J Biol Chem, 2000 Sep 1, 275(35), 26925 - 34 The TATA-binding protein-associated factor yTafII19p functionally interacts with components of the global transcriptional regulator Ccr4-Not complex and physically interacts with the Not5 subunit; Lemaire M et al.; The Saccharomyces cerevisiae HIS3 gene is a model system to characterize transcription initiation from different types of core promoters . The NOT genes were identified by mutations that preferentially increased transcription of the HIS3 promoter lacking a canonical TATA sequence . They encode proteins associated in a complex that also contains the Caf1 and Ccr4 proteins . It has been suggested that the Ccr4-Not complex represses transcription by inhibiting factors more specifically required for promoters lacking a TATA sequence . A potential target is the yTaf(II)19 subunit of TFIID, which, when depleted, leads to a preferential decrease of HIS3 TATA-less transcription . We isolated conditional taf19 alleles that display synthetic growth phenotypes when combined with not4 or specific not5 alleles . Inactivation of yTaf(II)19p by shifting these mutants to the restrictive temperature led to a more rapid and striking decrease in transcription from promoters that do not contain a canonical TATA sequence . We demonstrated by the two-hybrid assay and directly in vitro that yTaf(II)19p and Not5p could interact . Finally, we found by the two-hybrid assay that yTaf(II)19p also interacted with many components of the Ccr4-Not complex . Taken together, our results provide evidence that interactions between Not5p and yTaf(II)19p may be involved in transcriptional regulation by the Ccr4-Not complex. Circ Res, 2000 Jun 23, 86(12), E110 - 7 Nuclear factor-kappaB and cAMP response element binding protein mediate opposite transcriptional effects on the Flk-1/KDR gene promoter; Illi B et al.; -The vascular endothelial growth factor receptor Flk-1/KDR is highly expressed during development and almost disappears in adult tissues . Despite its biological relevance, little is known about the molecular mechanisms controlling its expression . In the present work, it is shown that cAMP response element binding protein (CREB) and nuclear factor-kappaB (NF-kappaB)-related antigens bind specific sequences in the Flk-1/KDR promoter . Functional studies demonstrate that cAMP represses whereas tumor necrosis factor-alpha, an activator of NF-kappaB, stimulates promoter activity . Histone acetyltransferases (HATs) P/CAF and CBP/p300 together with p65/RelA, the catalytic subunit of NF-kappaB, increase Flk-1/KDR promoter activity 10- to 20-fold . Consistently, inhibition by cAMP is reverted by increasing intracellular HATs and is completely abolished by site-specific mutagenesis of the cAMP response element . In contrast, specific mutations in the NF-kappaB response element abolish responsiveness to p65/RelA and HATs without affecting cAMP-dependent repression . These results suggest that opposing signaling pathways, activating NF-kappaB or CREB and requiring HAT molecules, control Flk-1/KDR promoter activity. J Mol Biol, 2000 Jun 30, 300(1), 11 - 6 A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase; Morgan CJ et al.; The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy . In the absence of denaturant, the hydrodynamic radius of 29.6(+/-0.25) A and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein . On the addition of 2 . 2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(+/-0.43) A, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state . In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(+/-0.25) A, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23 . 5 A) . The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species . Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent . Such a species could be important in the formation of dimeric TIM from its unfolded state . Nature, 2000 Jun 8, 405(6787), 701 - 4 Redundant roles for the TFIID and SAGA complexes in global transcription; Lee TI et al.; The transcription factors TFIID and SAGA are multi-subunit complexes involved in transcription by RNA polymerase II . TFIID and SAGA contain common TATA-binding protein (TBP)-associated factor (TAF(II)) subunits and each complex contains a subunit with histone acetyltransferase activity . These observations have raised questions about whether the functions of the two complexes in vivo are unique or overlapping . Here we use genome-wide expression analysis to investigate how expression of the yeast genome depends on both shared and unique subunits of these two complexes . We find that expression of most genes requires one or more of the common TAF(II) subunits, indicating that the functions of TFIID and SAGA are widely required for gene expression . Among the subunits shared by TFIID and SAGA are three histone-like TAF(II)s, which have been proposed to form a sub-complex and mediate a common function in global transcription . Unexpectedly, we find that the histone-like TAF(II)s have distinct roles in expression of the yeast genome . Most importantly, we show that the histone acetylase components of TFIID and SAGA (TAF(II)145 and Gcn5) are functionally redundant, indicating that expression of a large fraction of yeast genes can be regulated through the action of either complex. J Pept Res, 2000 May, 55(5), 398 - 408 Conformational characterization of a helix-nucleated bicyclic GCN4 decapeptide by proton NMR; Zhang M et al.; A bicyclic decapeptide, GCN4brM1, which was designed to be a helix-locked analog of the DNA-binding basic region from the yeast transcription factor GCN4, was synthesized and characterized using circular dichroism (CD) spectropolarimetry and 1H-NMR . This peptide has two Lys(i), Asp(i+4) side chain lactam bridges incorporated into its structure in overlapping positions in the peptide chain, linking residues 3 and 7 and residues 4 and 8 . CD spectra of GCN4brM1 in aqueous solution are consistent with the expected helical conformation, and indicate that this conformation is remarkably resistant to heat denaturation and is essentially unchanged by addition of 50% (v/v) trifluoroethanol (TFE) as cosolvent . NMR spectra measured in aqueous solution at -5 degrees C show long-range nuclear Overhauser effects (NOEs) that are consistent with an alpha-helical conformation throughout the peptide structure . The measured 3J(HN) coupling constants are also in agreement with an alpha-helical structure . Extremely slow proton-deuterium exchange rates measured for backbone amides in the middle of the peptide indicate that this helix is highly stabilized and rarely unfolds within the side chain bridged sequence . NOE-constrained molecular dynamics simulations gave rise to a single family of converged structures that are fully alpha-helical throughout the GCN4brM1 backbone, and show a single, well-defined conformation for the two side chain bridges . This study demonstrates that two overlapping Lys(i), Asp(i+4) lactam bridges, positioned in consecutive residue positions in a hexapeptide segment, form a rigid alpha-helical structure in aqueous solution that is propagated in both the N-terminal and C-terminal directions. J Cell Sci, 2000 Jul, 113 ( Pt 14), 2631 - 8 Sustained elevation in inositol 1,4,5-trisphosphate results in inhibition of phosphatidylinositol transfer protein activity and chronic depletion of the agonist-sensitive phosphoinositide pool; Speed CJ et al.; The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) in a signal-terminating reaction . We have utilised cell lines that stably underexpress the 43 kDa 5-phosphatase, as a model system to investigate whether Ins(1,4,5)P(3) can control the rate of its own formation by regulating the resupply of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) . A sustained 2.6-fold elevation in the basal concentration of Ins(1,4,5)P(3), in cell lines underexpressing the 43 kDa 5-phosphatase, correlated with a 32% reduction in the total cellular mass of PtdIns(4,5)P(2) . The depletion in cellular PtdIns(4,5)P(2) was confined to a Triton-insoluble cell compartment, enriched in caveolin . In resting cells with elevated Ins(1,4,5)P(3) concentrations resulting from underexpression of the 43 kDa 5-phosphatase, phosphatidylinositol (PtdIns) and phosphatidylinositol 4-phosphate (PtdIns(4)P) were depleted by 50% and PtdIns(4,5)P(2) by 61% in the caveolin-enriched Triton-insoluble compartment . Agonist stimulation resulted in the rapid turnover of phosphoinositides in the caveolin-enriched Triton-insoluble fraction of vector-transfected cells, but not in cells with high basal Ins(1,4,5)P(3) concentrations . Depletion of phosphoinositides from the caveolin-enriched Triton-insoluble pool in cells underexpressing the 43 kDa 5-phosphatase did not result from activation of phospholipase C isoenzymes, or inhibition of PtdIns 4-kinase or PtdIns(4)P 5-kinase activities . Significant inhibition of phosphatidylinositol transfer protein (PITP) activity (up to 70%) was observed in cells with elevated basal Ins(1,4,5)P(3) concentrations; however, no reduction in PITP(&agr;) protein expression was detected . These studies indicate that chronic elevation in cellular Ins(1,4,5)P(3) concentrations decreases the PITP-mediated resupply of phosphoinositides in the caveolin-enriched agonist-sensitive pool. Enzyme Microb Technol, 2000 Jun 1, 26(9-10), 715 - 723 Improved protocols for quantitative determination of metabolites from biological samples using high performance ionic-exchange chromatography with conductimetric and pulsed amperometric detection; Groussac E et al.; Simple and reliable protocols are described for an extensive analysis of metabolites in extracts from different biological sources . The separation was performed by high performance ionic-exchange chromatography (HPIC) at alkaline pH using two types of chromatography columns and two detection methods . Organic acids and inorganic anions were separated on an ionPac AS11 column using a 0.5 to 35 mM Na0H gradient . Detection limits in the range of milligrams per liter were achieved by use of a conductivity detector equipped with an anion self-regenerating suppressor . Twelve phosphorylated compounds belonging to the glycolytic and the pentose phosphate pathways could be resolved on a CarboPac PA1 column using a Na0H/Na-acetate gradient . Quantification was achieved by pulsed amperometry with detection limits in the micromolar range . Cell extracts obtained by extraction in boiling buffered ethanol described previously could be directly injected onto HPIC columns for the separation of metabolites because the extraction procedure affected neither the retention time nor the stability of most of the metabolites, and yielded very clean chromatograms . These improved protocols were applied for a dynamic analysis of intracellular metabolites in Saccharomyces cerevisiae in response to a glucose pulse. J Clin Invest, 2000 Jun, 105(12), 1819 - 25 Altered expression of fatty acid-metabolizing enzymes in aromatase-deficient mice; Nemoto Y et al.; Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen . In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production . These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism . To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid beta-oxidation . Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid beta-oxidation . In vitro assays of fatty acid beta-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished . Impaired gene expression and enzyme activities of fatty acid beta-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17beta-estradiol . Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element . These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid beta-oxidation and in maintaining hepatic lipid homeostasis. J Clin Invest, 2000 Jun, 105(12), 1711 - 21 Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase; Hallows KR et al.; The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl(-) channel that regulates other epithelial transport proteins by uncharacterized mechanisms . We employed a yeast two-hybrid screen using the COOH-terminal 70 residues of CFTR to identify proteins that might be involved in such interactions . The alpha1 (catalytic) subunit of AMP-activated protein kinase (AMPK) was identified as a dominant and novel interacting protein . The interaction is mediated by residues 1420-1457 in CFTR and by the COOH-terminal regulatory domain of alpha1-AMPK . Mutations of two protein trafficking motifs within the 38-amino acid region in CFTR each disrupted the interaction . GST-fusion protein pull-down assays in vitro and in transfected cells confirmed the CFTR-alpha1-AMPK interaction and also identified alpha2-AMPK as an interactor with CFTR . AMPK is coexpressed in CFTR-expressing cell lines and shares an apical distribution with CFTR in rat nasal epithelium . AMPK phosphorylated full-length CFTR in vitro, and AMPK coexpression with CFTR in XENOPUS: oocytes inhibited cAMP-activated CFTR whole-cell Cl(-) conductance by approximately 35-50% . Because AMPK is a metabolic sensor in cells and responds to changes in cellular ATP, regulation of CFTR by AMPK may be important in inhibiting CFTR under conditions of metabolic stress, thereby linking transepithelial transport to cell metabolic state. J Cell Biochem, 2000 Jun 12, 78(4), 533 - 40 Expression of replication factor C 40-kDa subunit is down-regulated during neonatal development in rat ventricular myocardium; Chim SS et al.; During neonatal development, cardiac myocytes undergo a transition from hyperplastic to hypertrophic growth . Whether these cells are terminally differentiated and permanently withdrawn from the cell cycle shortly after birth is controversial . Nevertheless, the clinical observation that functionally significant myocardial regeneration has not been documented in cardiovascular disease or injury during adulthood seems to support the notion that the vast majority of cardiac myocytes do not proliferate once they differentiate . Regardless of the controversy, the elucidation on how mitosis is blocked in cardiac myocytes may facilitate development of new cardiovascular therapies, based on the regeneration of the adult myocardium . To better understand postnatal myocardial development, we performed suppression subtractive hybridization to isolate genes that are differentially expressed in day one or day seven postnatal rat ventricular myocardium . Here we report the down-regulated mRNA expression of the 40-kDa subunit of replication factor C (RFC p40 or RFC2), which is an essential processive factor for proliferating cellular nuclear antigen-dependent DNA replication during neonatal myocardial development . Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7342 - 7 Isolation and characterization of BEN, a member of the TFII-I family of DNA-binding proteins containing distinct helix-loop-helix domains; Bayarsaihan D et al.; The transcriptional regulation of the Hoxc8 gene is controlled during early mouse embryogenesis by an enhanceosome-like control region, termed the early enhancer (EE), located 3 kb upstream from the Hoxc8 translation start site . The EE is involved in establishing the posterior expression pattern of Hoxc8 at embryonic day (E) 8.5-9 . 0 . Genetic and biochemical data have shown that nuclear factors interact with this region in a sequence-specific manner . We have used a yeast one-hybrid screen in a search for transcription factors that bind to EE motifs and have isolated a novel murine DNA-binding protein, termed BEN (binding factor for early enhancer) . The ORF of BEN encodes a protein of 1072 amino acids and contains six helix-loop-helix domains, a hydrophobic leucine zipper-like motif, and a serine-rich repeat . The murine BEN gene is structurally similar to the human gene TFII-I in that both genes encode unique 95-amino acid long helix-loop/span-helix domains . The BEN gene produces several major transcripts (3.6, 4.4, and 5.9 kb) present in most adult tissues and shows discrete spatial and temporal domains of expression in areas of epithelial-mesenchymal interaction during mouse embryogenesis from E9.5 to E12.5 . Several BEN-encoded polypeptides of different sizes ranging from 165 to 40 kDa were identified by Western blot analysis using BEN-specific polyclonal Abs . We propose, on the bases of sequence homology, that BEN is the mouse ortholog of the recently described human gene, WBSCR11, known also as GTF2IRD1, GTF3, Cream1, and MusTRD1 . This gene is deleted hemizygously in individuals with Williams Syndrome, an autosomal dominant genetic condition characterized by complex physical, cognitive, and behavioral traits resulting from a perturbed developmental process. Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7841 - 6 Hsp70 and hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils; Muchowski PJ et al.; The deposition of protein aggregates in neurons is a hallmark of neurodegenerative diseases caused by polyglutamine (polyQ) proteins . We analyzed the effects of the heat shock protein (Hsp) 70 chaperone system on the aggregation of fragments of huntingtin (htt) with expanded polyQ tracts . In vitro, Hsp70 and its cochaperone Hsp40 suppressed the assembly of htt into detergent-insoluble amyloid-like fibrils in an ATP-dependent manner and caused the formation of amorphous, detergent-soluble aggregates . The chaperones were most active in preventing fibrillization when added during the lag phase of the polymerization reaction . Similarly, coexpression of Hsp70 or Hsp40 with htt in yeast inhibited the formation of large, detergent-insoluble polyQ aggregates, resulting in the accumulation of detergent-soluble inclusions . Thus, the recently established potency of Hsp70 and Hsp40 to repress polyQ-induced neurodegeneration may be based on the ability of these chaperones to shield toxic forms of polyQ proteins and to direct them into nontoxic aggregates. Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7859 - 64 Molecular cloning and characterization of a lipid phosphohydrolase that degrades sphingosine-1- phosphate and induces cell death; Mandala SM et al.; Sphingosine and sphingosine-1-phosphate (SPP) are interconvertible sphingolipid metabolites with opposing effects on cell growth and apoptosis . Based on sequence homology with LBP1, a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast, we report here the cloning, identification, and characterization of a mammalian SPP phosphatase (mSPP1) . This hydrophobic enzyme, which contains the type 2 lipid phosphohydrolase conserved sequence motif, shows substrate specificity for SPP . Partially purified Myc-tagged mSPP1 was also highly active at dephosphorylating SPP . When expressed in yeast, mSPP1 can partially substitute for the function of LBP1 . Membrane fractions from human embryonic kidney HEK293 cells transfected with mSPP1 markedly degraded SPP but not lysophosphatidic acid, phosphatidic acid, or ceramide-1-phosphate . Enforced expression of mSPP1 in NIH 3T3 fibroblasts not only decreased SPP and enhanced ceramide levels, it also markedly diminished survival and induced the characteristic traits of apoptosis . Collectively, our results suggest that SPP phosphohydrolase may regulate the dynamic balance between sphingolipid metabolite levels in mammalian cells and consequently influence cell fate. Hum Mutat, 2000 Jun, 15(6), 577 - 8 Identification of four single nucleotide polymorphisms in DNA repair genes: XPA and XPB (ERCC3) in Polish population; Butkiewicz D et al.; A deficiency in DNA repair is associated with increased cancer risk . Inter-individual variations in DNA repair capacity observed in humans may result from genetic polymorphisms in DNA repair genes . In order to provide a basis for future functional and molecular epidemiology studies on cancer susceptibility, we screened 35 individuals for polymorphisms in coding regions of XPA and XPB genes involved in nucleotide excision repair (NER) . Relevant cDNA sequences were amplified by PCR, sequenced with fluorescently labeled terminators and analyzed with automated sequencer . Two polymorphisms in XPB were found: AAA-->AGA (445A>G; GenBank M31899) causing K117R substitution and GGC-->TGC (1299G>T; GenBank M31899) causing G402C exchange . Also, two polymorphisms in XPB were detected: CGA-->CAA (709G>A; GenBank D14533) causing R228Q exchange, and A-->G (23A>G; GenBank D14533) substitution in the 5' non-coding region of the gene . The three aforementioned amino acid substitutions were uncommon in this population (1.4%) . In contrast, the substitution located 4 nucleotides upstream of the ATG start codon of XPB was frequent (57%) . To our best knowledge this is the first report of these sequence variants . The location of these polymorphisms in evolutionary conserved regions suggest that they may be of functional significance . Genes Dev, 2000 Jun 15, 14(12), 1528 - 40 Xenopus cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading; Jares P et al.; The assembly and disassembly of protein complexes at replication origins play a crucial role in the regulation of chromosomal DNA replication . The sequential binding of the origin recognition complex (ORC), Cdc6, and the minichromosome maintenance (MCM/P1) proteins produces a licensed replication origin . Before the initiation of replication can occur, each licensed origin must be acted upon by S phase-inducing CDKs and the Cdc7 protein kinase . In the present report we describe the role of Xenopus Cdc7 (XCdc7) in DNA replication using cell-free extracts of Xenopus eggs . We show that XCdc7 binds to chromatin during G(1) and S phase . XCdc7 associates with chromatin only once origins have been licensed, but this association does not require the continued presence of XORC or XCdc6 once they have fulfilled their essential role in licensing . Moreover, XCdc7 is required for the subsequent CDK-dependent loading of XCdc45 but is not required for the destabilization of origins that occurs once licensing is complete . Finally, we show that CDK activity is not necessary for XCdc7 to associate with chromatin, induce MCM/P1 phosphorylation, or perform its essential replicative function . From these results we suggest a simple model for the assembly of functional initiation complexes in the Xenopus system. Trends Cell Biol, 2000 Jul, 10(7), 281 - 9 Drivers and passengers wanted! the role of kinesin-associated proteins; Manning BD et al.; Members of the kinesin superfamily of proteins participate in a wide variety of cellular processes . Although much attention has been devoted to the structural and biophysical properties of the force-generating motor domain of kinesins, the factors controlling the functional specificity of each kinesin have only recently been examined . Genetic and biochemical approaches have identified two classes of proteins that associate physically with the diverse non-motor domains of kinesins . These proteins can be divided into two general classes: first, those that form tight complexes with the kinesin and are instrumental in directing the distinct function of the motor (i.e . drivers) and, second, those proteins that might transiently interact with the motor or be an integral part of the motor's cargo (i.e . passengers) . Here, we discuss known kinesin-binding proteins, and how they might participate in the activity of their motor partners. EMBO J, 2000 Jun 15, 19(12), 3060 - 8 Overlapping roles for the histone acetyltransferase activities of SAGA and elongator in vivo; Wittschieben BO et al.; Elp3 and Gcn5 are histone acetyltransferases (HATs) that function in transcription as subunits of Elongator and SAGA/ADA, respectively . Here we show that mutations that impair the in vitro HAT activity of Elp3 confer typical elp phenotypes such as temperature sensitivity . Combining an elp3Delta mutation with histone H3 or H4 tail mutations confers lethality or sickness, supporting a role for Elongator in chromatin remodelling in vivo . gcn5Deltaelp3Delta double mutants display a number of severe phenotypes, and similar phenotypes result from combining the elp mutation with mutation in a gene encoding a SAGA-specific, but not an ADA-specific subunit, indicating that Elongator functionally overlaps with SAGA . Because concomitant active site alterations in Elp3 and Gcn5 are sufficient to confer severe phenotypes, the redundancy must be specifically related to the HAT activity of these complexes . In support of this conclusion, gcn5Deltaelp3Delta phenotypes are suppressed by concomitant mutation of the HDA1 and HOS2 histone deacetylases . Our results demonstrate functional redundancy among transcription-associated HAT and deacetylase activities, and indicate the importance of a fine-tuned acetylation-deacetylation balance during transcription in vivo. EMBO J, 2000 Jun 15, 19(12), 2834 - 44 Increased protein kinase or decreased PP2A activity bypasses sphingoid base requirement in endocytosis; Friant S et al.; Lipids have been implicated in signal transduction and in several stages of membrane trafficking, but these two functions have not been functionally linked . In yeast, sphingoid base synthesis is required for the internalization step of endocytosis and organization of the actin cytoskeleton . We show that inactivation of a protein phosphatase 2A (PP2A) or overexpression of one of two kinases, Yck2p or Pkc1p, can specifically suppress the sphingoid base synthesis requirement for endocytosis . The two kinases have an overlapping function because only a mutant with impaired function of both kinases is defective in endocytosis . An ultimate target of sphingoid base synthesis may be the actin cytoskeleton, because overexpression of the kinases and inactivation of PP2A substantially corrected the actin defect due to the absence of sphingoid base . These results suggest that sphingoid base controls protein phosphorylation, perhaps by activating a signal transduction pathway that is required for endocytosis and proper actin cytoskeleton organization in yeast. Biochim Biophys Acta, 2000 Jun 26, 1486(1), 97 - 107 Interorganelle transport of aminoglycerophospholipids; Voelker DR; The aminoglycerophospholipids of eukaryotic cells, phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho), can be synthesized by multiple pathways . The PtdSer pathway encompasses the synthesis of PtdSer, its decarboxylation to PtdEtn and subsequent methylation reactions to form PtdCho . The Kennedy pathways consist of the synthesis of PtdEtn and PtdCho from Etn and Cho precursors via CDP-Etn and CDP-Cho intermediates . The reactions along the PtdSer pathway are spatially segregated with PtdSer synthesis occurring in the endoplasmic reticulum or mitochondria-associated membrane (MAM), PtdEtn formation occurring in the mitochondria and Golgi/vacuole compartments and PtdCho formation occurring in the endoplasmic reticulum or MAM . The organelle-specific metabolism of the different lipids in the PtdSer pathway has provided a convenient biochemical means for defining events in the interorganelle transport of the aminoglycerophospholipids in intact cells, isolated organelles and permeabilized cells . Studies with both mammalian cells and yeast demonstrate many significant similarities in lipid transport processes between the two systems . Genetic experiments in yeast now provide the tools to create new strains with mutations along the PtdSer pathway that can be conditionally rescued by the Kennedy pathway reactions . The genetic studies in yeast indicate that it is now possible to begin to define genes that participate in the interorganelle transport of the aminoglycerophospholipids. Biochim Biophys Acta, 2000 Jun 26, 1486(1), 18 - 27 Transport of fatty acids and metabolites across the peroxisomal membrane; Hettema EH et al.; The peroxisomal membrane forms a permeability barrier for a wide variety of metabolites required for and formed during fatty acid beta-oxidation . To communicate with the cytoplasm and