sequential three-state model lumps all the roughness into a single trap . The "strange kinetics" model with logarithmic oscillations developed by Klafter and co-workers distributes the roughness over a larger number of states . Both models explain our data about equally well, but the coincidental values of rate constants in all of our double-exponential fits, and the absence of a spectroscopic signature distinct from the endpoints of the folding process favors more roughness than can be explained by just a single trap. J Mol Biol, 2003 Oct 10, 333(1), 75 - 85 Correlating protein-DNA and protein-protein interaction networks; Manke T et al.; Here, we search protein-DNA binding data for prevalent pairs and higher-order tuples of co-occurring transcription factors (TF) in Saccharomyces cerevisiae . While the identification of such modules is dependent on uncertainties of genome-wide data sets, we find several biologically meaningful examples, which allow putative annotation of yet unclassified genes . For the frequently occurring transcriptional module Mcm1-Fkh2-Ndd1, we identified several new target genes involved in cell-cycle control and filament formation . Using large-scale protein interaction data, we demonstrate a significant correlation between co-occurrence of TF binding sites and the vicinity in the protein interaction network . In particular we find that directly interacting transcription factors and those which are members of a protein complex are more likely to occur together as putative DNA-binding modules. J Microsc, 2003 Oct, 212(Pt 1), 53 - 61 Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples; Giddings TH; Specimen preparation methods based on high-pressure freezing and freeze-substitution have enabled significant advances in the quality of morphological preservation of biological samples for electron microscopy . However, visualization of a subset of cellular membranes, particularly the endoplasmic reticulum and cis Golgi, is often impaired by a lack of contrast . By contrast, some efforts to increase membrane staining may lead to excessively granular staining . No one freeze-substitution method has emerged that both overcomes these limitations and is suitable for all types of analysis . However, one or more of the following protocols, perhaps with minor modifications, should yield satisfactory results in most cases . Freeze-substitution in glutaraldehyde and uranyl acetate in acetone, followed by embedding in Lowicryl HM20, generates samples suitable for both immunolocalization and high-resolution structural studies . Membranes are typically lightly stained but very well defined . Initial freeze-substitution in tannic acid and glutaraldehyde in acetone prior to exposure to osmium tetroxide significantly enhanced contrast on mammalian cellular membranes . Finally, initial trials indicate that freeze-substitution in potassium permanganate in acetone can provide strong contrast on endoplasmic reticulum and Golgi as well as other membranes . The tendency of permanganate to degrade cytoskeletal elements and other proteins when employed in aqueous solutions at room temperature is apparently curtailed when it is used as a freeze-substitution reagent. J Microsc, 2003 Oct, 212(Pt 1), 26 - 33 An oscillating cryo-knife reduces cutting-induced deformation of vitreous ultrathin sections; Al-Amoudi A et al.; A new oscillating cryo-knife for producing uncompressed vitreous sections is introduced . The knife is a modified cryo diamond knife that is driven by a piezo translator . Optimal setting for the oscillation was found to be in the inaudible frequency range of 20-25 kHz . Yeast cells and polystyrene spheres were used as model systems to describe compression in the vitreous sections . We found that compression could be reduced by a factor of about 2 when the knife was oscillating . When the oscillator was turned off, sections were compressed by 40-45% . However, only 15-25% compression was obtained when the knife was oscillating . In some cases completely uncompressed sections of yeast cells were produced . It was also found that the amount of compression depends on the specimen itself and on its embedding medium . With the results shown here, we demonstrate that the oscillating knife can produce high-quality vitreous sections with minimum cutting artefacts. Biochem J, 2004 Jan 1, 377(Pt 1), 85 - 93 Murine bubblegum orthologue is a microsomal very long-chain acyl-CoA synthetase; Fraisl P et al.; It has been suggested that a gene termed bubblegum (Bgm), encoding an acyl-CoA synthetase, could be involved in the pathogenesis of the inherited neurodegenerative disorder X-ALD (X-linked adrenoleukodystrophy) . The precise function of the ALDP (ALD protein) still remains unclear . Aldp deficiency in mammals and Bgm deficiency in Drosophila led to accumulation of VLCFAs (very long-chain fatty acids) . As a first step towards studying this interaction in wild-type versus Aldp-deficient mice, we analysed the expression pattern of the murine orthologue of the Bgm gene . In contrast with the ubiquitously expressed Ald gene, Bgm expression is restricted to the tissues that are affected by X-ALD such as brain, testis and adrenals . During mouse brain development, Bgm mRNA was first detected by Northern-blot analysis on embryonic day 18 and increased steadily towards