PROTEIN IMPORT

A homology search using the SECI sequence (1) revealed a significant homology at the protein level with two other yeast genes, SLYI and SLPI (2). These three proteins are involved in protein secretion and intracellular transport in yeast, each at a different step: Slylp between the endoplasmic reticulum and Golgi, Slplp between Golgi and the vacuole and Seclp between Golgi and the plasma membrane. It is likely that these proteins serve a similar function, but each of them seems to display specificity for a given step in the secretory pathway. In order to further characterize the function of SECI two multicopy suppressors of secl-I temperature-sensitive muation were cloned from a cDNA library. Further analysis of these new yeast genes including DNA-sequencing and disruption of the chromosomal genes is under way and the results obtained will be presented.

of the substrate.Formation of the multiubiquitin chain is required for subsequent degradation of at least some N-end rule substrates.
To analyze the mechanism of ubiquitin-dependent protein degradation by the N-end rule pathway, we converted yeast triosephosphate isomerase (TIM), a homodimeric protein of known three-dimensional structure, into an N-end rule substrate.In vivo experiments demonstrated that conversion of any of the 21 lysine residues in each of the TIM monomers into arginines (which cannot be ubiquitinated) does not significantly inhibit the N-end rule-mediated degradation of the modified TIM.These and related findings indicated that at least a few of TIM'S lysines can serve as ubiquitin acceptors.We will also discuss the results of experiments to determine whether the targeting of a multiubiquitination-site lysine in an N-end rule substrate proceeds by a "scanning" mechanism (in which the binding of a targeting complex to a destabilizing N-terminal residue of a substrate is followed by a directional scanning of its polypeptide chain until N-terminus-proximal lysine is encountered) or whether a "stochastic capture" mechanism is involved (in which the capture of a relevant lysine depends on its mobility and spufial proximity to the substrate's N-terminal residue).The attachment of N-linked carbohydrates to proteins is thought to occur during or shortly after translocation of the nascent polypeptide chain into the lumen of the endopladc reticulum (1,2).Transfer of the oligosaccharide to the polypeptide backbone requires the acceptor sequence Asn-X-Thr/Ser on the peptide chain.The presence of an acceptor site, however, does not always lead to glycosylation.Analysis of sequence differences between glycosylated and non-glycosylated secretory proteins could not find obvious sequence features in the vicinity of acceptor sites that would correlate with the absence or presence of N-linked glycosylation ( 3 ) .On the assmption that glycosylation occurs cotranslocationally, it has been suggested that the locat$on of the acceptor site on the nascent peptide chain could correlate with accessibility for glycosylation, in particular the probability of glycosylation would be strongly reduced for C-terminally located sites.
In an attenpt to obtain steric distortions along the molecule of human IL-lp secreted by S.cerevisiae (4), we have introduced the consensus sequence for N-linked glycosylation at six different positions of the IL-1p molecule.Since the crystal structure of this protein has been determined ( 5 ) and six of the exposed loops of the molecule contain an Asn residue, the mutant acceptor sites were created by substituting amino acids at position t2 from Asn residues with Thr or Ser.Analysis of secreted recombinant protein, produced in S.cerevisiae transformed with the six different constructs, showed that only three mutant acceptor sites were glycosylated.Structural characterization of the mutated proteins by computer modelling revealed that the three non-glycosylated sites had the side chain of the t2 residue (Thr/Ser) hidden within the folded structure, Exchange between acceptor sites did not affect the glycosylation pattern thus indicating that position in the structure rather than sequence "per sen determines the modification.
A portion of the Saccharomyces cerevisae Mnnl protein was purified as a TrpE fusion from E. coli and used to prepared a polyclonal antisera to this protein.By immunoprecipitation from labeled cells, we found that the antisera recognized a single polypeptide from wild-type yeast cells, which was overproduced 50 fold in a strain harboring the M N N l gene on a 2~ based vector, and was absent from a strain bearing a chromosomal deletion of the M N N Z gene.The mnnl mutant lacks the Golgi al->3 mannosyltransferase activity suggesting a direct relationship between this gene and enzyme.
To test this, we have purified the Mnnl protein by native immunoprecipitation from the MNNZ overexpressing strain and assayed this protein for al->3 mannosyltransferase activity.The immune pellet was able to catalyze the transfer of mannose from UDP-mannose to a MansGlcNacz substrate (prepared from alg3,secld glycoproteins synthesized at the nonpermissive temperature) in an a1 ->3 linkage.Immune pellets prepared from a AmnnZ strain, or using pre-immune serum, did not exhibit any mannosyltransferase activity.