mitochondria, peroxisomes need dedicated proteins to transport such hydrophilic molecules across their membranes . Genetic and biochemical studies in the yeast Saccharomyces cerevisiae have identified enzymes for redox shuttles as well as the first peroxisomal membrane transporter . This peroxisomal ATP-binding cassette transporter (Pat) is highly homologous to the gene mutated in X-linked adrenoleukodystrophy (X-ALD) . The yeast Pat is required for import of activated fatty acids into peroxisomes suggesting that this is the primary defect in X-ALD. Biochim Biophys Acta, 2000 Jun 26, 1486(1), 1 - 17 Fatty acid import into mitochondria; Kerner J et al.; The mitochondrial carnitine system plays an obligatory role in beta-oxidation of long-chain fatty acids by catalyzing their transport into the mitochondrial matrix . This transport system consists of the malonyl-CoA sensitive carnitine palmitoyltransferase I (CPT-I) localized in the mitochondrial outer membrane, the carnitine:acylcarnitine translocase, an integral inner membrane protein, and carnitine palmitoyltransferase II localized on the matrix side of the inner membrane . Carnitine palmitoyltransferase I is subject to regulation at the transcriptional level and to acute control by malonyl-CoA . The N-terminal domain of CPT-I is essential for malonyl-CoA inhibition . In liver CPT-I activity is also regulated by changes in the enzyme's sensitivity to malonyl-CoA . As fluctuations in tissue malonyl-CoA content are parallel with changes in acetyl-CoA carboxylase activity, which in turn is under the control of 5'-AMP-activated protein kinase, the CPT-I/malonyl-CoA system is part of a fuel sensing gauge, turning off and on fatty acid oxidation depending on the tissue's energy demand . Additional mechanism(s) of short-term control of CPT-I activity are emerging . One proposed mechanism involves phosphorylation/dephosphorylation dependent direct interaction of cytoskeletal components with the mitochondrial outer membrane or CPT-I . We have proposed that contact sites between the outer and inner mitochondrial membranes form a microenvironment which facilitates the carnitine transport system . In addition, this system includes the long-chain acyl-CoA synthetase and porin as components. Endocrine, 2000 Feb, 12(1), 69 - 76 Isolation and characterization of the androgen receptor mutants with divergent transcriptional activity in response to hydroxyflutamide; Wang C et al.; A yeast genetic screening was developed to isolate androgen receptor (AR) mutants with divergent transactivation characteristics in response to hydroxyflutamide (HF), an active metabolite of flutamide used for prostate cancer treatment . Two mutants carrying the substitution C685Y or E708K were isolated and characterized . Substitution of C685Y for wild-type AR (wtAR) rendered the receptor supersensitive to androgenic activity from HF and female hormones such as 17beta-estradiol (E2) and progesterone (P) . Similar effects were observed in the AR mutant, named T876AAR, isolated from LNCaP cells . Surprisingly, we found that C685YAR7, but not T876AAR7, could be activated by casodex (bicalutamide), a nonsteroidal pure antiandrogen, with an induction fold 3- to 5-fold times higher than that for wild type or T876AAR . By contrast, although replacement of E708K for wtAR showed little effect on dihydrotestosterone-mediated transactivation, E708KAR lost its transcriptional response from many other ligands . The effects of ligands on E708KAR could be controlled at the DNA-binding level owing to the finding of a significant decrease in the DNA-binding ability once E708KAR was bound to HF, E2, or P . Together, these results suggest that C685YAR can be a novel tool for assaying the androgenic activity from antiandrogens, and the mechanism revealed from E708KAR could provide a possible explanation for the partial androgen insensitivity syndrome in men with a natural E708KAR mutation. Chromosoma, 2000, 109(1-2), 123 - 32 Localisation of RAD50 and MRE11 in spermatocyte nuclei of mouse and rat; Eijpe M et al.; Synaptonemal complexes (SCs) are zipperlike structures that are assembled between homologous chromosomes during meiotic prophase . They consist of two axial elements (AEs) (one along each of the two homologous chromosomes), which, in mature SCs, are connected by numerous transverse filaments along their length . Several proteins involved in the later steps of meiotic recombination most probably function in close association with the AEs of SCs, because the proteins involved in these steps have all been localised along AEs or SCs by immunocytochemical methods . It is not known at which step in meiotic recombination this association with the AEs is established . In order to shed some light on this issue, we analysed the localisation of two proteins that are involved in early steps of meiotic recombination, RAD50 and MRE11, relative to AEs and SCs by immunofluorescence labelling of paraffin sections of the mouse testis, using affinity-purified polyclonal antibodies against RAD50 and MRE11, and monoclonal and polyclonal antibodies against SC components . The localisation patterns of MRE11 and RAD50 within spermatocytes were very similar . MRE11 and RAD50 appeared in high abundance in preleptotene spermatocytes, just before SC components could be detected . From preleptotene until early zygotene they were present throughout the nucleus . In mid and late zygotene, MRE11 and RAD50 concentrated in distinct areas; in early pachytene the two proteins had almost disappeared from the nucleus, except from the sex vesicle (the chromatin of the XY bivalent), where they persisted in high abundance until diplotene . We propose that MRE11 and RAD50, together with other proteins, prepare chromatin throughout the early meiotic prophase nucleus for the initiation of meiotic recombination . Possibly, only a small fraction of the RAD50- and MRE11-containing (pre)recombination complexes associates transiently with AEs, where further steps in meiotic recombination can take place. Chromosoma, 2000, 109(1-2), 94 - 102 EXO1 and MSH4 differentially affect crossing-over and segregation; Khazanehdari KA et al.; The 5'-3' exonuclease Exo1p from Saccharomyces cerevisiae is required for wild-type levels of meiotic crossing-over and normal meiotic chromosome segregation as is the meiosis-specific MutS homologue, Msh4p . Mutations in both genes reduce crossing-over by approximately two-fold, but deltamsh4 strains have significantly lower viability and a higher frequency of meiosis I non-disjunction . Epistasis analysis indicates a complex interaction between the two genes . Although crossing-over was not detectably lower in the double mutant, viability was significantly worse than either single mutant . Such a result suggests that the two genes are affecting meiotic viability by distinct mechanisms . We propose that deltaexo1 affects chromosome segregation by reducing crossing-over, while deltamsh4 affects both the frequency and distribution of crossovers . Mutation in EXO1 reduces gene conversion frequencies significantly at some but not all loci, suggesting that other enzymes are also involved in DNA resection . We propose that Exo1p plays an early role in establishing some recombination intermediates by generating single-stranded tails . The role of Msh4p is suggested to be in determining whether some recombination intermediates are resolved as crossover events and in generating crossover interference . The synergistic effect of deltaexo1deltamsh4 on spore viability suggests that the two genes have partially compensatory roles in a process affecting meiotic success. Chromosoma, 2000, 109(1-2), 44 - 9 mei-41 is required for precocious anaphase in Drosophila females; McKim KS et al.; This paper reports on a new role for mei-41 in cell cycle control during meiosis . This function is revealed by the requirement of mei-41 for the precocious anaphase observed in crossover-defective mutants . Normally in Drosophila oocytes, tension on the meiotic spindle causes a metaphase I arrest . This tension results because crossovers, and the resulting chiasmata, hold homologs together that are being pulled by kinetochore microtobules toward opposite spindle poles . In the absence of tension, such as in a recombination-defective mutant, metaphase arrest is not observed and meiosis proceeds through the two divisions . Here we show that in some recombination-defective mutants, the precocious anaphase requires the mei-41 gene product . For example, metaphase arrest is not observed in mei-218 mutants because of the severe reduction in crossing over . In mei-41 mei-218 double mutants, however, metaphase arrest was restored . The effect of mei-41 is dependent on double-strand break formation . Thus, in mutants that fail to initiate meiotic recombination the absence of mei-41 has no effect. Chromosoma, 2000, 109(1-2), 10 - 26 Sister chromatid cohesion and recombination in meiosis; van Heemst D et al.; Sister chromatids are associated from their formation until their disjunction . Cohesion between sister chromatids is provided by protein complexes, of which some components are conserved across the kingdoms and between the mitotic and meiotic cell cycles . Sister chromatid cohesion is intimately linked to other aspects of chromosome behaviour and metabolism, in particular chromosome condensation, recombination and segregation . Recombination, sister chromatid cohesion and the relation between the two processes must be regulated differently in mitosis and meiosis . In meiosis, cohesion and recombination are modified in such a way that reciprocal exchange and reductional segregation of homologous chromosomes are ensured. Chromosoma, 2000, 109(1-2), 3 - 9 Hanging on to your homolog: the roles of pairing, synapsis and recombination in the maintenance of homolog adhesion; Walker MY et al.; Homologous chromosomes initially undergo weak alignments that bring homologous sequences into register during meiosis . These alignments can be facilitated by two types of mechanisms: interstitial homology searches and telomere-telomere alignments . As prophase (and chromatin compaction) proceeds, these initial pairings or alignments need to be stabilized . In at least some organisms, such as Saccharomyces cerevisiae and S . pombe, these pairings can apparently be maintained by the creation of recombination intermediates . In contrast, synapsis during zygotene may be able to facilitate and/or maintain chromosome pairing even in the absence of exchange in several higher organisms . It thus seems possible that the synaptonemal complex plays a role both in maintaining homolog adhesion during meiotic prophase and, more speculatively, in facilitating meiotic exchange. J Immunol Methods, 2000 Jun 23, 240(1-2), 185 - 95 Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama; van der Linden R et al.; Functional heavy chain immunoglobulins have, so far, only been found in camels and llamas . Antigen-specific fragments of these heavy chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable and can be produced at high level by the yeast Saccharomyces cerevisiae . The work described in this paper was conducted to determine whether llamas (Lama glama) are a practical source of antigen-specific V(HH) fragments . Llamas were immunised with various types of antigens and the antibody responses were examined during the course of immunisation . Both, conventional and heavy chain IgG antibodies were produced in response to each of the antigens . The heavy chain IgG repertoire displayed a recognition pattern different to that of conventional llama IgGs, resulting in the expansion of the accessible epitope repertoire . Llamas have a lower proportion of heavy chain IgG antibodies in their serum than have camels . To enable the specific and efficient isolation of V(HH) genes from peripheral blood B-cells, the long and short-hinge sequences of Lama glama heavy chain IgGs were determined, revealing the presence of a novel subclass of short-hinge heavy chain IgG . Long and short-hinge specific PCR primers were designed to be used in the construction of llama V(HH) libraries . We conclude that, using the techniques described, antigen-specific V(HH) antibody fragments are readily accessible from the llama, thus providing highly valuable binding molecules for a variety of applications. FEBS Lett, 2000 Jun 9, 475(1), 43 - 6 Detection of the absorption of glucose molecules by living cells using atomic force microscopy; de Souza Pereira R; A very small electrode (nanobiosensor) was constructed by immobilizing enzyme (glucose oxidase or hexokinase) on the surface of the cantilever of the atomic force microscope in order to detect the absorption of glucose molecules by living cells . If glucose is present, the nanobiosensor deflects, probably due to the reaction heat evolved in the process . Nanobiosensors built with inactivated enzyme or cantilevers without immobilized enzyme were not capable of producing this type of signal (deflection) . This technique will be very useful in detecting the passage of specific molecules through a cell wall (or a cell membrane for other types of cells). Nat Cell Biol, 2000 Jun, 2(6), E96 - 8 It's a kar9ochore to capture microtubules; Bloom K; Microtubule orientation to cortical spatial cues is essential for the fidelity of asymmetric cellular processes . A cortical microtubule-capture site, composed of Bim1 and Kar9, has now been identified in yeast . Bim1 is the yeast homologue of EB1, a binding partner of the adenomatous polyposis coli (APC), indicating that important features of this complex may be highly conserved. Nat Cell Biol, 2000 Jun, 2(6), 318 - 25 Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis; Matsuyama S et al.; Mitochondria trigger apoptosis by releasing caspase activators, including cytochrome c (cytC) . Here we show, using a pH-sensitive green fluorescent protein (GFP), that mitochondria-dependent apoptotic stimuli (such as Bax, staurosporine