adulthood, whereas the highest level of Ald mRNA was found on embryonic day 12 and decreased gradually during differentiation . Protein fractionation and confocal laser imaging of Bgm-green fluorescent protein fusion proteins revealed a microsomal localization that was different from peroxisomes (where Aldp is detected), endoplasmic reticulum and Golgi . Mouse Bgm showed acyl-CoA synthetase activity towards a VLCFA substrate in addition to LCFAs, and this activity was enriched in the microsomal compartment . Speculating that Bgm expression could be regulated by Ald deficiency, we compared the abundance of Bgm mRNA in wild-type and Ald knockout mice but observed no difference . Although mouse Bgm is capable of activating VLCFA, we conclude that a direct interaction between the mouse Bgm and the Aldp seems unlikely. J Biol Chem, 2003 Dec 19, 278(51), 51937 - 49 Epub 2003 Sep 26. Molecular basis for the rapid dissociation of nuclear localization signals from karyopherin alpha in the nucleoplasm; Gilchrist D et al.; The yeast karyopherin heterodimer Kap60p.Kap95p facilitates nuclear import of proteins bearing a classic nuclear localization signal (NLS) . The alpha subunit Kap60p binds to the NLS of cargo molecules in the cytoplasm, forming stable complexes that must ultimately dissociate in the nucleoplasm . Although Kap60p can release NLSs on its own using an autoinhibitory sequence (AIS) motif that can occupy the NLS binding site, that mechanism is too slow to support rapid nuclear import . We previously showed that the nuclear basket nucleoporin Nup2p and the exportin complex Cse1p.Gsp1p.GTP function as karyopherin release factors (KaRFs) because they can accelerate the rate of dissociation of NLSs from Kap60p . Here we dissect the molecular mechanics of their KaRF activity . We show that Cse1p accelerates dissociation of Kap60p.NLS-cargo complexes and Kap60p.Nup2p complexes by increasing the affinity of Kap60p for its AIS motif . In contrast, Nup2p uses a conserved sequence motif (VMXXRKIA) coupled to an AIS-like motif to accelerate dissociation of Kap60p.NLS complexes in a vectorial reaction mechanism . Mutation of either motif in Nup2p leads to a loss of KaRF activity and to the accumulation of Kap60p.NLS-cargo complexes in the nucleoplasm of yeast . We discuss a model whereby Nup2p, Cse1p, and Gsp1p cooperate to establish directionality in the movement of Kap60p and NLS-cargos across the nuclear pore complex. J Biol Chem, 2003 Dec 12, 278(50), 49732 - 42 Epub 2003 Sep 26. Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases; Bernard DG et al.; Heme attachment to the apoforms of fungal mitochondrial cytochrome c and c1 requires the activity of cytochrome c and c1 heme lyases (CCHL and CC1HL), which are enzymes with distinct substrate specificity . However, the presence of a single heme lyase in higher eukaryotes is suggestive of broader substrate specificity . Here, we demonstrate that yeast CCHL is active toward the non-cognate substrate apocytochrome c1, i.e . CCHL promotes low levels of apocytochrome c1 conversion to its holoform in the absence of CC1HL . Moreover, that the single human heme lyase also displays a broader cytochrome specificity is evident from its ability to substitute for both yeast CCHL and CC1HL . Multicopy and genetic suppressors of the absence of CC1HL were isolated and their analysis revealed that the activity of CCHL toward cytochrome c1 can be enhanced by: 1) reducing the abundance of the cognate substrate apocytochrome c, 2) increasing the accumulation of CCHL, 3) modifying the substrate-enzyme interaction through point mutations in CCHL or cytochrome c1, or 4) overexpressing Cyc2p, a protein known previously only as a mitochondrial biogenesis factor . Based on the functional interaction of Cyc2p with CCHL and the presence of a putative FAD-binding site in the protein, we hypothesize that Cyc2p controls the redox chemistry of the heme lyase reaction. J Biol Chem, 2003 Dec 12, 278(50), 49920 - 8 Epub 2003 Sep 26. Coupling of the transcriptional regulation of glutathione biosynthesis to the availability of glutathione and methionine via the Met4 and Yap1 transcription factors; Wheeler GL et al.; Depletion of the cellular pool of glutathione is detrimental to eukaryotic cells and in Saccharomyces cerevisiae leads to sensitivity to oxidants and xenobiotics and an eventual cell cycle arrest . Here, we show that the Yap1 and Met4 transcription factors regulate the expression of gamma-glutamylcysteine synthetase (GSH1), encoding the rate-limiting enzyme in glutathione biosynthesis to prevent the damaging effects of glutathione depletion . Transcriptional profiling of a gsh1 mutant indicates that glutathione depletion leads to a general activation of Yap1 target genes, but the expression of Met4-regulated genes remains unaltered . Glutathione depletion appears to result in Yap1 activation via oxidation of thioredoxins, which normally act to down-regulate the Yap1-mediated response . The requirement for Met4 in regulating GSH1 expression is lost in the absence of the centromere-binding protein Cbf1 . In contrast, the Yap1-mediated effect is unaffected, indicating that Met4 acts via Cbf1 to regulate the Yap1-mediated induction of GSH1 expression in response to glutathione depletion . Furthermore, yeast cells exposed to the xenobiotic 