The sequence of the M N N Z gene (V.MacKay, in preparation) predicts a type 11 transmembrane protein of 85 kDa with four sites for N-linked oligosaccharide addition.In pulse-chase experiments we found that the al->3 mannosyltransferase was initially synthesized as a 98.5 kDa protein.Treatment with endoglycosidase H reduced the molecular weight to 92.5 kDa suggesting that only three of four Asn-X-Ser/Thr sites are used.The protein was very stable, with a half life of -5 hours.Following synthesis, the al->3 mannosyltransferase underwent a slow, incremental increase in molecular weight (creepup) such that by three hours the relative mobility had increased to 107 kDa.This relative increase in mass was retained after removal of N-linked oligosaccharides with endoglycosidase H.As the protein increased in size, it was precipitated more efficiently with an antibody to al->3 mannose residues.These results suggest that the slow modification is at least partly due to self-modification of 0-linked oligosaccharides with al->3 linked mannose.The al->3 mannosyltransferase did not creep up in molecular weight in secZ2 or secld mutants at the nonpermissive temperature.The modification was partially blocked in a sec7 mutant and was apparently unaffected in a secl4 or s e d mutant at the nonpermissive temperatures.The subcellular distribution of the al->3 mannosyltransferase was assessed by indirect immunofluorescent staining of yeast cells using the antibody directed to the Mnnlp.We see a punctate distribution of fluorescent staining that is similar to what has been seen with Kex2p (Redding et al., JCB 113, 527-538) and Sec7p (Franzusoff et al., JCB 112,27-37).Together, these results indicate that the al->3 mannosyltransferase is a resident protein of the yeast Golgi.This is consistent with our previous work in which the modification catalyzed by the a l -> 3 mannosyltransferase was proposed to occur in a distinct medial Golgi compartment (Graham and Emr, JCB 114,207-218).We are studying the mechanistic and quantitative aspects of the secretion of heterologous proteins by S. cerevisiue, using guar a-galactosidase as a model enzyme.
To determine the effect of the signal sequence on the secretion, we fused the yeast-derived invertase and prepro-a-factor signal sequences to the a-galactosidase gene.Using a 2pm-based plasmid and the constitutive PGK promoter to express these gene fusions in yeast, we found no differences in terms of production level (40 m d ) , but the secretion efficiency was higher in the case of the preproa-factor signal sequence (98%) when compared to the invertase signal sequence (90%).Western blot analysis and N-terminal protein sequencing showed that, when secretion was directed by the prepro-sequence of the a-factor, removal of the Glu-Ala dipeptides by the STEl3 gene product did not occur.Western blot analysis also demonstrated that, when secretion was directed by the invertase signal sequence, the intracellular enzyme is only core-glycosylated, suggesting that it is present in the ER.This localisation was confirmed by immunogold electron microscopy, and identifies the ER-Golgi transport step as the rate-limiting step in secretion of a-galactosidase directed by the invertase signal sequence.However, using the prepro-a-factor signal sequence, the intracellular enzyme was outerchain glycosylated and processed by the KEX2 gene product, showing that it had left the ER.Null mutants of the PMRl gene are known to give higher secretion efficiencies for a number of different heterologous proteins.Therefore, we also tested the secretion of a-galactosidase fused to either signal sequcnce in a pmrl disruption mutant.Surprisingly, the pmrl disruption resulted in a twofold increase in the level of secreted a-galactosidase.This indicates that some of the a-galactosidase expressed in the wild-type strain is degraded, whereas, in a pmrl mutant, this degradation is reduced.To explain the pmrl phenotype, Rudolph et al. [Cell 58 (1989), 133-1451 postulated a bypass hypothesis.According to the most extreme version of this hypothesis, vesicles derived from the ER do not fuse with the first Golgi compartment, but fuse directly with the plasma membrane, bypassing multiple Golgi compartments.However, we found that processing by the KEX2 gene product.which is known to be localised in a late Golgi compartment, is intact in apmrl mutant, which contradicts the hypothesis of Rudolph et al.The constitutive secretory pathway of Sacchammyces cerevkiue has proven to be an efficient system for production of many biopharmaceuticals, including insulin, interferon and several human rowth factors.The advantages of heterologous protein expression in yeast include the abfiity to attain high levels of protein expression, the ability to easily high culture densities, and the ability to perform extensive genetic mani ulations.% : absolute levels of heterologous protein secretion are often high, the {nal 'eld of the desired product can be compromised by the presence of improperly foldedlmolecules.Since a number of yeast roteins have been recently implicated in rotein folding of nascent pol eptides in tfe endoplasmic reticulum, it is now ossigle to genetically secretion.