and ultraviolet irradiation) induce rapid, Bcl-2-inhibitable mitochondrial alkalinization and cytosol acidification, followed by cytC release, caspase activation and mitochondrial swelling and depolarization . These events are not induced by mitochondria-independent apoptotic stimuli, such as Fas . Activation of cytosolic caspases by cytC in vitro is minimal at neutral pH, but maximal at acidic pH, indicating that mitochondria-induced acidification of the cytosol may be important for caspase activation; this finding is supported by results obtained from cells using protonophores . Cytosol acidification and cytC release are suppressed by oligomycin, a FoF1-ATPase/H +-pump inhibitor, but not by caspase inhibitors . Ectopic expression of Bax in wild-type, but not FoF1/H+-pump-deficient, yeast cells similarly results in mitochondrial matrix alkalinization, cytosol acidification and cell death . These findings indicate that mitochondria-mediated alteration of intracellular pH may be an early event that regulates caspase activation in the mitochondrial pathway for apoptosis. J Neurochem, 2000 Jul, 75(1), 109 - 16 Isolation and characterization of novel presenilin binding protein; Kashiwa A et al.; Approximately 50% of familial Alzheimer's disease (AD) cases are linked to the presenilin (PS) gene . This suggests that an altered function of mutated PSs accounts for a fundamental process leading to AD . Here we identify a new PS binding protein, PBP, which is highly expressed in cerebral cortex and hippocampus . immunohistochemical studies and cell fractionation analysis show that PBP redistributes from cytoplasm to membranes in the presence of PS . In addition, PBP is deficient in the soluble fraction of sporadic AD brains. J Soc Biol, 1999, 193(1), 35 - 40 {Structure and evolution of human sub-telomeric regions}; Vergnaud G; Recent progress in the field of human genome analysis has led to the development of new concepts in the definition of subtelomeric domains . Analysis of DNA sequences from human and yeast chromosome ends have shown that short stretches of degenerate TTAGGG are found at a distance from the telomeric repeats . These stretches define a boundary between two structurally different regions . The distal domain is characterised by numerous, short segments of interrupted homology to many other human telomeric regions and to a number of ESTs . The proximal domain shows much longer uninterrupted homology to a few chromosome ends . This domain evolved quickly within primates at least, as demonstrated by the detailed study of locus DNF92 which spread very recently in humans from 17 qter to at least ten other chromosome ends . At the different sites, presence-absence polymorphisms are observed within humans . The region remained single locus at the paralogous site in higher primates . Conversely, a human and orangutan single locus telomeric domain occupies multiple chromosome ends in chimpanzee . Balanced translocation is the likely mechanism through which the spreading occurred . Some members of the olfactory receptor gene family show a similar behaviour: multiple telomeric locations, and presence-absence polymorphism . Strikingly, the set of chromosome ends occupied by the two regions is identical, except for the two ancestral sites . Moreover, the relative frequency of detection of the region at the different sites indicates some kind of competition between the two regions . Consequently, these two regions represent major new tools to investigate recent human genome evolution and human genome diversity in different populations. Curr Opin Microbiol, 2000 Jun, 3(3), 303 - 8 Systematic and large-scale two-hybrid screens; Uetz P et al.; The increasing rate at which complete genome sequences become available necessitates rapid and robust methods for investigating the functions of their encoded proteins . Efforts have been made to study protein function by systematically screening large sets of proteins using the two-hybrid method . Analyses of the complete proteomes of baceriophage T7, the mammalian viruses hepatitis C and vaccinia, as well as of several protein complexes including RNA splicing proteins and RNA polymerase III from yeast, have been undertaken . Saccharomyces cerevisiae has been studied extensively by two-hybrid methods, with more than 2500 protein-protein interactions described . Systematic studies on metazoan proteomes are, however, still in their infancy. J Virol, 2000 Jul, 74(13), 6168 - 72 Interaction of human immunodeficiency virus type 2 Vpx and invariant chain; Pancio HA et al.; Vpx is a virion-associated protein of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency viruses . The yeast two-hybrid system was used to identify invariant chain (Ii) as a cellular protein that interacts with HIV-2 Vpx . Vpx-Ii interaction was confirmed in cell-free reactions using bacterially expressed glutathione S-transferase fusion proteins and by coimmunoprecipitation in transfected and infected cells . In chronically infected cells expressing Vpx, Ii levels were markedly decreased, presumably due to enhanced degradation . These findings suggest that Vpx may disrupt major histocompatibility complex class II antigen presentation. J Virol, 2000 Jul, 74(13), 6096 - 104 Expression of human herpesvirus 6B rep within infected cells and binding of its gene product to the TATA-binding protein in vitro and in vivo; Mori Y et al.; We have characterized the human herpesvirus 6B (HHV-6B) rep gene, which is a homologue of the adeno-associated virus type 2 rep and is unique in the herpesvirus family . Three transcripts, 9.0, 5.0, and 2 . 7 kb (the major transcript), were detected by Northern blotting using an HHV-6B rep probe under late conditions . We investigated the expression kinetics of the rep gene using cycloheximide (CHX) and phosphonoformic acid (PFA), which are inhibitors of protein synthesis and viral DNA synthesis, respectively . The 5.2-kb transcript was mainly detected in the absence of protein biosynthesis upon infection, and none of the 9.0-, 5.0-, and 2.7-kb transcripts detected under the late conditions were detected in the presence of CHX and PFA . Sequences obtained from a cDNA library showed that the 5.0- and 2.7-kb transcripts were spliced from two and three exons, respectively, and the 2.7-kb transcript was more abundant . Immunohistochemistry using an antibody raised against the HHV-6 rep gene product (REP) revealed that REP was mainly present in the nucleus of MT-4 cells within 24 h after infection with HHV-6B . Using pull-down assays, coimmunoprecipitation, and a mammalian two hybrid system, we showed that HHV-6 REP binds to a transcription factor, human TATA-binding protein, through its N-terminal region. Plant Cell Physiol, 2000 Apr, 41(4), 523 - 33 Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors; Athwal GS et al.; The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds . The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543 . Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner . Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req) . As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity . Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D) . At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand . The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity. J Neurosci, 2000 Jun 15, 20(12), 4524 - 34 Interaction of the postsynaptic density-95/guanylate kinase domain-associated protein complex with a light chain of myosin-V and dynein; Naisbitt S et al.; NMDA receptors interact directly with postsynaptic density-95 (PSD-95), a scaffold protein that organizes a cytoskeletal- signaling complex at the postsynaptic membrane . The molecular mechanism by which the PSD-95-based protein complex is trafficked to the postsynaptic site is unknown but presumably involves specific motor proteins . Here we demonstrate a direct interaction between the PSD-95-associated protein guanylate kinase domain-associated protein (GKAP) and dynein light chain (DLC), a light chain subunit shared by myosin-V (an actin-based motor) and cytoplasmic dynein (a microtubule-based motor) . A yeast two-hybrid screen with GKAP isolated DLC2, a novel protein 93% identical to the previously cloned 8 kDa dynein light chain (DLC1) . A complex containing PSD-95, GKAP, DLC, and myosin-V can be immunoprecipitated from rat brain extracts . DLC colocalizes with PSD-95 and F-actin in dendritic spines of cultured neurons and is enriched in biochemical purifications of PSD . Immunogold electron microscopy reveals a concentration of DLC in the postsynaptic compartment of asymmetric synapses of brain in which it is associated with the PSD and the spine apparatus . We discuss the possibility that the GKAP/DLC interaction may be involved in trafficking of the PSD-95 complex by motor proteins. Protein Sci, 2000 May, 9(5), 1011 - 23 Nonpolar contributions to conformational specificity in assemblies of designed short helical peptides; Boon CL et al.; A series of designed short helical peptides was used to study the effect of nonpolar interactions on conformational specificity . The consensus sequence was designed to obtain short helices (17 residues) and to minimize the presence of interhelical polar interactions . Furthermore, the sequence contained a heptad repeat (abcdefg), where positions a and d were occupied by hydrophobic residues Leu, Ile, or Val, and positions e and g were occupied by Ala . The peptides were named according to the identities of the residues in the adeg positions, respectively . The peptides llaa, liaa, ilaa, iiaa, ivaa, viaa, lvaa, vlaa, and vvaa were synthesized, and their characterization revealed marked differences in specificity . An experimental methodology was developed to study the nine peptides and their pairwise mixtures . These peptides and their mixtures formed a vast array of structural states, which may be classified as follows: helical tetramers and pentamers, soluble and insoluble helical aggregates, insoluble unstructured aggregates, and soluble unstructured monomers . The peptide liaa formed stable helical pentamers, and iiaa and vlaa formed stable helical tetramers . Disulfide cross-linking experiments indicated the presence of an antiparallel helix alignment in the helical pentamers and tetramers . Rates of amide proton exchange of the tetrameric form of vlaa were 10-fold slower than the calculated exchange rate for unfolded vlaa . In other work, the control of specificity has been attributed to polar interactions, especially buried polar interactions; this work demonstrated that subtle changes in the configuration of nonpolar interactions resulted in a large variation in the extent of conformational specificity of assemblies of designed short helical peptides . Thus, nonpolar interactions can have a significant effect on the conformational specificity of oligomeric short helices. Nature, 2000 Jun 1, 405(6786), 579 - 83 Peptides accelerate their uptake by activating a ubiquitin-dependent proteolytic pathway; Turner GC et al.; Protein degradation by the ubiquitin system controls the intracellular concentrations of many regulatory proteins . A protein substrate of the ubiquitin system is conjugated to ubiquitin through the action of three enzymes, E1, E2 and E3, with the degradation signal (degron) of the substrate recognized by E3 (refs 1-3) . The resulting multi-ubiquitylated substrate is degraded by the 26S proteasome . Here we describe the physiological regulation of a ubiquitin-dependent pathway through allosteric modulation of its E3 activity by small compounds . Ubr1, the E3 enzyme of the N-end rule pathway (a ubiquitin-dependent proteolytic system) in Saccharomyces cerevisiae mediates the degradation of Cup9, a transcriptional repressor of the peptide transporter Ptr2 (ref . 5) . Ubr1 also targets proteins that have destabilizing amino-terminal residues . We show that the degradation of Cup9 is allosterically activated by dipeptides with destabilizing N-terminal residues . In the resulting positive feedback circuit, imported dipeptides bind to Ubr1 and accelerate the Ubr1-dependent degradation of Cup9, thereby de-repressing the expression of Ptr2 and increasing the cell's capacity to import peptides . These findings identify the physiological rationale for the targeting of Cup9 by Ubr1, and indicate that small compounds may regulate other ubiquitin-dependent pathways. Mol Cells, 2000 Apr 30, 10(2), 232 - 5 Random changes of amino acid residues with expected frequency by saturated point mutagenesis; Kim SJ et al.; The yeast transcriptional activator protein, Gcn4p from Saccharomyces cerevisiae binds to the specific sequence in the promoters of many amino acid biosynthetic genes for general control . A new random saturation mutagenesis method was developed to isolate Gcn4p derivatives with only one or two mutations in the DNA binding domain without using radioactive isotope . This will be used to identify the amino acids of Gcn4p involved in protein-protein interactions . Saturation mutagenesis in the DNA binding domain of Gcn4p was performed using spiked degenerate oligonucleotides containing randomized codon bases designed specifically for only one or two base changes in the mutagenized area . These oligonucleotides were synthesized to have two flanking restriction enzyme sites for cloning to the appropriate vector . The 3' ends were mutually primed after hybridization via the palindromic sequences of the restriction enzyme sites . These molecules were then converted to double stranded DNA upon treatment with DNA polymerase . Here, a library collection of 100,680 in an altered Gcn4p pool was generated by cloning a mixed-base oligonucleotide in the place of the sequence coding for the DNA binding domains . The quality of the library was examined by DNA sequencing and found to be in good agreement with the expected statistical values . Calculated mutation frequency was 66% of mutant nucleotide rate and actual sequencing data