1-chloro-2,4-dintrobenzene are rapidly depleted of glutathione, accumulate oxidized thioredoxins, and elicit the Yap1/Met4-dependent transcriptional response of GSH1 . The addition of methionine, which promotes Met4 ubiquitination and inactivation, specifically represses GSH1 expression after 1-chloro-2,4-dintrobenzene exposure but does not affect Yap1 activation . These results indicate that the Yap1-dependent activation of GSH1 expression in response to glutathione depletion is regulated by the sulfur status of the cell through a specific Met4-dependent mechanism. Yakugaku Zasshi, 2003 Sep, 123(9), 805 - 9 {Determination of the optical purity of N-nitrosofenfluramine found in the Chinese slimming diet}; Nakadai A et al.; From 2001 to the summer of 2002, more than 800 cases of liver damage were reported in Japan among people taking Chinese diet aids . The Japanese Ministry of Health, Labor and Welfare has recently announced that N-nitrosofenfluramine was the hepatotoxic compound contained in the diet aids based on animal experiments performed by the National Institute of Health Sciences . Although N-nitrosofenfluramine is a derivative of fenfluramine, a previously used antiobesity drug, neither pharmacologic nor toxicologic properties have been reported for N-nitroso fenfluramine . It should be noted that N-nitrosofenfluramine has two optical isomers, although it is not yet known which isomer damages the liver and other organs . The Japanese Ministry of Health, Labor and Welfare has not commented on this point . Pursuing this question, 10 types of Chinese slimming aid samples including those obtained from patients with fulminating hepatitis were analyzed by NMR, GC/MS, and a newly established HPLC method using a chiral separation column . It was found that the N-nitrosofenfluramine in all of the toxic diet aids was the (S)-isomer form . No (R)-isomer was detected . These results strongly suggest that the nitroso-compound in the diets must be prepared from pharmacologically active (S)-fenfluramine (dexfenfluramine) . Thus the pharmacologic and toxicologic properties of each isomer should be investigated. Melanoma Res, 2003 Oct, 13(5), 503 - 9 Discrimination between gene expression patterns in the invasive margin and the tumour core of malignant melanomas; Roesch A et al.; Genes that determine the invasive capacity of the invasive front of malignant melanomas (MM) have not yet been systematically investigated in vivo . Therefore, we combined laser pressure catapulting (LPC) microdissection with cDNA microarray technology (DermArray, Research Genetics, representing about 5700 genes) to systematically analyse differences in gene expression profiles between the invasive margin and the tumour centre in nine cases of vertical growth phase MM . Signal-to-noise statistical algorithms combined with hierarchical clustering were performed to determine class-separating genes . The gene encoding phosphoenolpyruvate carboxykinase 1 (PEPCK), the Homo sapiens gene similar to Saccharomyces cerevisiae SSM4 (TEB4), the gene encoding ribosomal protein L19, the Homo sapiens gene similar to the Aspergillus nidulans SudD (a suppressor of the bimD6 homologue), the gene encoding the interleukin-3 receptor alpha subunit, the gene encoding the inositol 1,4,5-triphosphate 3-kinase isoenzyme, and three anonymous expressed sequence tags were identified as class-separating genes . These genes significantly discriminate between the invasive front and the tumour centre . Using this set of genes, 15 out of 18 LPC-dissected MM regions could be grouped correctly . We conclude that the candidate genes identified could spark further research on MM progression and may provide novel prognostic parameters. Cell Cycle, 2003 Nov-Dec, 2(6), 568 - 72 Multiple roles of replication forks in S phase checkpoints: sensors, effectors and targets; Pasero P et al.; There is mounting evidence that replication defects are the major source of spontaneous genomic instability in the cell and that S phase checkpoints are the principle defense against such instability . In Saccharomyces cerevisiae, S phase checkpoints can be provoked by either depletion of dNTPs or DNA damage . In both cases the checkpoint kinases Mec1 and Rad53 act to suppress late origin firing, stabilize slowed or stalled replication forks and prevent S phase progression until conditions are appropriate for the resumption of DNA replication . The present review highlights recent work emphasizing the central importance of replication forks, not just as targets, but also as sensors and primary effectors of checkpoint responses, and identifies the roles played by specific fork-associated factors in these processes. Science, 2003 Sep 26, 301(5641), 1908 - 11 An age-induced switch to a hyper-recombinational state; McMurray MA et al.; There is a strong correlation between age and cancer, but the mechanism by which this phenomenon occurs is unclear . We chose Saccharomyces cerevisiae to examine one of the hallmarks of cancer--genomic instability--as a function of cellular age . As diploid yeast mother cells aged, an approximately 100-fold increase in loss of heterozygosity (LOH) occurred . Extending life-span altered neither the onset nor the frequency of age-induced LOH; the switch to hyper-LOH appears to be on its own clock . In young cells, LOH occurs by