We have been studying the expression of human insulin-like growth factor I (IGFI) in S. cerevkiue.Authentic IGFI is made as a prohormone, which is post-translationally rocessed to a polypeptide of 70 amino acids and contains three intramolecular disulfide gonds.Expression of the mature portion of IGFI in yeast results in the secretion of correctly folded IGFI as well as an isomeric b -product which contains two of the three disulfide bonds interchanged (scrambled IGFII and higher molecular weight aggregates which are disulfide linked. In the present study, we have asked whether manipulations of the intracellular concentration of protein disulfide isomerase (PDI) can reduce the levels of scrambled and aggregated IGFI molecules.Protein disulfide isomerase is a ubi uitous protein of the ER structure is highly conserved between species and the yeast homolo e has been recently cloned by several grou s.To elevate expression levels of yeast FDI, the PDI coding sequences were placeain front of a strong, constitutive promoter (glyceraldehyde-3phosphate dehydrogenase -This construct was either integrated into the chromosome or placed on a high-copy number yeast plasmid, providing two different levels of PDI overexpression.PDI expression levels were compared in yeast strains containing either (1) the wild-PDI gene (2) a single copy of PDI under the GAPH promoter or (3) multico ies of I under the GAPH promoter.Both yeast strains containin the GAPH-PD?construction demonstrated elevated levels of PDI activity in vim.BGFI production was also analyzed in these various strains to determine the effect of PDI overexpression on the production of scrambled and aggregated IGFI.manipulate Eese proteins to optimize S. cerevkiue for high-leve P heterologous protein which catalyzes the in vitro isomerization of intramolecular disu 7 fide bonds.Its primary GAPH).We have identified a heterocyclic compound which inhibits vacuole biosynthesis in Saccharomyces cerevisiae.We have generated mutants which are resistant to this compound and prepared a gene library from one of them.By transfwning the gene library into a sensitive strain we could identify the gene responsible for this resistance phenotype.
The gene codes for a protein of 183 amino acids and shows considerable homologies to gene SEC 18.A number of the S. cermisiae vacuolar protein sorting (ups) mutants exhibit an unique vacuolar morphology.Unlike wild-type cells that contain 1-5 large vacuolar structures, these class B mutant cells contain 20-40 smaller vacuole-like compartments.Previous studies have demonstrated that the class B "fragmented vacuoles" accumulate dyes used-to stain wild-type vacuoles and appear to have a similar acidic pH.However, these mutant organelles do not serve as appropriate localization targets for the soluble vacuolar hydrolase carboxypeptidase Y (CPY).It is unclear if the organelles accumulated in the class B mutants are intermediates in vacuole formation or by-products of vacuole disassembly.
To explore the role of VPS gene products in vacuole formation/stability and vacuolar protein localization, the wild-type allele of one of the class B mutants, up&, was cloned by complementation of the vacuolar protein sorting defect exhibited by ups5 mutants.The VPS5 gene is predicted to encode a very hydrophilic protein of 675 amino acids that lacks any significant sequence homology with other known proteins.A TrpE-Vps5 fusion protein has been used to generate polyclonal antiserum directed against the VPS5 gene product.This antiserum detects a single, VPS5 specific, cytoplasmic protein that is not modified with N-linked oligosaccharides, but is phosphorylated specifically on a serine residue(s).Subcellular fractionation studies indicate, that Vps5p is associated with a large, sedimentable complex.This association appears to involve protein-protein interactions and is saturable; when Vpdp is overproduced, the vast majority of the protein remains in a soluble fraction.Initial biochemical cross-linking studies have potentially identified at least one other protein component of this complex.
Gene disruption experiments have shown that the VPS5 gene product is not essential for cell viability.However, cells carrying the null allele contain fragmented vacuoles and exhibit differential defects in vacuolar protein sorting.>95% of CPY was secreted from these cells in its Golgi modified p2 precursor form.Another soluble vacuolar hydrolase, proteinase A, was largely retained by the cells and 30% was present in its mature vacuolar form.The vacuolar membrane protein, alkaline phosphatase, was completely retained in AUpS5 cells, with >95% present as the mature vacuolar form.These results indicate that at least some aspect(s) of the vacuolar protein sorting and/or delivery system may remain intact in AVpS5 cells.This differential vacuolar protein sorting phenotype is not limited to ups5 mutants, but is shared by a small group of ups mutants (some of which exhibit a even more pronounced differential sorting defect).The VPS gene product functions perturbed in these mutants appear to be required for the vacuolar delivery of only a subset of soluble proteins.Based in these observations, we propose that alternative pathways may exist for the sorting and/or delivery of soluble proteins to the vacuole.The mitochondrial genome encodes only a small fraction of the total mitochondrial proteins.T o reach the mitochondrial matrix, nuclear-encoded precursor proteins have to cross t h e outer and the inner mitochondrial membranes.A few of the proteins involved in translocation across the outer membrane have been identified (Baker and Schatz, 1991).In contrast, very little is known about translocation across the inner mitochondrial membrane.
Our laboratory has evidence that the inner mitochondrial membrane c o n t a i n s a protein translocation machinery that is distinct from the translocation system of the o u t e r membrane.T h e s e two translocation machineries appear to act in tandem (Geli and Glick, 1990).
Import of radiolabelled artificial precursor proteins into right-side-out inner membrane vesicles is inhibited by antisera against inner membrane proteins; antisera against o u t e r ' membranes o r against s p e c i f i c outer membrane components like ISP42 (Vestweber et al., 1989) do not inhibit.
In order to identify inner membrane proteins involved in protein import, inner membranes were solubilized and then fractionated by anion e x c h a n g e chromatography.T h e proteins present in each fraction were coupled to cyanogen bromide-activated Sepharose beads.These affinity beads were then used to affinity-purify antibodies from the complex antiserum.The affinity purified antibodies were tested for their ability to inhibit the import of artificial precursor proteins into inner membrane vesicles.