revealed 68% mutant nucleotide rates from the sequenced library . Thus, among 21 mutants, 16 had one point mutations and 5 had two point mutations . This approach appears to be an effective and general tool for creating proteins with one or two amino acid change(s) in their molecules. Appl Biochem Biotechnol, 2000 Spring, 84-86, 1079 - 86 Sonic wave separation of invertase from a dilute solution to generated droplets; Tanner RD et al.; It has previously been shown that a droplet fractionation process, simulated by shaking a separatory funnel containing a dilute protein solution, can generate droplets richer in protein than present in the original dilute solution . In this article, we describe an alternative method that can increase the amount of protein transferred to the droplets . The new method uses ultrasonic waves, enhanced by a bubble gas stream to create the droplets . The amount of protein in these droplets increases by about 50% . In this method, the top layer of the dilute protein solution (of the solution-air interface) becomes enriched in protein when air is bubbled into the solution . This concentrating procedure is called bubble fractionation . Once the protein has passed through the initial buildup, this enriched protein layer is transferred into droplets with the aid of a vacuum above the solution at the same time that ultrasonic waves are introduced . The droplets are then carried over to a condenser and coalesced . We found that this new method provides an easier way to remove the protein-enriched top layer of the dilute solution and generates more droplets within a shorter period than the separatory funnel droplet generation method . The added air creates the bubbles and carries the droplets, and the vacuum helps remove the effluent airstream from the condenser . The maximum partition coefficient, the ratio of the protein concentration in the droplets to that in the residual solution (approx 8.5), occurred at pH 5.0. Adv Exp Med Biol, 2000, 475, 143 - 52 Regulation of CREB by moderate hypoxia in PC12 cells; Beitner-Johnson D et al.; The mechanisms by which excitable cells adapt and respond to changes in O2 levels remain largely unknown . We have investigated the effect of hypoxia on the cyclic AMP response element binding protein (CREB) transcription factor . PC12 cells were exposed to moderate levels of hypoxia (5% O2) for various times between 20 min and 6 hr . We found that hypoxia rapidly and persistently induced ser133 phosphorylation of CREB . This effect was more robust than that produced by exposing PC12 cells to either forskolin, KCl, or NGF . This effect was not due to activation of any of the previously known CREB kinases, including PKA, CaMK, PKC, p70s6k, or MAPKAP kinase-2 . Thus, hypoxia may induce activation of a novel CREB kinase . To test whether phosphorylation of CREB was associated with an activation of CRE-dependent gene expression, cells were transfected with wild type and mutated regions of the 5'-flanking region of the tyrosine hydroxylase (TH) gene fused to a CAT reporter gene . Mutation of the CRE element in a TH reporter gene reduced, but did not abolish, the effects of hypoxia on TH gene expression . However, hypoxia did not induce transactivation of a GAL4-luciferase reporter by a GAL4-CREB fusion protein . Thus, the mechanism by which hypoxia regulates CREB is distinct, and more complex, than that induced by forskolin, depolarization, or nerve growth factor. J Biol Chem, 2000 Jun 16, 275(24), 18099 - 107 A1 functions at the mitochondria to delay endothelial apoptosis in response to tumor necrosis factor; Duriez PJ et al.; Tumor necrosis factor (TNF) does not cause endothelial apoptosis unless the expression of cytoprotective genes is blocked . We have previously demonstrated that one of the TNF-inducible cytoprotective genes is the Bcl-2 family member, A1 . A1 is induced by the action of the transcription factor, NFkappaB, in response to inflammatory mediators . In this report we demonstrate that, as with other cell types, inhibition of NFkappaB initiates microvascular endothelial apoptosis in response to TNF . A1 is able to inhibit this apoptosis over 24 h . We demonstrate that A1 is localized to and functions at the mitochondria . Whereas A1 is able to inhibit mitochondrial depolarization, loss of cytochrome c, cleavage of caspase 9, BID, and poly(ADP-ribose) polymerase, it does not block caspase 8 or caspase 3 cleavage . In contrast, A1 is not able to prevent endothelial apoptosis by TNF over 72 h, when NFkappaB signaling is blocked . On the other hand, the caspase inhibitor, benzyloxycarbonyl-VAD-formylmethyl ketone, completely blocks TNF-induced endothelial apoptosis over 72 h . Our findings indicate that A1 is able to maintain temporary survival of endothelial cells in response to TNF by maintaining mitochondrial viability and function . However, a mitochondria-independent caspase pathway eventually results in endothelial death despite mitochondrial protection by A1. J Biol Chem, 2000 Aug 25, 275(34), 26484 - 91 Stress-activated protein kinases (JNK and p38/HOG) are essential for vascular endothelial growth factor mRNA stability; Pages G et al.; Stability of the vascular endothelial growth factor (VEGF) mRNA is tightly regulated through its 3'-untranslated region (3'-UTR) . Here, we demonstrate that VEGF mRNA levels are increased by anisomycin, a strong activator of stress-activated protein kinases . Hence, VEGF mRNA induction is inhibited by SB202190, an inhibitor of JNK and p38/HOG kinase . Furthermore, VEGF mRNA expression is increased in cells that overexpress JNK and p38/HOG by an increase in its stability . We show by two different approaches that anisomycin exerts its effect on the VEGF mRNA 3'-UTR . First, by using an in vitro mRNA degradation assay, the half-life of the VEGF mRNA 3'-UTR region transcript was found to be increased when incubated with extracts from anisomycin-treated cells; and second, the 3'-UTR was also sufficient to confer mRNA instability to the Nhe3 (Na(+)/H(+) exchanger 3) heterologous reporter gene, and anisomycin treatment stabilized the chimeric mRNA (Nhe3 fused to the VEGF mRNA 3'-UTR) . This chimeric mRNA is also more stable in cells overexpressing p38/HOG and JNK that have been stimulated by anisomycin . We show that such regulation is mediated through an AU-rich region of the 3'-UTR contained within a stable hairpin structure . By RNA electrophoretic mobility shift assays, we show that this region binds proteins specifically induced by anisomycin treatment . These findings clearly demonstrate a major role of stress-activated protein kinases in the post-transcriptional regulation of VEGF. Eur J Biochem, 2000 Jun, 267(12), 3801 - 11 A bifunctional delta-fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus . A new member of the cytochrome b5 superfamily; Sperling P et al.; Many plant genes have been cloned that encode regioselective desaturases catalyzing the formation of cis-unsaturated fatty acids . However, very few genes have been cloned that encode enzymes catalyzing the formation of the functional groups found in unusual fatty acids (e.g . hydroxy, epoxy or acetylenic fatty acids) . Here, we describe the characterization of an acetylenase from the moss Ceratodon purpureus with a regioselectivity differing from the previously described Delta12-acetylenase . The gene encoding this protein, together with a Delta6-desaturase, was cloned by a PCR-based approach with primers derived from conserved regions in Delta5-, Delta6-fatty-acid desaturases and Delta8-sphingolipid desaturases . The proteins that are encoded by the two cloned cDNAs are likely to consist of a N-terminal extension of unknown function, a cytochrome b5-domain, and a C-terminal domain that is similar to acyl lipid desaturases with characteristic histidine boxes . The proteins were highly homologous in sequence to the Delta6-desaturase from the moss Physcomitrella patens . When these two cDNAs were expressed in Saccharomyces cerevisiae, both transgenic yeast cultures desaturated Delta9-unsaturated C16- and C18-fatty acids by inserting an additional Delta6cis-double bond . One of these transgenic yeast clones was also able to introduce a Delta6-triple bond into gamma-linolenic and stearidonic acid . This resulted in the formation of 9,12,15-(Z,Z,Z)-octadecatrien-6-ynoic acid, the main fatty acid found in C . pupureus . These results demonstrate that the Delta6-acetylenase from C . pupureus is a bifunctional enzyme, which can introduce a Delta6cis-double bond into 9,12,(15)-C18-polyenoic acids as well as converting a Delta6cis-double bond to a Delta6-triple bond. Mol Biol Cell, 2000 Jun, 11(6), 2103 - 15 Mitochondria-to-nuclear signaling is regulated by the subcellular localization of the transcription factors Rtg1p and Rtg3p; Sekito T et al.; Cells modulate the expression of nuclear genes in response to changes in the functional state of mitochondria, an interorganelle communication pathway called retrograde regulation . In yeast, expression of the CIT2 gene shows a typical retrograde response in that its expression is dramatically increased in cells with dysfunctional mitochondria, such as in rho(o) petites . Three genes control this signaling pathway: RTG1 and RTG3, which encode basic helix-loop-helix leucine zipper transcription factors that bind as heterodimer to the CIT2 upstream activation site, and RTG2, which encodes a protein of unknown function . We show that in respiratory-competent (rho(+)) cells in which CIT2 expression is low, Rtg1p and Rtg3p exist as a complex largely in the cytoplasm, and in rho(o) petites in which CIT2 expression is high, they exist as a complex predominantly localized in the nucleus . Cytoplasmic Rtg3p is multiply phosphorylated and becomes partially dephosphorylated when localized in the nucleus . Rtg2p, which is cytoplasmic in both rho(+) and rho(o) cells, is required for the dephosphorylation and nuclear localization of Rtg3p . Interaction of Rtg3p with Rtg1p is required to retain Rtg3p in the cytoplasm of rho(+) cells; in the absence of such interaction, nuclear localization and dephosphorylation of Rtg3p is independent of Rtg2p . Our data show that Rtg1p acts as both a positive and negative regulator of the retrograde response and that Rtg2p acts to transduce mitochondrial signals affecting the phosphorylation state and subcellular localization of Rtg3p. Mol Biol Cell, 2000 Jun, 11(6), 2085 - 102 The peroxin pex3p initiates membrane assembly in peroxisome biogenesis; Ghaedi K et al.; Rat cDNA encoding a 372-amino-acid peroxin was isolated, primarily by functional complementation screening, using a peroxisome-deficient Chinese hamster ovary cell mutant, ZPG208, of complementation group 17 . The deduced primary sequence showed approximately 25% amino acid identity with the yeast Pex3p, thereby we termed this cDNA rat PEX3 (RnPEX3) . Human and Chinese hamster Pex3p showed 96 and 94% identity to rat Pex3p and had 373 amino acids . Pex3p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol . A homozygous, inactivating missense mutation, G to A at position413, in a codon (GGA) for Gly(138) and resulting in a codon (GAA) for Glu was the genetic cause of peroxisome deficiency of complementation group 17 ZPG208 . The peroxisome-restoring activity apparently required the full length of Pex3p, whereas its N-terminal part from residues 1 to 40 was sufficient to target a fusion protein to peroxisomes . We also demonstrated that Pex3p binds the farnesylated peroxisomal membrane protein Pex19p . Moreover, upon expression of PEX3 in ZPG208, peroxisomal membrane vesicles were assembled before the import of soluble proteins such as PTS2-tagged green fluorescent protein . Thus, Pex3p assembles membrane vesicles before the matrix proteins are translocated. Mol Biol Cell, 2000 Jun, 11(6), 1947 - 57 The Kex2p proregion is essential for the biosynthesis of an active enzyme and requires a C-terminal basic residue for its function; Lesage G et al.; The Saccharomyces cerevisiae prohormone-processing enzyme Kex2p is biosynthesized as an inactive precursor extended by its N-terminal proregion . Here we show that deletion of the proregion renders Kex2p inactive both in vivo and in vitro . Absence of the proregion impaired glycosylation and stability and resulted in the retention of the enzyme in the endoplasmic reticulum . These phenotypes were partially complemented by expression of the proregion in trans . Trans complementation was specific to Kex2p proregion because expression of any of the seven mammalian prohormone convertase propeptides had no effect . These data are consistent with a model whereby Kex2p proregion functions as an intramolecular chaperone and indicate that covalent linkage to the protein is not an absolute requirement for proregion function . Furthermore, extensive mutagenesis revealed that, in addition to their function as proteolytic recognition sites, C-terminal basic residues play an active role in proregion-dependent Kex2p activation. Mol Cell Biol, 2000 Jul, 20(13), 4948 - 57 Ribosomal DNA replication fork barrier and HOT1 recombination hot spot: shared sequences but independent activities; Ward TR et al.; In the ribosomal DNA of Saccharomyces cerevisiae, sequences in the nontranscribed spacer 3' of the 35S ribosomal RNA gene are important to the polar arrest of replication forks at a site called the replication fork barrier (RFB) and also to the cis-acting, mitotic hyperrecombination site called HOT1 . We have found that the RFB and HOT1 activity share some but not all of their essential sequences . Many of the mutations that reduce HOT1 recombination also decrease or eliminate fork arrest at one of two closely spaced RFB sites, RFB1 and RFB2 . A simple model for the juxtaposition of RFB and HOT1 sequences is that the breakage of strands in replication forks arrested at RFB stimulates recombination . Contrary to this model, we show here that HOT1-stimulated recombination does not