reciprocal recombination, whereas LOH in old cells was nonreciprocal, occurring predominantly in the old mother's progeny . Thus, nuclear genomes may be inherently unstable with age. Science, 2003 Sep 26, 301(5641), 1859 - 60 Cell biology . An age of instability; Sinclair DA; It is well established that as we age our cancer risk increases dramatically . As Sinclair explains in his Perspective, the link between cancer and aging is now solidified by new work in budding yeast (McMurray and Gottschling) . As yeast cells age there is a marked increase in their genetic instability (a hallmark of cancer), which is independent of the mechanism that determines their life-span. Mol Endocrinol, 2003 Dec, 17(12), 2554 - 65 Epub 2003 Sep 25. Transcriptional control in male germ cells: general factor TFIIA participates in CREM-dependent gene activation; De Cesare D et al.; Regulation of gene expression in haploid male germ cells follows a number of specific rules that differ from somatic cells . In this physiological context, transcriptional control mediated by the activator CREM (cAMP-responsive element modulator) represents an established paradigm . In somatic cells activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CBP (cAMP response element binding protein-binding protein) . In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, ACT (activator of CREM in testis), which confers a powerful, phosphorylation-independent activation capacity . In addition to specialized transcription factors and coactivators, a variety of general factors of the basal transcriptional machinery, and their distinct tissue-specific isoforms, are highly expressed in testis, supporting the general notion that testis-specific gene expression requires specialized mechanisms . Here, we describe that CREM interacts with transcription factor IIA (TFIIA), a general transcription factor that stimulates RNA polymerase II-directed transcription . This association was identified by a two-hybrid screen, using a testis-derived cDNA library, and confirmed by coimmunoprecipitation . The interaction is restricted to the activator isoforms of CREM and does not require Ser117 . Importantly, CREM does not interact with TFIIAtau-ALF, a testis-specific TFIIA homolog . CREM and TFIIA are expressed in a spatially and temporally coordinated fashion during the differentiation program of germ cells . The two proteins also colocalize intracellularly in spermatocyte and spermatid cells . These findings contribute to the understanding of the highly specialized rules of transcriptional regulation in haploid germ cells. Bioinformatics, 2003 Sep 22, 19(14), 1787 - 99 CLICK and EXPANDER: a system for clustering and visualizing gene expression data; Sharan R et al.; MOTIVATION: Microarrays have become a central tool in biological research . Their applications range from functional annotation to tissue classification and genetic network inference . A key step in the analysis of gene expression data is the identification of groups of genes that manifest similar expression patterns . This translates to the algorithmic problem of clustering genes based on their expression patterns . RESULTS: We present a novel clustering algorithm, called CLICK, and its applications to gene expression analysis . The algorithm utilizes graph-theoretic and statistical techniques to identify tight groups (kernels) of highly similar elements, which are likely to belong to the same true cluster . Several heuristic procedures are then used to expand the kernels into the full clusters . We report on the application of CLICK to a variety of gene expression data sets . In all those applications it outperformed extant algorithms according to several common figures of merit . We also point out that CLICK can be successfully used for the identification of common regulatory motifs in the upstream regions of co-regulated genes . Furthermore, we demonstrate how CLICK can be used to accurately classify tissue samples into disease types, based on their expression profiles . Finally, we present a new java-based graphical tool, called EXPANDER, for gene expression analysis and visualization, which incorporates CLICK and several other popular clustering algorithms . AVAILABILITY: http://www.cs.tau.ac.il/~rshamir/expander/expander.html J Nat Prod, 2003 Sep, 66(9), 1221 - 4 New bioactive diterpene polyesters from Euphorbia decipiens; Ahmad VU et al.; A reinvestigation with a modified extraction procedure of Euphorbia decipiens resulted in the isolation and structure elucidation of three new myrsinane-type diterpene esters (1-3) . The structures of compounds 1-3 were elucidated by spectroscopic data interpretation . Compound 1 showed inhibitory activity against prolyl endopeptidase (PEP), whereas compound 2 exhibited DNA-damaging activity in a mutant yeast bioassay. Nucleic Acids Res Suppl, 2003, (3), 305 - 6 Sequence specific transcription factor, JDP2 interacts with histone and inhibits p300-mediated histone acetylation; Pan J et al.; Jun dimerization protein 2 (JDP2) is a novel member of AP-1 family and acts as a general repressor of a variety of transcription . JDP2 is able to bind to specific sites in target gene such as c-jun by forming homodimer or heterodimers with a Jun/ATF family member to counteract their transcriptional activity . Previously we showed that