T h e polypeptides recognized by the inhibiting a n t i b o d i e s were identified by Western blot analysis.One of the affinity-purified antibodies recognized a protein which was only found in the column fractions used to affinity purify them.This protein has a molecular weight of 40,000 and can quench the inhibitory capacity of the antibody.O u r preliminary results suggest that the 40 kDa protein is a component of the mitochondrial inner m e m b r a n e translocation machinery.The secretory system of eukaryotic cells consists of a series of membrane-bound organelles (including the Endoplasmic Reticulum (ER) and Golgi apparatus) through which proteins destined for secretion from the cell move sequentially.
The fungal toxin Brefeldin A (BFA) has been shown to dramatically alter the morphology of certain secretory organelles in mammalian cells and to block protein transport.The first observed morphological effect of BFA on mammalian cells is the complete disappearance of the Golgi apparatus as a distinct organelle, and the appearance of normally Golgi-localized proteins in the ER.It has recently been shown that BFA also affects other organelles within the cell.In the presence of BFA, the TGN (trans Golgi network) fuses with the endosomes via microtubule-dependent tubular processes, and traffic from the endosome system to the lysosome is disrupted.In addition, the lysosome breaks down into tubular structures.
These and other data indicate that there are multiple targets of BFA within the cell, and also that there is a general mechanism for maintenance of organelle structure and protein trafficking within the cell.Identification of the targets of BFA will be very important in elucidating this m e c h a n i s m .
We have been attempting to identify the targets of BFA by a molecular genetic approach using S .cerevisiae.A wild type strain of S. cerevisiae is resistant to very high levels of BFA, so the first step in the project was the identification of a mutant strain sensitive to BFA.We demonstrated that treatment of this strain with BFA at a concentration of 100pg/ml blocks cell division completely, and leads to a viability of only 10% after 8 hours of treatment.Preliminary evidence indicates that at least two secretory proteins (a-factor and carboxy-peptidase Y) are accumulated in the ER when sensitive cells are treated with BFA.These results indicate that in S .cerevisiae, as in mammalian cells, BFA blocks protein transport from the ER to the Golgi apparatus.
Our approach to identifying BFA targets has been to express a yeast genomic library carried within a 2-micron vector in BFA-sensitive cells, and to select BFAresistant clones.This approach has been used previously to identify the targets in S .cerevisiae of at least three other drugs.We have identified one good candidate gene for a target of BFA.This gene present on a multicopy vector results in a level of resistance to the lethal effects of BFA that is 30-fold higher than a strain carrying the vector alone.In addition, this gene in multiple copies suppresses to a limited extent the temperature-sensitive lethality of two mutants defective in ER to Golgi transport.This result suggests that the gene identified is involved in ER to Golgi transport and hence that its protein product may be a target of BFA involved at this stage of the secretory p a t h w a y .indicates that the phosphatidylcholine analog is metabolized in the vacuole and that this metabolism is dependent on the intemalmd via a non-endocyric route, and are tnulsported by this m t e to the nuclear envelope and mitochondria.This m t e functions with a lower cellular energy charge (ATP levels 80% depleted) than the docytic mute, and is not dependent on cells with N-ethylmalehide, and thus bears resemblance to the ptein-mediated flipping of phospholipid analogs that has been previcusly shdied in mammalian systems (M&LQ and WR.; J. Biol.Chem.phosphatidylcholine f l i p activity in the yeast plasma membrane.We have also studied the internahtion of a fluomcent phosphatidylethanolamine analog and find that this analog is transported to the nuclear envelope and mitochondria, but not to the vacuole.We therefore infer that the phosphatidylethanolatnine analog is inkmalid exclusively through a non-endocytic pathway, presumably through the action of a flippase.The putative phosphatidyledmnolamine flippase differs from its phosphatidylcholine c in that it is partially inhibited by depleted cellular ATP levels.By using the assays for lipid interdm ' tion described here, we are now isolating mutants in both the endocytic and non-endocytic pathways in order to identify and clone the responsible genes.
gene, or on the gene encoding the clathrin heavy chain, w.How does a polar protein cross a lipidic and continuous membrane ?An instance of such a phenomenon is provided in eukaryotes by the translocation of polypeptides from their site of synthesis, the cytoplasm, to the lumen of the endoplasmic reticulum.We have approached this process in Saccharomyces cerevisiae through the study of a gene, SSS1, that restores at elevated copy number a wild-type phenotype in the translocation mutant sec61.
SSSl is an essential gene that encodes a 8,900 dalton basic protein (Ssslp).Although most of Sssl p is hydrophilic, with only two short hydrophobic stretches close to its c a b x yterminus, it fractionates after gentle cell lysis in a 1O.OOOg pellet.This association with a rapidly sedimenting material is resistant to treaments with high salt, urea, carbonate or hydroxylamine.