require the arrest of forks at the RFB . Therefore, while HOT1 activity is independent of replication fork arrest, HOT1 and RFB require some common sequences, suggesting the existence of a common trans-acting factor(s). Mol Cell Biol, 2000 Jul, 20(13), 4838 - 48 Bypass of a meiotic checkpoint by overproduction of meiotic chromosomal proteins; Bailis JM et al.; The Saccharomyces cerevisiae zip1 mutant, which exhibits defects in synaptonemal complex formation and meiotic recombination, triggers a checkpoint that causes cells to arrest at the pachytene stage of meiotic prophase . Overproduction of either the meiotic chromosomal protein Red1 or the meiotic kinase Mek1 bypasses this checkpoint, allowing zip1 cells to sporulate . Red1 or Mek1 overproduction also promotes sporulation of other mutants (zip2, dmc1, hop2) that undergo checkpoint-mediated arrest at pachytene . In addition, Red1 overproduction antagonizes interhomolog interactions in the zip1 mutant, substantially decreasing double-strand break formation, meiotic recombination, and homologous chromosome pairing . Mek1 overproduction, in contrast, suppresses checkpoint-induced arrest without significantly decreasing meiotic recombination . Cooverproduction of Red1 and Mek1 fails to bypass the checkpoint; moreover, overproduction of the meiotic chromosomal protein Hop1 blocks the Red1 and Mek1 overproduction phenotypes . These results suggest that meiotic chromosomal proteins function in the signaling of meiotic prophase defects and that the correct stoichiometry of Red1, Mek1, and Hop1 is needed to achieve checkpoint-mediated cell cycle arrest at pachytene. Mol Cell Biol, 2000 Jul, 20(13), 4806 - 13 Identification of amino acid residues in the Caenorhabditis elegans POU protein UNC-86 that mediate UNC-86-MEC-3-DNA ternary complex formation; Rockelein I et al.; The POU homeodomain protein UNC-86 and the LIM homeodomain protein MEC-3 are essential for the differentiation of the six mechanoreceptor neurons in the nematode Caenorhabditis elegans . Previous studies have indicated that UNC-86 and MEC-3 bind cooperatively to at least three sites in the mec-3 promoter and synergistically activate transcription . However, the molecular details of the interactions of UNC-86 with MEC-3 and DNA have not been investigated so far . Here we used a yeast system to identify the functional domains in UNC-86 required for transcriptional activation and to characterize the interaction of UNC-86 with MEC-3 in vivo . Our results suggest that transcriptional activation is mediated by the amino terminus of UNC-86, whereas amino acids in the POU domain mediate DNA binding and interaction with MEC-3 . By random mutagenesis, we identified mutations that only affect the DNA binding properties of UNC-86, as well as mutations that prevent coactivation by MEC-3 . We demonstrated that both the POU-specific domain and the homeodomain of UNC-86, as well as DNA bases adjacent to the proposed UNC-86 binding site, are involved in the formation of a transcriptionally active complex with MEC-3 . These data suggest that some residues involved in the contact of UNC-86 with MEC-3 also contribute to the interaction of the functionally nonrelated POU protein Oct-1 with Oca-B, whereas other positions have different roles. Mol Cell Biol, 2000 Jul, 20(13), 4691 - 8 Evidence for an interaction between ubiquitin-conjugating enzymes and the 26S proteasome; Tongaonkar P et al.; The targeting of proteolytic substrates is accomplished by a family of ubiquitin-conjugating (E2) enzymes and a diverse set of substrate recognition (E3) factors . The ligation of a multiubiquitin chain to a substrate can promote its degradation by the proteasome . However, the mechanism that facilitates the translocation of a substrate to the proteasome in vivo is poorly understood . We have discovered that E2 proteins, including Ubc1, Ubc2, Ubc4, and Ubc5, can interact with the 26S proteasome . Significantly, the interaction between Ubc4 and the proteasome is strongly induced by heat stress, consistent with the requirement for this E2 for efficient stress tolerance . A catalytically inactive derivative of Ubc4 (Ubc4(C86A)), which causes toxicity in yeast cells, can also bind the proteasome . Purified proteasomes can ligate ubiquitin to a test substrate without the addition of exogenous E2 protein, suggesting that the ubiquitylation of some proteolytic substrates might be directly coupled to degradation by the proteasome. Mol Cell Biol, 2000 Jul, 20(13), 4614 - 25 Hsl1p, a Swe1p inhibitor, is degraded via the anaphase-promoting complex; Burton JL et al.; Ubiquitination and subsequent degradation of critical cell cycle regulators is a key mechanism exploited by the cell to ensure an irreversible progression of cell cycle events . The anaphase-promoting complex (APC) is a ubiquitin ligase that targets proteins for degradation by the 26S proteasome . Here we identify the Hsl1p protein kinase as an APC substrate that interacts with Cdc20p and Cdh1p, proteins that mediate APC ubiquitination of protein substrates . Hsl1p is absent in G(1), accumulates as cells begin to bud, and disappears in late mitosis . Hsl1p is stabilized by mutations in CDH1 and CDC23, both of which result in compromised APC activity . Unlike Hsl1p, Gin4p and Kcc4p, protein kinases that have sequence homology to Hsl1p, were stable in G(1)-arrested cells containing active APC . Mutation of a destruction box motif within Hsl1p (Hsl1p(db-mut)) stabilized Hsl1p . Interestingly, this mutation also disrupted the Hsl1p-Cdc20p interaction and reduced the association between Hsl1p and Cdh1p in coimmunoprecipitation studies . These findings suggest that the destruction box motif is required for Cdc20p and, to a lesser extent, for Cdh1p to target Hsl1p to the APC for ubiquitination . Hsl1p has been previously shown to inhibit Swe1p, a protein kinase that negatively regulates the cyclin-dependent kinase Cdc28p, by promoting Swe1p degradation via SCF(Met30) in a bud morphogenesis checkpoint . Results of the present work indicate that Hsl1p is degraded in an APC-dependent manner and suggest a link between the SCF (Skp1-cullin-F box) and APC-proteolytic systems that may help to coordinate the proper progression of cell cycle events. Mol Cell Biol, 2000 Jul, 20(13), 4591 - 603 Upf1p control of nonsense mRNA translation is regulated by Nmd2p and Upf3p; Maderazo AB et al.; Upf1p, Nmd2p, and Upf3p regulate the degradation of yeast mRNAs that contain premature translation termination codons . These proteins also appear to regulate the fidelity of termination, allowing translational suppression in their absence . Here, we have devised a novel quantitative assay for translational suppression, based on a nonsense allele of the CAN1 gene (can1-100), and used it to determine the regulatory roles of the UPF/NMD gene products . Deletion of UPF1, NMD2, or UPF3 stabilized the can1-100 transcript and promoted can1-100 nonsense suppression . Changes in mRNA levels were not the basis of suppression, however, since deletion of DCP1 or XRN1 or high-copy-number can1-100 expression in wild-type cells caused an increase in mRNA abundance similar to that obtained in upf/nmd cells but did not result in comparable suppression . can1-100 suppression was highest in cells harboring a deletion of UPF1, and overexpression of UPF1 in cells with individual or multiple upf/nmd mutations lowered the level of nonsense suppression without affecting the abundance of the can1-100 mRNA . Our findings indicate that Nmd2p and Upf3p regulate Upf1p activity and that Upf1p plays a critical role in promoting termination fidelity that is independent of its role in regulating mRNA decay . Consistent with these relationships, Upf1p, Nmd2p, and Upf3p were shown to be present at 1, 600, 160, and 80 molecules per cell, levels that underscored the importance of Upf1p but minimized the likelihood that these proteins were associated with all ribosomes or that they functioned as a stoichiometric complex. Mol Cell Biol, 2000 Jul, 20(13), 4483 - 93 Testing cyclin specificity in the exit from mitosis; Jacobson MD et al.; Cyclical inactivation of B-type cyclins has been proposed to be required for alternating DNA replication and mitosis . Destruction box-dependent Clb5p degradation is strongly increased in mitotic cells, and constitutive overexpression of Clb5p lacking the destruction box resulted in rapid accumulation of inviable cells, frequently multiply budded, with DNA contents ranging from unreplicated to apparently fully replicated . Loss of viability correlated with retention of nuclear Clb5p at the time of nuclear division . CLB2-Deltadb overexpression that was quantitatively comparable to CLB5-Deltadb overexpression with respect to Clb protein production and Clb-associated kinase activity resulted in a distinct phenotype: reversible mitotic arrest with uniformly replicated DNA . Simultaneous overexpression of CLB2-Deltadb and CLB5-Deltadb overexpressers similarly resulted in a uniform arrest with replicated DNA, and this arrest was significantly more reversible than that observed with CLB5-Deltadb overexpression alone . These results suggest that Clb2p and not Clb5p can efficiently block mitotic completion . We speculate that CLB5-Deltadb overexpression may be lethal, because persistence of high nuclear Clb5p-associated kinase throughout mitosis leads to failure to load origins of replication, thus preventing DNA replication in the succeeding cell cycle. Cell, 2000 Apr 28, 101(3), 249 - 58 Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation; Travers KJ et al.; The unfolded protein response (UPR) regulates gene expression in response to stress in the endoplasmic reticulum (ER) . We determined the transcriptional scope of the UPR using DNA microarrays . Rather than regulating only ER-resident chaperones and phospholipid biosynthesis, as anticipated from earlier work, the UPR affects multiple ER and secretory pathway functions . Studies of UPR targets engaged in ER-associated protein degradation (ERAD) reveal an intimate coordination between these responses: efficient ERAD requires an intact UPR, and UPR induction increases ERAD capacity . Conversely, loss of ERAD leads to constitutive UPR induction . Finally, simultaneous loss of ERAD and the UPR greatly decreases cell viability . Thus, the UPR and ERAD are dynamic responses required for the coordinated disposal of misfolded proteins even in the absence of acute stress. Anal Biochem, 2000 May 15, 281(1), 36 - 44 A high-performance liquid chromatographic method to measure sphingosine 1-phosphate and related compounds from sphingosine kinase assays and other biological samples; Caligan TB et al.; Sphingosine 1-phosphate is an intermediate of sphingosine catabolism as well as a potent signaling compound . Conditions were established for the extraction and analysis of sphingosine 1-phosphate and other sphingoid base 1-phosphates from in vitro sphingosine kinase assays and other biological samples . The sphingoid base 1-phosphates were extracted in high yield (85%) using small C-18 reverse-phase columns (LiChroprep RP-18) . After the extracts were treated with 0.1 N KOH to remove glycerolipids, the sphingoid base 1-phosphates were converted to fluorescent o-phthalaldehyde derivatives that were separated by HPLC using C-18 columns with a mobile phase of methanol:10 mM potassium phosphate (pH 7.2):1 M tetrabutylammonium dihydrogen phosphate (in water) (83:16:1, v/v/v) . The o-phthalaldehyde derivative of sphingosine 1-phosphate was reasonably stable (t(1/2) > or = 18 h) when EDTA was present and could be detected in picomole amounts . The HPLC retention time of the sphingoid base 1-phosphates could be shifted by adjusting the mobile phase to pH 5.5, which is useful in separating overlapping compounds (such as sphingosine 1-phosphate and 4-D-hydroxysphinganine) and in confirming the identity of sphingoid base 1-phosphates in biological samples . The extraction procedure and HPLC method facilitated assays of sphingosine kinase with different sphingoid bases as substrates and/or inhibitors and enabled the quantitation of sphingoid base 1-phosphates in human plasma, serum, and platelets as well as in strains of Saccharomyces cerevisae with mutations in sphingolipid metabolism. J Biol Chem, 2000 Aug 25, 275(34), 26492 - 9 Interaction between transmembrane domains five and six of the alpha -factor receptor; Dube P et al.; The alpha-factor pheromone receptor (STE2) activates a G protein signal pathway that induces conjugation of the yeast Saccharomyces cerevisiae . Previous studies implicated the third intracellular loop of this receptor in G protein activation . Therefore, the roles of transmembrane domains five and six (TMD5 and -6) that bracket the third intracellular loop were analyzed by scanning mutagenesis in which each residue was substituted with cysteine . Out of 42 mutants examined, four constitutive mutants and two strong loss-of-function mutants were identified . Double mutants combining Cys substitutions in TMD5 and TMD6 gave a broader range of phenotypes . Interestingly, a V223C mutation in TMD5 caused constitutive activity when combined with the L247C, L248C, or S251C mutations in TMD6 . Also, the L226C mutation in TMD5 caused constitutive activity when combined with either the M250C or S251C mutations in TMD6 . The residues affected by these mutations are predicted to fall on one side of their respective helices, suggesting that they may interact . In support of this, cysteines substituted at position 223 in TMD5 and position 247 in TMD6 formed a disulfide bond, providing the first direct evidence of an interaction between these transmembrane domains in the alpha-factor receptor . Altogether, these results identify an important region of interaction between conserved hydrophobic regions at the base of TMD5 and TMD6 that is required for the proper regulation of receptor signaling. J Cell Biochem, 2000 May, 78(2), 179 - 85 Recruitment of