JDP2 inhibits the retinoic acid (RA) dependent transcription by recruiting a histone deacetylase 3 (HDAC3) complex to the promoter region of the target genes . We present here that JDP2 has an inhibitory activity of acetylation of all core histones mediated by histone acetyltransferase (HAT) both of p300 and PCAF in vitro . The studies of both histone-binding and HAT-inhibitory activity using a variety of recombinant JDP2(s) indicated that JDP2 might target histone itself through the histone-binding domain of JDP2, which is essential but not sufficient for the inhibition of acetylation . Therefore, our data suggested that HAT-inhibitory activity of JDP2 could in part explain the transcriptional repression of several target genes. Curr Genet, 2003 Oct, 44(1), 19 - 25 Epub 2003 Jul 09. Repression of transcription by Rgt1 in the absence of glucose requires Std1 and Mth1; Lakshmanan J et al.; In the yeast Saccharomyces cerevisiae, glucose induces expression of the hexose transporter ( HXT) genes by inhibiting the repressor function of the transcription factor Rgt1 . We have previously shown that Rgt1 binds to the HXT gene promoters only in the absence of glucose . In the presence of glucose, Rgt1 becomes phosphorylated and is unable to bind to the HXT promoters and repress their transcription . We report that Rgt1 interacts with Std1 and Mth1 in a yeast two-hybrid assay and co-immunoprecipitates with both proteins in vivo only when glucose is absent . In addition, we demonstrate that repression of HXT gene expression by Rgt1 is abolished in the std1 mth1 double mutant . While Rgt1 is normally phosphorylated only in the presence of high concentrations of glucose, it is constitutively modified in the std1 mth1 double mutant . Based on these data, we conclude that, in the absence of glucose, Rgt1 associates with Std1 and Mth1 to repress HXT gene expression. Curr Genet, 2003 Dec, 44(4), 173 - 83 Epub 2003 Sep 24. Clustered-charge to alanine scanning mutagenesis of the Mal63 MAL-activator C-terminal regulatory domain; Danzi SE et al.; The MAL-activator genes of Saccharomyces cerevisiae encode regulatory proteins required for the expression of the structural genes encoding maltose permease and maltase . Residues within the C-terminal region of the Mal63 protein required for negative regulation were previously identified . Evidence suggested that the C-terminal domain is also involved in positive regulatory functions, such as inducer responsiveness and transactivation in the context of a full-length protein . Charged-cluster to alanine scanning mutagenesis of the regulatory domain of MAL63 and the constitutive MAL43-C were undertaken to identify distinct regions within Mal63p involved in positive functions and to define their roles in induction . Mutations that affect the ability to activate transcription in the inducible MAL63 but have no effect in the constitutive MAL43-C define regions that function in induction . Those that affect both the inducible and constitutive alleles define regions involved in activation more generally . Mutations in MAL63 fell into three classes, those that have little or no impact on activity, those that decrease activity, and those that enhance function . Mutations from these classes mapped to distinct regions of the protein, identifying a region of approximately 90 residues (residues 331-423) involved in maltose sensing and an approximately 50-residue region at the extreme C-terminus (residues 420-470) required for activation, such as the formation and/or maintenance of an active state . These studies support a model for MAL-activator function which involves complex protein-protein interactions and overlapping negative and positive regulatory regions. Biochim Biophys Acta, 2003 Sep 30, 1606(1-3), 153 - 62 A tomato alternative oxidase protein with altered regulatory properties; Holtzapffel RC et al.; We have investigated the expression and regulatory properties of the two alternative oxidase (Aox) proteins that are expressed in tomato (Lycopersicon esculentum L . Mill cv . Sweetie) after storage of green fruit at 4 degrees C . Four Aox genes were identified in the tomato genome, of which two (LeAox1a and LeAox1b) were demonstrated to be expressed in cold-treated fruit . The activity and regulatory properties of LeAox1a and LeAox1b were assayed after expression of each protein in yeast cells (Saccharomyces cerevisiae), proving that each is an active Aox protein . The LeAox1b protein was shown to have altered regulatory properties due to the substitution of a Ser for the highly conserved Cys(I) residue . LeAox1b could not form inactive disulfide-linked dimers and was activated by succinate instead of pyruvate . This is the first example of a dicot species expressing a natural Cys(I)/Ser isoform . The implications of the existence and expression of such Aox isoforms is discussed in the light of the hypothesised role for Aox in plant metabolism. J Biol Chem, 2003 Dec 5, 278(49), 48524 - 8 Epub 2003 Sep 23. Metazoan origin selection: origin recognition complex chromatin binding is regulated by CDC6 recruitment and ATP hydrolysis; Harvey KJ et al.; Using a plasmid competition assay, we have measured the stability of origin recognition complex (ORC) associated with sperm chromatin under physiological conditions . Under conditions in which pre-RCs are formed, both ORC and CDC6 dissociate from sperm chromatin with a relatively fast t(1/2) of 15 min . ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis . The t(1/2) for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present . Strikingly, the presence of a non-hydrolyzable ATP derivative, ATPgammaS, not only increases both ORC and CDC6 t(1/2) but also inhibits the loading of MCM . The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences. J Biol Chem, 2003 Dec 12, 278(50), 49868 - 73 Epub 2003 Sep 23. Methylthioadenosine phosphorylase regulates ornithine decarboxylase by production of downstream metabolites; Subhi AL et al.; The gene encoding methylthioadenosine phosphorylase (MTAP), the initial enzyme in the methionine salvage pathway, is deleted in a variety of human tumors and acts as a tumor suppressor gene in cell culture (Christopher, S . A., Diegelman, P., Porter, C . W., and Kruger, W . D . (2002) Cancer Res . 62, 6639-6644) . Overexpression of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is frequently observed in tumors and has been shown to be tumorigenic in vitro and in vivo . In this paper, we demonstrate a novel regulatory pathway in which the methionine salvage pathway products inhibit ODC activity . We show that in Saccharomyces cerevisiae the MEU1 gene encodes MTAP and that Meu1delta cells have an 8-fold increase in ODC activity, resulting in large elevations in polyamine pools . Mutations in putative salvage pathway genes downstream of MTAP also cause elevated ODC activity and elevated polyamines . The addition of the penultimate salvage pathway compound 4-methylthio-2-oxobutanoic acid represses ODC levels in both MTAP-deleted yeast and human tumor cell lines, indicating that 4-methylthio-2-oxobutanoic acid acts as a negative regulator of polyamine biosynthesis . Expression of MTAP in MTAP-deleted MCF-7 breast adenocarcinoma cells results in a significant reduction of ODC activity and reduction in polyamine levels . Taken together, our results show that products of the methionine salvage pathway regulate polyamine biosynthesis and suggest that MTAP deletion may lead to ODC activation in human tumors. J Biol Chem, 2003 Dec 5, 278(49), 48727 - 34 Epub 2003 Sep 23. DNA damage response-mediated degradation of Ho endonuclease via the ubiquitin system involves its nuclear export; Kaplun L et al.; Yeast mating switch Ho endonuclease is rapidly degraded by the ubiquitin system and this depends on the DNA damage response functions, MEC1, RAD9, and CHK1 . A PEST sequence marks Ho for degradation . Here we show that the novel F-box receptor, Ufo1, recruits phosphorylated Ho for degradation . Mutation of PEST residue threonine 225 stabilizes Ho, yet HoT225A still binds Ufo1 in vitro . Stable HoT225A accumulates within the nucleus, whereas HoT225E is degraded . Deletion of the nuclear exportin Msn5 traps native Ho in the nucleus and extends its half-life . These experiments suggest that Ho is degraded in the cytoplasm . In mec1 mutants stable Ho accumulates within the nucleus; Ho produced in mec1 cells does not bind Ufo1 . Thus the MEC1 pathway has functions both in phosphorylation of Thr-225 for nuclear export and in additional phosphorylations for binding Ufo1 . Cells with HO under its genomic promoter, but stabilized by deletion of the Msn5 exportin, proliferate, but are multibudded . These experiments elucidate some of the links between the DNA damage response and degradation of Ho by the ubiquitin system. J Exp Biol, 2003 Nov, 206(Pt 21), 3869 - 75 Trypsin-modulating oostatic factor: a potential new larvicide for mosquito control; Borovsky D; Trypsin-modulating oostatic factor (TMOF), a mosquito decapeptide, terminates trypsin biosynthesis in the mosquito gut . The hormone is secreted from the ovary, starting 18 h after the blood meal, circulates in the hemolymph, binds to a gut receptor and stops trypsin biosynthesis by exerting a translational control on trypsin mRNA . Because of the unique primary amino acid sequence of the hormone (YDPAPPPPPP) and its stable three-dimensional conformation, TMOF is not degraded by gut proteolytic enzymes and can traverse the gut epithelial cells into the hemolymph of adults and larvae . Using this unique property, hormone fed to different species of mosquito larvae stops food digestion and causes larval mortality . To determine the shortest amino acid sequence that can bind to the gut receptor and still cause high larval mortality, 25 analogues of TMOF were synthesized and tested . The tetrapeptide (YDPA) was as effective as the decapeptide, indicating that the binding to the gut receptor is at the N-terminus of the molecule . Cloning and expressing the hormone on the coat protein of tobacco mosaic virus (TMV) in Chlorella sp . and Saccharomyces cerevisiae cells and feeding the recombinant cells to mosquito larvae caused larval mortality . These results indicate that TMOF can be used as a new biorational insecticide against mosquito larvae. Insect Biochem Mol Biol, 2003 Oct, 33(10), 959 - 70 The C-terminus of the Hermes transposase contains a protein multimerization domain; Michel K et al.; Transposase activity that mediates the mobility of class II transposable elements, is most commonly initiated by the assembly of higher order synaptic complexes, called transpososomes . The formation of these