Dissociation occurs in presence of 0.5% Triton X-100.This behavior indicates that Ssslp is an integral membrane protein that probably localizes in the endoplasmic reticulum.Analysis of the vicinity of Sssl p by chemical crosslinking with bifunctional reagents and immunoprecipitation has indeed confirmed this view.Furthermore, it revealed that this protein is in close spatial proximity to the components of the translocation machinery encoded by SEC61, SEC62and SEC63.Does the SSSl gene play a direct role in translocation, as suggested by the localization of its product and by its interaction with the SEC61 gene ?To address this possibility, a conditional SSSl expression mutant was constructed so as to express the gene at wild-type levels under permissive conditions, i.e. on galactose medium.SSSl expression and cell growth are stopped by a shift to glucose medium.Precursor forms of secretory proteins start to accumulate within the cells 2-4 hours after a shift to these non-permissive conditions.This phenotype indicates that SSSl functions in preprotein translocation to the endoplasmic reticulum.The cell wall of yeast consists of an internal glucan layer probably interwoven with mannoproteins and a n external layer of mannoproteins.Most of the wall proteins can be extracted with hot detergent (SDS-extractable proteins): the remaining wall proteins can be set free by enzymatically dissolving the glucan layer indicating that they are intimately associated with the glucan layer.Although the glucanase-extractable proteins are less numerous than the SDS-extractable wall proteins, they represent the bulk of the mannoprotein layer in terms of mass.
Recently, we have shown Wan FUnsum et al. 1991) that one or more of the glucanase-extractable mannoproteins possess a unique type of side-chain characterized by the presence of beta-linked glucose residues.This raises the question if this sidechain is synthesized (1) at the cell surface by means of a transferase capable of attaching glucan chains to carbohydrate side-chains of the mannoproteins or (2)  intracellularly in one of the compartments of the secretion route through glucosyltransferases.To answer this question, the ts mutant sec6-4, in which secretion vesicles accumulate at the restictive temperature, was used.The plasma membrane fraction and the secretion vesicles were isolated by gel filtration Walworth and Novick 1987).and extracted with sodium carbonate to remove the luminal proteins and the peripheral membrane proteins.Then their sugar compositions were determined.Glucose Sodium carbonate-extracted membrane vesicles from the secretion route contain several glycolipid-anchored mannoproteins (Conzelmann et al. 1990).We propose, therefore, that the glucose found in sodium carbonate-extracted secretion vesicles (Table 1) belongs to glycolipid-anchored glucomannoproteins that presumably are precursors of the glucomannoproteins in the cell wall.This would also imply that the glucosylation of glucomannoproteins.at least partially, occurs intracellularly.The lower glucose content of the plasma membrane fraction is probably due to enzymic degradation of the glucose-containing side-chains by Zymolyase during spheroplasting.Lipoxygenases (EC 1.13.11.12) catalyze the oxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid into reactive cis-trans hydroperoxidiene intermediates which then sexve as substrates for other enzymes leading to the production of a variety of secondary metabolites.In order to explore the characteristics of the individual lipoxygenase isoenzymes in more detail larger amounts of the pure enzymes are needed and their production in an heterologous host is therefore desirable.Full length cDNAs encoding pea seed lipoxygenase isoenzymes 2 and 3 (1,2) were expressed in Saccharomyes cerrvirine with the aid of yeast-E.colishuttle vectors.Expression of the cDNA for lipoxygenase 2 under the control of the constitutive phosphoglycerate kinase (PGK) gene promoter yielded significant amounts of active enzyme inside the cell, both with yeast transformants carrying the cDNA gene on high copy number plasmids or integrated into chromosome V. Addition of the yeast invertase signal sequence in front of the pea lipoxygenase 3 yielded secreted active pea seed lipoxygenase in the medium, hut large amounts of inactive lipoxygenase 3 remained inside the yeast cell.Expression of LOX3 cDNA can be achieved either constitutively with the PGK promoter or inducibly with the GAL1 promoter (3).A change of the signal peptide could perhaps improve the level of secreted protein.Therefore, high copy and integration vectors were constructed containing the pre-pro-sequence from the yeast a-factor mating hormone in front of the LOX2 cDNA under the control of the PGK promoter and terminator.Southern blot experiments demonstrated 'that the transformed yeast strains contain the corresponding plasmid in a correct way.Although lipoxygenase 2 specific RNA was detected by Northern blot analysis neither intranor extracellular lipoxygenase protein could be detected.One possible explanation could be that after the addition of the approx.90 amino acids of the signal peptide to the already large protein proper folding is inhibited and the protein is degraded.Recently, it has been demonstrated that a lipoxygenase protein accumulates in the vacuoles of paraveinal mesophyll cells of soybean leaves (4).Also in the cotyledons of soybean seeds the lipoxygenase isoenzymes are located in storage protein bodies (5,6) which in legumes as well as in other plants are deposited in the vacuoles of cells in developing cotyledons during seed maturation.Since previous studies of plant lipoxygenases have failed to uncover signal sequences or associations with the endoplasmic reticulum and Golgi vesicles, it has been suggested that lipoxygenases are synthesized on free ribosomes and delivered through the cytosol and the tonoplast into the vacuoles.Experiments have been initiated to address the question of the site of synthesis and route for vacuole deposition.Vacuoles of transformed yeast strains were isolated on a discontinuous Ficoll-gradient and presence of pea lipoxygenase in them demonstrated by Western blot analysis.The SEC14 homologous gene has been disrupted in several haploid strains from differenr origins.The SGC14 delcted nilitants wcre viable but were unable to fomi true mycelium in liquid rich incdiuixi They form snmth colonies on solid rich mcdiuiii ill ~uiiuiist with rough colonies fonned by wild-type strains.