chromatin remodeling machines; Peterson CL et al.; The assembly of eukaryotic DNA into folded nucleosomal arrays has drastic consequences for many nuclear processes that require access to the DNA sequence, including RNA transcription, DNA replication, recombination, and repair . Two types of highly conserved chromatin remodeling enzymes have been implicated as regulators of the repressive nature of chromatin structure: ATP-dependent remodeling complexes and nuclear histone acetyltransferases (HATs) . Recent studies indicate that both types of enzymes can be recruited to chromosomal loci through either physical interactions with transcriptional activators or via the global accessibility of chromatin during S phase of the cell cycle . Here we review these recent observations and discuss the implications for gene-specific regulation by chromatin remodeling machines . Bioessays, 2000 Jun, 22(6), 545 - 53 A look at the Caenorhabditis elegans Kex2/Subtilisin-like proprotein convertase family; Thacker C et al.; Significant advances have recently been made in our understanding of the mechanisms of activation of proteins that require processing . Often this involves endoproteolytic cleavage of precursor forms at basic residues, and is carried out by a group of serine endoproteinases, termed the proprotein convertases . In mammals, seven different convertases have been identified to date . These act in both the regulated secretory pathway for the processing of prohormones and proneuropeptides and in the constitutive secretory pathway, in which a variety of proproteins are activated endoproteolytically . The recently completed sequence of the nematode Caenorhabditis elegans genome affords a unique opportunity to examine the entire proprotein convertase family in a multicellular organism . Here we review the nature of the family, emphasising the structural features, characteristic of the four nematode genes, that supply all of the necessary functions unique to this group of serine endoproteinases . Studies of the C . elegans genes not only provide important information about the evaluation of this gene family but should help to illuminate the roles of these proteins in mammalian systems . BioEssays 22:545-553, 2000 . Bioessays, 2000 Jun, 22(6), 503 - 6 Building a protein interaction map: research in the post-genome era; Chen Z et al.; With the extensive amount of information generated by genome-wide sequencing, the entire set of gene products in an organism can now be predicted . The challenge of understanding the function of each gene in the genome has led to the development of many large-scale and high-throughput experimental techniques . Recently, two papers, Walhout et al.(1) and Uetz et al.,(2) have described studies that add a new functional dimension to research conducted on a genome-wide scale . These two groups have utilized the yeast two-hybrid system to identify interactions among the entire complement of proteins encoded by the Caenorhabditis elegans and the Saccharomyces cerevisiae genomes, respectively . Using a set of 29 genes that have been previously characterized, Walhout et al . demonstrated the feasibility and efficiency of this technique by building an interaction matrix among a large number of proteins . On an even larger scale, Uetz et al . conducted two-hybrid analyses using proteins that represent over 87% of the total gene products in yeast and identified interactions for about 15% of the total yeast proteins . BioEssays 22:503-506, 2000 . J Comp Neurol, 2000 Jun 19, 422(1), 66 - 94 Neuroanatomy of cells expressing clock genes in Drosophila: transgenic manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker neurons and their projections; Kaneko M et al.; Subsets of brain neurons expressing the clock genes period (per) and timeless (tim) are involved in the generation of circadian behavioral rhythms . However, current knowledge of projection patterns of these neurons is limited to those immunoreactive to an antibody against a crustacean neuropeptide . The GAL4-expression system was utilized to visualize neuronal processes from all per and tim-expressing neurons in the central nervous system . Each of two types of GAL4-driver fusion genes, per-gal4 or tim-gal4, was combined in transgenic flies with marker genes-lacZ, and sequences encoding green fluorescent protein or TAU protein-under the control of the GAL4-responsive element UAS . This allowed visualization of the cytoplasm of GAL4-expressing cells . Thus, neurites of clock neurons in the adult brain as well as those of larvae and pupae were revealed . Among the anatomical patterns revealed by per-gal4- or tim-gal4-driven marker expression were a previously unknown, dorsally located neuronal cluster, along with the projections of these cells and of other dorsal neurons characterized in earlier studies only by the location of their perikarya . The similarity of projections from PER- or TIM-containing neurons during development to those in the adult implies that these features of mature clock neurons are established by the larval stages . Neurons that have never been identified as PER- or TIM-immunoreactive were also visualized in this assay system, indicating promoter activity of the clock genes in these cells and suggesting that their products cannot accumulate to detectable levels in certain neurons . Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6306 - 10 Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and transcription factor interaction target; Todone F et al.; Eukaryotic nuclei contain three different types of RNA polymerases (RNAPs), each consisting of 12-18 different subunits . The evolutionarily highly conserved RNAP subunit RPB5 is shared by all three enzymes and therefore represents a key structural/functional component of all eukaryotic RNAPs . Here we present the crystal structure of the RPB5 subunit from Saccharomyces cerevisiae . The bipartite structure includes a eukaryote-specific N-terminal domain and a C-terminal domain resembling the archaeal RNAP subunit H . RPB5 has been implicated in direct protein-protein contacts with transcription factor IIB, one of the components of the RNAP(II) basal transcriptional machinery, and gene-specific activator proteins, such as the hepatitis B virus transactivator protein X . The experimentally mapped regions of RPB5 involved in these interactions correspond to distinct and surface-exposed alpha-helical structures. J Biol Chem, 2000 Aug 4, 275(31), 23421 - 4 Activation of heterotrimeric G-protein signaling by a ras-related protein . Implications for signal integration; Cismowski MJ et al.; Utilizing a functional screen in the yeast Saccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion . One of the identified activators, termed AGS1 (for activator of G-protein signaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily . Expression of AGS1 in yeast and in mammalian cells results in specific activation of Galpha(i)/Galpha(o) heterotrimeric signaling pathways . In addition, the in vivo and in vitro properties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for Galpha(i)/Galpha(o) . AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways. Biochem J, 2000 Jun 15, 348 Pt 3, 667 - 73 Activity of phosphatidylinositol transfer protein is sensitive to ethanol and membrane curvature; Komatsu H et al.; Phosphatidylinositol transfer protein (PITP) is critical for many cellular signalling and trafficking events that are influenced by ethanol . The influence of ethanol and membrane curvature on the activity of recombinant mouse PITP-alpha in vitro is evaluated by monitoring the transfer of phosphatidylinositol (PtdIns) from rat hepatic microsomes to unilamellar vesicles . Acute exposure to pharmacological levels of ethanol enhanced the function of PITP . Chloroform shared a similar ability to enhance function when both drug concentrations were normalized to their respective octanol/water partition coefficients, indicating that the effect is not unique to ethanol and might be common to hydrophobic solutes . Neither the PITP activity nor its ethanol enhancement was altered by using thermally pretreated (denatured) or protease-treated microsomes, indicating that the native microsomal protein structure was unlikely to be a determinant of transfer . Kinetic analyses indicated that ethanol acted by increasing the PITP-mediated flux of PtdIns from both microsomal and liposomal surfaces . The activity of PITP was strongly dependent on the lipid structure, with a steep dependence on the expressed curvature of the membrane . Activity was greatest for small, highly curved sonicated vesicles and decreased markedly for large, locally planar unilamellar vesicles . Ethanol enhanced PITP-mediated PtdIns transfer to all vesicles, but its effect was much smaller than the enhancement due to curvature, which is consistent with ethanol's comparatively modest ability to perturb membrane lipids . The ethanol efficacy observed is as pronounced as any previously described lipid-mediated ethanol action . In addition, these observations raise the possibility that PITP specifically delivers PtdIns to metabolically active membrane domains of convex curvature and/or low surface densities of lipid. Microbiol Mol Biol Rev, 2000 Jun, 64(2), 435 - 59 Acetylation of histones and transcription-related factors; Sterner DE et al.; The state of chromatin (the packaging of DNA in eukaryotes) has long been recognized to have major effects on levels of gene expression, and numerous chromatin-altering strategies-including ATP-dependent remodeling and histone modification-are employed in the cell to bring about transcriptional regulation . Of these, histone acetylation is one of the best characterized, as recent years have seen the identification and further study of many histone acetyltransferase (HAT) proteins and their associated complexes . Interestingly, most of these proteins were previously shown to have coactivator or other transcription-related functions . Confirmed and putative HAT proteins have been identified from various organisms from yeast to humans, and they include Gcn5-related N-acetyltransferase (GNAT) superfamily members Gcn5, PCAF, Elp3, Hpa2, and Hat1: MYST proteins Sas2, Sas3, Esa1, MOF, Tip60, MOZ, MORF, and HBO1; global coactivators p300 and CREB-binding protein; nuclear receptor coactivators SRC-1, ACTR, and TIF2; TATA-binding protein-associated factor TAF(II)250 and its homologs; and subunits of RNA polymerase III general factor TFIIIC . The acetylation and transcriptional functions of these HATs and the native complexes containing them (such as yeast SAGA, NuA4, and possibly analogous human complexes) are discussed . In addition, some of these HATs are also known to modify certain nonhistone transcription-related proteins, including high-mobility-group chromatin proteins, activators such as p53, coactivators, and general factors . Thus, we also detail these known factor acetyltransferase (FAT) substrates and the demonstrated or potential roles of their acetylation in transcriptional processes. Biol Chem, 2000 Apr, 381(4), 337 - 42 Genomic organization, expression analysis, and chromosomal localization of the mouse PEX3 gene encoding a peroxisomal assembly protein; Muntau AC et al.; The peroxin Pex3p has been identified as an integral peroxisomal membrane protein in yeast where pex3 mutants lack peroxisomal remnant structures . Although not proven in higher organisms, a role of this gene in the early peroxisome biogenesis is suggested . We report here the cDNA cloning and the genomic structure of the mouse PEX3 gene . The 2 kb cDNA encodes a polypeptide of 372 amino acids (42 kDa) . The gene spans a region of 30 kb, contains 12 exons and 11 introns and is located on band A of chromosome 10 . The putative promoter region exhibits characteristic housekeeping features . PEX3 expression was identified in all tissues analyzed, with the strongest signals in liver and in testis, and could not be induced by fenofibrate . The data presented may be useful for the generation of a mouse model defective in PEX3 in order to clarify the yet unknown functional impact of disturbances in early peroxisomal membrane assembly. J Radiat Res (Tokyo), 2000 Mar, 41(1), 9 - 17 Positional cloning and functional analysis of the gene responsible for Nijmegen breakage syndrome, NBS1; Tauchi H; Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, combined immunodeficiency, and a high incidence of lymphoid tumor . Cells from NBS patients show chromosomal instability, hypersensitivity to ionizing radiation and abnormal p53-mediated cell cycle regulation . We cloned the underlying gene for NBS, designated NBS1, by complementation-assisted positional cloning from the candidate region 8q21 . Large genomic sequencing, as well as a search using computer programs, provides a powerful approach for identifying the underlying gene for a disease . The NBS1 gene encodes a protein of 754 amino acids that has FHA and BRCT domains which often are conserved in cell-cycle checkpoint proteins . The gene has weak homology to the yeast (Saccharomyces cerevisiae) Xrs2 protein in the N-terminus region . Like yeast Xrs2, the NBS1 protein forms a complex with hRAD50/hMRE11, and the complex is condensed as foci in the nucleus after irradiation, indicative that this triple-complex is a crucial factor in DNA repair . Functional analysis of the NBS1 protein is in progress and it should provide further clues to understanding the repair mechanism of radiation-induced DNA double-strand breaks. Mol Biochem Parasitol, 2000 May, 108(2), 237 - 47 Plasmodium falciparum erythrocyte membrane protein 1 is anchored to the actin-spectrin junction and knob-associated histidine-rich protein in the erythrocyte skeleton; Oh SS et al.; A distinctive pathological feature of Plasmodium falciparum malaria is the endothelial attachment of erythrocytes infected with mature asexual-stage parasites in microvessels of the major organs . Electron-dense protrusions described as knobs are displayed on the surface of parasitized erythrocytes and act as attachment points in cytoadherence . Parasite-encoded knob-associated histidine-rich protein (KAHRP) is a major component of knobs found on the cytoplasmic side of the host cell membrane . P . falciparum erythrocyte membrane protein 1 (PfEMP1) is a family of parasite-encoded cytoadherence receptors localized to knobs on the surface of parasitized erythrocytes . Despite its high antigenic diversity, PfEMP1 has a remarkably conserved cytoplasmic domain . We demonstrate in this study that the cytoplasmic domain of PfEMP1 (VAR(CD)) binds to host spectrin and actin and to full-length KAHRP in vitro . Apparent dissociation constants determined for VAR(CD)/F-actin and VAR(CD)/KAHRP interactions are 44.9+/-6.4 and 10 . 