complexes, that contain the transposable element's DNA as well as two or more molecules of the transposase, is dependent on interactions between transposase molecules . Using the yeast Two-Hybrid system, we were able to identify three regions mediating multimerization of the Hermes transposase, an element used for germline transformation of insects belonging to the hAT family of transposable elements . One region facilitating protein binding of Hermes transposase molecules was found within the first 252 amino acids of the transposase . The second region was located at the C-terminus of the transposase, and was found to be specific for Hermes transposase multimerization . Amino acids 551-569 were not only required for multimerization but were also necessary for transposition of the element . The third region was located between amino acids 253 and 380 and was found to eliminate the non-specific protein binding ability of the N-terminal protein interaction region but was required for the specific protein binding ability of the C-terminal region of the transposase . Five point mutations affecting the structural integrity of the C-terminal multimerization region abolished or significantly reduced transpositional activity . The same region had been previously identified to mediate dimerization in Activator (Ac), another hAT element, indicating that hAT transposase multimerization is likely to be a prerequisite for mobility of their elements. Cell, 2003 Sep 19, 114(6), 689 - 99 AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding; Strack B et al.; HIV-1 and other retroviruses exit infected cells by budding from the plasma membrane, a process requiring membrane fission . The primary late assembly (L) domain in the p6 region of HIV-1 Gag mediates the detachment of the virion by recruiting host Tsg101, a component of the class E vacuolar protein sorting (Vps) machinery . We now show that HIV Gag p6 contains a second region involved in L domain function that binds AIP1, a homolog of the yeast class E Vps protein Bro1 . Further, AIP1 interacts with Tsg101 and homologs of a subunit of the yeast class E Vps protein complex ESCRT-III . AIP1 also binds to the L domain in EIAV p9, and this binding correlates perfectly with L domain function . These observations identify AIP1 as a component of the viral budding machinery, which serves to link a distinct region in the L domain of HIV-1 p6 and EIAV p9 to ESCRT-III. Bioprocess Biosyst Eng, 2002 Jun, 25(2), 69 - 78 Epub 2002 Apr 16. Effect of experimental error on the efficiency of different optimization methods for bioprocess media optimization; Milavec P et al.; Four optimization methods (Simplex, Rosenbrock, iterative factorial experimental design (IFED) and genetic algorithms) for the optimization of the biotechnological media composition under conditions where the measured quantities are subjected to the experimental error were compared . The computer simulations were performed on some of the selected two- to six- parameter biotechnological models . The optimization process was modified in such a way that the experimental error was considered . The results show that the optimization efficiency increases when this new termination criteria is implemented . In addition, the method efficiency becomes independent of the experimental error . In general, Simplex and Rosenbrock methods need fewer experiments and their distribution of necessary experiments is narrower than for IFED and genetic algorithms . The increase of model parameters that need to be optimized results in a decrease in the method efficiency and in an increase of the average number of required experiments . The results were further verified on the cultivation of Saccharomyces cerevisiae and were found to be in good agreement with the results obtained from the computer simulations. J Biol Chem, 2003 Dec 5, 278(49), 48563 - 9 Epub 2003 Sep 22. CYP17 mutation E305G causes isolated 17,20-lyase deficiency by selectively altering substrate binding; Sherbet DP et al.; Cytochrome p450c17 (CYP17) converts the C21 steroids pregnenolone and progesterone to the C19 androgen precursors dehydroepiandrosterone (DHEA) and androstenedione, respectively, via sequential 17alpha-hydroxylase and 17,20-lyase reactions . Disabling mutations in CYP17 cause combined 17alpha-hydroxylase/17,20-lyase deficiency, but rare missense mutations cause isolated loss of 17,20-lyase activity by disrupting interactions of redox partner proteins with CYP17 . We studied an adolescent male with clinical and biochemical features of isolated 17,20-lyase deficiency, including micropenis, hypospadias, and gynecomastia, who is homozygous for CYP17 mutation E305G, which lies in the active site . When expressed in HEK-293 cells or Saccharomyces cerevisiae, mutation E305G retains 17alpha-hydroxylase activities, converting pregnenolone and progesterone to 17alpha-hydroxysteroids . However, mutation E305G lacks 17,20-lyase activity for the conversion of 17alpha-hydroxypregnenolone to DHEA, which is the dominant pathway to C19 steroids catalyzed by human CYP17 (the delta5-steroid pathway) . In contrast, mutation E305G exhibits 11-fold greater catalytic efficiency (kcat/Km) for the cleavage of 17alpha-hydroxyprogesterone to androstenedione compared with wild-type CYP17 . We conclude that mutation E305G selectively