Reference%
The alkaline protease is an cxtncellolar enzyme that is synthesized as a p p p t i d e and then processed to mature forni probably in the Golgi appnrntris.We observed by irnniunoprccipitatioii experiments that the non-processed precursor is secrctcd in the ~vlture medium by the SEC14 homolog disruptant.Alteration of the Golgi appantus membrane conyosition could account for this phcnotypc.We are invcstigating the secretion of several proteins in this mutant.
Morphological and swrciion phenotypes were shown to be revened by introduction in the deletcd s h i n either the entire Y.lipoZyricu genc or its $.cerevisiae homologous fragment.We have cloned and characterized the W 1 5 0 -g e n e of Saccharomvces cerevisiae.It encodes a protein of 412 amino acids (hspl50), which is secreted efficiently and fast via the secretory pathway to the growth medium.During secretion the polypeptide chain is extensively O-glycosylated and processed by the kex2-protease to two subunits of 53 and 341 amino acids.According to Northern analysis, transcription of the -HSP150-gene is activated by heat shock.Consequently, the synthesis of the hspl50-protein is increased substantially and abruptly, whereafter heat-induction starts to decline.The w 1 5 0 -g e n e is preceeded by several heat shock element-like sequences (HSE).To study whether they are responsible for heat-regulation, the chromosomal M 1 5 0 -g e n e was disrupted, and the disruptant was transformed with plasmids where the coding region of W 1 5 0 , or a m150-&Z fusion gene, was preceeded with m 1 5 0 promotor regions of different lengths.Northern analysis showed that a single HSE, downstream of the TATA-box, was sufficient to confer heat shock-induced transcription.When this HSE was destroyed by site-directed mutagenesis, heat-regulation was lost.In addition to heat shock, the transcription of the W 1 5 0 -g e n e is regulated by the availability of nutrients in the growth medium.Transcription is activated under nitrogen-starvation conditions in the absence of heat shock.The promotor element responsible for nutrient-dependent regulation is within the first 137 nucleotides upstream of the structuralml5C-gene.This region bears some resemblance to the promotor region involved in nutrient-dependent regulation of the m 1 -g e n e .The SRP54 homologue from Y. lipolytica was cloned and sequenced in order to determine its in vivo role in the synthesis and secretion of secretory proteins.Degenerate primers designed based on the sequences the the SRP54 genes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mouse were used in a polymerase chain reaction to synthesize a probe for isolation of the SRP54 gene from a Y. lipolytica 1\ Charon 4 library.Sequencing of 2467 nucleotides revealed one long open reading frame of 1608 nucleotides coding for a polypeptide of 59 kilodaltons.The predicted polypeptide has 55%, 57% and 50% sequence identity to the SRP54 homologues of S. cerevisiae, S. pombe, and mouse, respectively.Like these SRP54 polypeptides, the Y. lipolytica SRP54 homologue has two domains -an N-terminal domain with a highly conserved GTP-binding site (G-domain) and a methionine rich C-terminal domain (M-domain).The SRP54 coding region has been deleted/disrupted in a diploid, and preliminary results suggest that the SRP54 gene is at least essential for spore germination.Next conditional mutations in the SRP54 gene will be isolated and their effects on synthesis and secretion of the alkaline extracellular protease of Y. liplolytica will be examined.A fragment containing prochymosin cDNA in which 13 nucleotides coding N-terminal sequence were replaced by short sequence derived from 1 inker was sandwiched between promoter-preprosequence of MFa ( S .cerevisiae) and MFa terminator in shuttle vector pJDBa containing in addition LEU;! and fragment of 2p DNA plasmid from S.cerevisiae, and ori and amp' from pBR322 (E.coli).The structure of recombinant DNA designed pJDBaCHE was confirmed by restriction mapping and analysis of phenotype of selected E.coli HB101, S.cerevisiae CRF18 (a, his3, leu21 and AH215 (a, his3, leu2) transformants.A proper phase of translation of fused gene-preproa-prochymosin cDNA was proved by immunological, biochemical and electrophoretic analysis of products secreted from S.cerevisiae transformants.
S.pombe LP36 (h-=, leul-32) and SA5-3c ( h ' " , leul-32) were transformed with pJDBaCHE.Chymosin proteins secreted from transformants selected on the basis of complementation of S.pombe LEU1 function by LEU2 ( S .cerevisiae) in pJDBaCHE were detected by milk-clotting assay, inhibition effect of pepstatin (the inhibitor of aspartyl proteases including chymosin) (no inhibition w a s observed after application of inhibitor of serine proteases-PKF), and imunoassay, using antibodies against chymosin.The results of these experiments demonstrate that (a) promoter-prepro sequence of MFa is accepted by transcription, translation and secretion system of S.pombe, (b) the expression of genes under the control of MFa promoter which is a-mating type specific in S.cerevisiae is not cross-regulated by S.pombe mating type specific genes, (c) S.cerevisiae E X 2 protease-like enzyme is likely to be also in S.pombe because an autocatalytic cleavage of chymosin precursor secreted from S.pombe would not occur from prolonged prosequence (KaSpar et al., Gene 67: 131, 1988).