7+/-2.2 nM, respectively . Further, we provide evidence that KAHRP polypeptides self-associate in solution to form structures similar to knobs and show binding of self-associated KAHRP clusters to spectrin-actin-protein 4.1 complexes . Findings in this study suggest that PfEMP1 is localized to the knob in P . falciparum-infected erythrocytes by binding to the host spectrin-actin junction and to self-associated KAHRP through its conserved cytoplasmic domain. Reprod Toxicol, 2000 May-Jun, 14(3), 193 - 205 Detection of dopaminergic modulators in a tier I screening battery for identifying endocrine-active compounds (EACs); O'Connor JC et al.; Apomorphine (APO; D(2) receptor agonist), haloperidol (HAL; D(2) receptor antagonist), and reserpine (RES; a dopamine depletor that acts to lower brain dopamine levels by depleting central nervous system monoamines via disrupting storage vesicle function) have been examined in a Tier I screening battery, which has been designed to detect endocrine-active compounds (EACs) . The Tier I battery incorporates two short-term in vivo tests (a 5-day ovariectomized female battery and a 15-day intact male battery using Sprague-Dawley rats) and an in vitro yeast transactivation system (YTS) . In addition, two blood collection procedures were evaluated for their utility in detecting HAL-induced increases in serum prolactin (PRL) levels (i.e., the stress associated with each procedure) . In the in vivo female battery, both HAL and RES increased serum PRL concentrations as expected, although the increase caused by RES was marginal . Increases in serum PRL levels are enhanced when daily dosages are administered via multiple-daily dosing of the test compound, which results in higher sustained blood levels of the test compounds . APO failed to decrease serum PRL concentrations in the female battery . In the in vivo male battery, HAL increased serum PRL concentrations as expected . However, APO and RES failed to affect serum PRL concentrations . The blood collection comparison experiment demonstrated that possible confounding of the data can occur with serum PRL concentrations when animals are exposed to stress . Basal levels of PRL were approximately fourfold higher in animals that were bled via the tail vein procedure when compared to PRL levels from animals that were bled under CO(2) anesthesia at euthanization . As a result of the higher basal PRL levels, the HAL-induced increase in serum PRL concentrations was completely attenuated in the tail-vein bled animals (1.3-fold) . In contrast, HAL produced a fivefold increase in serum PRL in animals where blood was collected under CO(2) anesthesia at euthanization . Hence, collection of blood from animals under CO(2) anesthesia at euthanization is an acceptable approach for detection of compounds that increase PRL . In summary, HAL-like compounds would be identified in the Tier I male and female battery primarily via increased serum PRL concentrations . RES-like compounds would be identified in the Tier I male battery via decreased gonadotropins and steroids and possibly in the Tier I female battery by a minimal increase in serum PRL concentrations . Compounds that produce a marginal increase in serum PRL when administered using single daily dosing can also be confirmed in an in vivo female battery with multiple dosing because this regimen increases the magnitude of the PRL increase . APO, a D(2) receptor agonist, was not detected in the in vivo male or female batteries, but in both instances the top dosage produced minimal decreases in body weight (99 to 96% of control) . Hence, the proposed Tier I battery needs to be further evaluated with higher dosages of APO and other D(2) receptor agonists to determine whether it is capable of detecting such agents. J Biol Chem, 2000 Jun 9, 275(23), 17536 - 40 MTABC3, a novel mitochondrial ATP-binding cassette protein involved in iron homeostasis; Mitsuhashi N et al.; Atm1p, a mitochondrial half-type ATP-binding cassette (ABC) protein in Saccharomyces cerevisiae, transports a precursor of the iron-sulfur (Fe/S) cluster from mitochondria to the cytosol . We have identified a novel half-type human ABC protein, designating it MTABC3 (mammalian mitochondrial ABC protein 3) . MTABC3 mRNA is ubiquitously expressed in all of the rat and human tissues examined . MTABC3 protein is shown to be present in the mitochondria, as assessed by immunoblot analysis and confocal microscopic analysis of subcellular fractions of Chinese hamster ovary cells stably expressing MTABC3 . Accumulation of iron in the mitochondria, mitochondrial DNA damage, and respiratory dysfunction in the yeast ATM1 mutant strain (atm1-1 mutant cells) were almost fully reversed by expressing MTABC3 in these mutant cells . These results indicate that MTABC3 is a novel ortholog of the yeast and suggest an important role in mitochondrial function . Interestingly, the human MTABC3 gene has been mapped to chromosome 2q36, a region within the candidate locus for lethal neonatal metabolic syndrome, a disorder of the mitochondrial function associated with iron metabolism, indicating that MTABC3 is a candidate gene for this disorder. J Biol Chem, 2000 Aug 18, 275(33), 25840 - 9 Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting; Scott SV et al.; We have been studying protein components that function in the cytoplasm to vacuole targeting (Cvt) pathway and the overlapping process of macroautophagy . The Vac8 and Apg13 proteins are required for the import of aminopeptidase I (API) through the Cvt pathway . We have identified a protein-protein interaction between Vac8p and Apg13p by both two-hybrid and co-immunoprecipitation analysis . Subcellular fractionation of API indicates that Vac8p and Apg13p are involved in the vesicle formation step of the Cvt pathway . Kinetic analysis of the Cvt pathway and autophagy indicates that, although Vac8p is essential for Cvt transport, it is less important for autophagy . In vivo phosphorylation experiments demonstrate that both Vac8p and Apg13p are phosphorylated proteins, and Apg13p phosphorylation is regulated by changing nutrient conditions . Although Apg13p interacts with the serine/threonine kinase Apg1p, this protein is not required for phosphorylation of either Vac8p or Apg13p . Subcellular fractionation experiments indicate that Apg13p and a fraction of Apg1p are membrane-associated . Vac8p and Apg13p may be part of a larger protein complex that includes Apg1p and additional interacting proteins . Together, these components may form a protein complex that regulates the conversion between Cvt transport and autophagy in response to changing nutrient conditions. J Biol Chem, 2000 Oct 20, 275(42), 32966 - 73 Aut7p, a soluble autophagic factor, participates in multiple membrane trafficking processes; Legesse-Miller A et al.; Aut7p, a protein recently implicated in autophagic events in the yeast Saccharomyces cerevisiae, exhibits significant homology to a mammalian protein, p16, herein termed GATE-16 (Golgi-associated ATPase Enhancer of 16 kDa), a novel intra-Golgi transport factor . Here we provide evidence for the involvement of Aut7p in different membrane trafficking processes . Aut7p largely substitutes for the activity of GATE-16 in mammalian intra-Golgi transport in vitro . In vivo, AUT7 interacts genetically with endoplasmic reticulum to Golgi SNAREs, specifically with BET1 and SEC22 . Aut7p interacts physically with the following two v-SNAREs: Bet1p, which is involved in endoplasmic reticulum to Golgi vesicular transport, and Nyv1p, implicated in vacuolar inheritance . We suggest that, in addition to its role in autophagocytosis, Aut7p has pleiotropic effects and participates in at least two membrane traffic events. Curr Biol, 2000 Jun 1, 10(11), 675 - 8 Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function; Brady DM et al.; The spindle checkpoint delays the metaphase to anaphase transition in response to defects in kinetochore-microtubule interactions in the mitotic apparatus (see {1} {2} {3} {4} for reviews) . The Mad and Bub proteins were identified as key components of the spindle checkpoint through budding yeast genetics {5} {6} and are highly conserved {3} . Most of the spindle checkpoint proteins have been localised to kinetochores, yet almost nothing is known about the molecular events which take place there . Mad1p forms a tight complex with Mad2p {7}, and has been shown to recruit Mad2p to kinetochores {8} . Similarly, Bub3p binds to Bub1p {9} and may target it to kinetochores {10} . Here, we show that budding yeast Mad1p has a regulated association with Bub1p and Bub3p during a normal cell cycle and that this complex is found at significantly higher levels once the spindle checkpoint is activated . We find that formation of this complex requires Mad2p and Mps1p but not Mad3p or Bub2p . In addition, we identify a conserved motif within Mad1p that is essential for Mad1p-Bub1p-Bub3p complex formation . Mutation of this motif abolishes checkpoint function, indicating that formation of the Mad1p-Bub1p-Bub3p complex is a crucial step in the spindle checkpoint mechanism. Curr Biol, 2000 Jun 1, 10(11), 667 - 70 Genetic identification of neurons controlling a sexually dimorphic behaviour; Sylvain G et al.; In the fruit fly Drosophila melanogaster, locomotor activity is sexually dimorphic: female flies constantly modulate their activity pattern whereas males show a steadier, stereotyped walking pace {1} . Here, we mapped the area of the brain controlling this behavioural dimorphism . Adult male Drosophila expressing a dominant feminising transgene in a small cluster of neurons in the pars intercerebralis exhibited a female-like pattern of locomotor activity . Genetic ablation of these neurons prevented the feminisation of the locomotor activity of transgenic males . The results suggest that this cluster of neurons modulates sex-specific activity, but is not involved in initiating fly locomotion . Nor does it control male courtship behaviour, because feminisation of courtship was not correlated with the feminisation of locomotor activity. Curr Biol, 2000 Jun 1, 10(11), 630 - 9 Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required for MAP kinase signaling in vivo; Drogen F et al.; BACKGROUND: Many signals are transduced from the cell surface to the nucleus through mitogen-activated protein (MAP) kinase cascades . Activation of MAP kinase requires phosphorylation by MEK, which in turn is controlled by Raf, Mos or a group of structurally related kinases termed MEKKs . It is not understood how MEKKs are regulated by extracellular signals . In yeast, the MEKK Ste11p functions in multiple MAP kinase cascades activated in response to pheromones, high osmolarity and nutrient starvation . Genetic evidence suggests that the p21-activated protein kinase (PAK) Ste20p functions upstream of Ste11p, and Ste20p has been shown to phosphorylate Ste11p in vitro . RESULTS: Ste20p phosphorylated Ste11p on Ser302 and/or Ser306 and Thr307 in yeast, residues that are conserved in MEKKs of other organisms . Mutating these sites to non-phosphorylatable residues abolished Ste11p function, whereas changing them to aspartic acid to mimic the phosphorylated form constitutively activated Ste11p in vivo in a Ste20p-independent manner . The amino-terminal regulatory domain of Ste11p interacted with its catalytic domain, and overexpression of a small amino-terminal fragment of Ste11p was able to inhibit signaling in response to pheromones . Mutational analysis suggested that this interaction was regulated by phosphorylation and dependent on Thr596, which is located in the substrate cleft of the catalytic domain . CONCLUSIONS: Our results suggest that, in response to multiple extracellular signals, phosphorylation of Ste11p by Ste20p removes an amino-terminal inhibitory domain, leading to activation of the Ste11 protein kinase . This mechanism may serve as a paradigm for the activation of mammalian MEKKs. Curr Biol, 2000 Jun 1, 10(11), R407 - 9 Membrane transport: tethers and TRAPPs; Lowe M; Transport vesicles are tethered to their target membrane prior to the interaction of v-SNAREs and t-SNAREs across the membrane junction . Recent evidence suggests tethering is a complex process requiring multiple components. RNA, 2000 May, 6(5), 730 - 43 Mtt1 is a Upf1-like helicase that interacts with the translation termination factors and whose overexpression can modulate termination efficiency; Czaplinski K et al.; Translation termination is the final step that completes the synthesis of a polypeptide . Premature translation termination by introduction of a nonsense mutation leads to the synthesis of a truncated protein . We report the identification and characterization of the product of the MTT1 gene, a helicase belonging to the Upfl-like family of helicases that is involved in modulating translation termination . MTT1 is homologous to UPF1, a factor previously shown to function in both mRNA turnover and translation termination . Overexpression of MTT1 induced a nonsense suppression phenotype in a wild-type yeast strain . Nonsense suppression is apparently not due to induction of {PSI+}, even th