impairs 17,20-lyase activity for DHEA synthesis despite an increased capacity to form androstenedione . Mutation E305G provides genetic evidence that androstenedione formation from 17alpha-hydroxyprogesterone via the minor delta4-steroid pathway alone is not sufficient for complete formation of the male phenotype in humans. J Biol Chem, 2003 Nov 28, 278(48), 48445 - 52 Epub 2003 Sep 22. Atg23 is essential for the cytoplasm to vacuole targeting pathway and efficient autophagy but not pexophagy; Tucker KA et al.; Cells must regulate both biosynthesis and degradation to ensure proper homeostasis of cellular organelles and proteins . This balance is demonstrated in a unique way in the yeast Saccharomyces cerevisiae, which possesses two distinct, yet mechanistically related trafficking routes mediating the delivery of proteins from the cytoplasm to the vacuole: the biosynthetic cytoplasm to vacuole targeting (Cvt) and the degradative autophagy pathways . Several components employed by these two transport routes have been identified, but their mechanistic interactions remain largely unknown . Here we report a novel gene involved in these pathways, which we have named ATG23 . Atg23 localizes to the pre-auto-phagosomal structure but also to other cytosolic punctate compartments . Our characterization of the Atg23 protein indicates that it is required for the Cvt pathway and efficient autophagy but not pexophagy . In the absence of Atg23, cargo molecules such as prApe1 are correctly recruited to a pre-autophagosomal structure that is unable to give rise to Cvt vesicles . We also demonstrate that Atg23 is a peripheral membrane protein that requires the presence of Atg9/Apg9 to be specifically targeted to lipid bilayers . Atg9 transiently interacts with Atg23 suggesting that it participates in the recruitment of this protein. Eur J Drug Metab Pharmacokinet, 2003 Jan-Mar, 28(1), 59 - 65 In vitro metabolism of perospirone in rat, monkey and human liver microsomes; Mizuno Y et al.; In vitro metabolism of perospirone was examined with rat, monkey and human liver S9, human liver microsomes and yeast microsomes expressing human P450, using 14C labeled perospirone . With rat liver S9, the major metabolites were MX9 and ID-11614, produced by cleavage at the butylene chain . However, some butylene non-cleavage and hydration of the cyclohexane ring were found, although limited in extent . Unknown metabolites accounted for about 10% of the total . After incubation for 10 minutes with monkey liver S9, the major metabolites were ID-15036 and MX11, hydrated in the cyclohexane ring . After incubation for 60 minutes, ID-15001, i.e . the butylene chain cleavage type increased . Unknown metabolites accounted for about 20% . After incubation for 10 minutes with human liver S9, the major metabolite was ID-15036, hydrated in the cyclohexane ring . In addition, MX11 and many unknown metabolites were evident . After incubation for 60 minutes, the butylene chain cleavage type and unknown metabolites increased . Individual differences were found in the metabolic reaction rate . With human liver microsomes . MX11, ID-15001 and unknown metabolites were again the major metabolites . With yeast microsomes expressing human P450 subtypes, CYP1A1, 2C8, 2D6, 3A4 were responsible for the metabolism in particular, and CYP3A4 contributes greatly . Therefore it is unlikely that genetic polymorphism will arise a present a problem with regard to the clinical drug . The results demonstrated that the main metabolic pathway in human liver S9 and liver microsomes involve oxidation at cyclohexane, oxidative cleavage of the butylene side chain and S-oxidation . The same was the case in rat and monkey S9, but species differences were found in the proportions of the metabolites produced. Nat Cell Biol, 2003 Oct, 5(10), 921 - 7 Epub 2003 Sep 21. The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila; Pantalacci S et al.; Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously . The salvador (sav) gene, which encodes a scaffold protein, has been shown to restrict cell number by coordinating cell-cycle exit and apoptosis during Drosophila development . Here we identify Hippo (Hpo), the Drosophila orthologue of the mammalian MST1 and MST2 serine/threonine kinases, as a partner of Sav . Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype . Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1, overexpression of Hpo destabilizes DIAP1 in cell culture . We show that DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and that Hpo can phosphorylate DIAP1 in vitro . Thus, Hpo may promote apoptosis by reducing cellular amounts of DIAP1 . In addition, we show that Sav is an unstable protein that is stabilized by Hpo . We propose that Hpo and Sav function together to restrict tissue growth in vivo. Nat Cell Biol, 2003 Oct, 5(10), 914 - 20 Epub 2003 Sep 21. Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway; Udan RS et al.; Proliferation and apoptosis must be precisely regulated to form organs with appropriate cell numbers and to avoid tumour growth . Here we show that Hippo (Hpo), the Drosophila homologue of the mammalian Ste20-like kinases, MST1/2, promotes proper termination of cell proliferation and stimulates apoptosis during development . hpo mutant tissues are larger than normal because mutant cells continue to