SDS-PAAG electrophoretic analysis of products secreted from transformed cells at pH 3.6 combined with immunoblotting demonstrated the presence of two major bands (very likely pseudochymosin and/or chymosin and slower prochymosin) secreted also from S. cerevisiae CRFl8 (pJDBaCHE).The electrophoretic mobility of faster band was equal to chymosin standard (Mw=36 -1).
In addition several minor bands of prochymosin derived proteins secreted only from S.pombe(pJDBaCHE) were observed showing that posttranslational processing of preproa-prochymosin in S.pombe may proceed in slightly different way than in S.cerevisiae.Preliminary results of experiments in which proteins secreted from S. cerevisiae CRF18(pJDBaCHE) or S. pombe (pJDBaCHE) under various pH were separated by electrophoresis and "stained" with chymosin antibodies or concanavalin-peroxidase are supporting this view and showing that differences between the species in their protein glycosylation pathways should also be taken into account.Glaxo Institute for Molecular Biology , Chemin des Aulx , 1228 Planles -Ouates , Geneva, Switzerland.
We have previously described a temperaturesensitive mutant of Saccharomyces cerevisiae designated pmi40 which is defective in glycosylation and secretion due to a thermolabile phosphomannose isomerase (PMI) activity.Inactivation of PMI at the restrictive temperature of 370 C prevents synthesis of dolicholphosphate-mannose required for a number of critical mannosyl transfer reactions and results in cell death.Here we report the isolation of the PMZ 40 gene by complementation of the corresponding mutation.The PMZ 40 gene contains an efficiently spliced intron which differs from the majority of those so far identified in S .cerevisiae in that it is short and the branch forming structure has an AACTAAC motif replacing the highly conserved consensus TACTAAC.Deletion of the PMI coding sequence results in a strain which requires D-mannose for growth.The gene is located on chromosome V and its transcription is induced 12 fold when cells are grown on D-mannose as sole carbon source rather than D-glucose.Under both conditions , however, the level of protein measured either as specific enzyme activity or as immunocross-reactive material on Western blots is identical.The yeast Saccharomyces cerevisiae contains two forms of cytochrome c, iso-1-and iso-2-cytochrome c which are encoded by the nuclear genes CYCl and CYC7, respectively.The apo forms the iso-cytochromes c are synthesized in the cytosol, imported into mitochondria, and subsequently modified by covalent attatchment with heme through the action of cytochrome c heme lyase, which is encoded by CYC3 (1,2).C y d -mutants deficient in heme lyase do not appreciably import cytochrome c and completely lack the mature holo cytochromes c (3). Pulse-chase labelling of apo-iso-1-and apo-iso-2-cytochrome c in cyc3mutants demonstrated that apo-iso-1-cytochrome c has a very short half-life, as compared to the relatively stable apo-iso-2-cytochrome c, which is located primarily in the cytosol (4).It was hypothesized that a specific protease degrades apo-iso-1-cytochrome c but not apo-iso-2-cytochrome c.CYC2 encodes a factor required for efficient mitochondrial import of cytochrome c; consequently cyc2-A strains contain only approximately 30% of the normal level of iso-1-cytochrome c (5). Reversion of a CYCI' cyc2-A cyc7-A strain allowed us to select mutants with increased levels of holo-iso-1-cytochrome c.The increase could be due to overproduction or stabilization of iso-1-cytochrome c, or to a mutation of a gene required for degradation of apo-iso-1-cytochrome c.A single mutant gene, denoted cycl2, was found to be responsible for an increase in holo-iso-I-cytochrome c to 70% of the normal levels.The cycl2 gene was unlinked to CYCI, and cycl2-strains contained normal levels of CYCl mRNA.The stability of apo-iso-l-

cytochrome c was assessed in CYC12' and 171212-strains lacking heme lyase (cyc3-A CYCl+ cyc2-A cyc7-A).
Because the cycl2-strains had a significantly higher level of apo-iso-1-cytochrome c compared to CYCI2' strains, CYCl2 either encodes the specific protease, or a factor controlling its synthesis or activity.Carboxypeptidase Y (CPY) is a vacuolar protein which has been extensively studied as a model for intracellular protein transport and targeting in eukaryotes.The gene for CPY, P R C l , encodes a preproenzyme with a signal peptide of 20 amino acid residues followed by a pro-peptide of 91 residues.A four amino acid segment of the pro-peptide serves as signal for vacuolar targeting.The signal peptide is cleaved off in the endoplasmic reticulum where folding also takes place; pro-CPY is activated in the vacuole by removal of the propeptide in a process that depends on the vacuolar endopeptidase, proteinase A.
It has recently been demonstrated that the pro-peptide of CPY is necessary for correct and efficient folding of the enzyme in vitro.The aim of this work is to investigate the function of the CPY pro-peptide in the folding and transport of the enzyme in vivo.
Using the enzyme Ba131 we have constructed in-frame deletions within the region encoding the CPY pro-peptide without affecting the vacuolar targeting signal.These mutations encode zymogens which lack between 15 and 58 amino acids and have been expressed from a single copy plasmid in a Aprcl strain.While small deletions (< 30 residues) reduced the CPY activity to various degrees, large ones resulted in total loss of CPY activity.In addition to size, position of the deletion is important, but efficient folding is not dependent on a single, small sequence element.We have measured the rate of transport of the enzymes by pulse-labeling and immunoprecipitation of the lysates followed by SDS-PAGE.The transport through the secretory pathway of t h e truncated pro-enzymes is slower than that of the wild-type enzyme, and no secretion was observed.Assuming that transport from the endoplasmic reticulum is dependent on correct folding, these results suggest that the pro-peptide of CPY is necessary for the correct folding of the enzyme in vivo.The STE6 gene of Saccharomyces cerevisiae encodes a transporter which mediates the extracellular transport of the a-factor pheromone, a modified oligopeptide required for mating.The Ste6 transporter is composed of two homologous halves, each containing six predicted transmembrane domains and an intracellular loop with a consensus sequence for an ATP-binding motif.This unique architecture is the hallmark of a superfamily of evolutionary conserved transport proteins, designated the "ATP-binding cassette" (ABC) transporter superfamily.This superfamily includes the mammalian P-glycoprotein (Pgp), a drug efflux pump that prevents accumulation of a variety of cytotoxic compounds in multidrug resistant cells; the pfmdrl gene product of Plasmodium fakiparum, assoclated with chloroquine resistance; and the cystic fibrosis transmembrane conductance regulator (CFTR) protein, mutations in which cause cystic fibrosis in humans.Ste6 and Pgp are highly homologous (up to 57% amino acid sequence homology, including conservative substitutions) and have similar length, predicted secondary structure and membrane topology.To determine if the structural similarity between the yeast and the mammalian transporters translates into a functional homology, we asked whether Pgp could substitute for Ste6 in transporting a-factor.We have shown that expression of a cDNA for the mouse mdr3 gene in a yeast sre6 deletion strain confers to the sterile cells the ability to export afactor and to mate.In addition, an amino acid substitution (SeP9+Phem9) located in predicted transmembrane domain 11 (TM-11) and known to affect the activity and drug specificity of the mdr3 gene product, completely abolishes its ability to complement the yeast sre6 deletion.This suggests that Pgp transports a-factor in yeast cells by mechanisms similar to those involved in drug transport in mammalian cells, and also that TM-11 may be involved in substrate recognition and transport.This complementation system in yeast allows us to analyze both the yeast Ste6 and mammalian Pgp transporters.This analysis should yield interesting insights into the mode of action of the ABC superfamily of transport proteins.We have determined that amino acids 95-102 (KKSKKKRC) are necessary for the nuclear localization of m:Gtase.

0749--503X
Changing the lysines at positions 95, 96, 98, 99, and 100 to glutamic acids (EESEEERC) does not affect the activity of the enzyme as measured in vitro, but alters the indirect immunofluorescent staining pattern from one that decorates the nuclear periphery to a pattern that is largely if not wholly cytoplasmic.tRNAs isolated from cells containing mutant mZGtase have the same amount of m$G as wild type tRNAs suggesting that mature tRNAs can serve as substrates in the cytoplasm.
A fusion protein consisting of amino acids 95-102 in frame with R-galactosidase is active and is located in both the nucleus and the cytoplasm.
These results demonstrate that the KKSKKKRC sequence which is necessary for nuclear targeting is also sufficient for targeting a portion of the fusion protein to nuclei.Nuclear targeting of this fusion is, however, less efficient than that of a previously described fusion that contains the first 213 amino acids of m$Gtase in frame with B-galactosidase.nuclear staining pattern unlike the peripheral staining seen with native m:Gtase.These observations are consistent with the hypothesis that information necessary for the subnuclear localization of m$Gtase is located after amino acid 213.
The peripheral staining pattern seen with the wild type protein suggests that the enzyme is associated with the nuclear membrane.Antibodies to mzGtase and to the known nuclear membrane pore protein, WPl' were used simultaneously and while both show nuclear rim staining the "UP1 signal appears punctate while the m$Gtase signal does not.been suggested that tRNA biosynthetic enzymes may be located together in the nucleus6, biochemical fractionations are underway to determine whether m:Gtase cofractionates with the integral membrane protein tRNA-splicing endonuclease' or whether it behaves like the tRNA-splicing ligase6.To answer these questions, a reporter system is being developed that will allow us to screen for translocation defective temperature sensitive mutants and to track the cellular localization of chimeric proteins, consisting of BO sequences fused to the reporter protein.S. pombe's invertase and two heterofogous proteins, a rice amylase Cundidu tropicalis is able to secrete an acid protease (ACP) when grown in the presence of exogenous proteins as nitrogen sources.ACP is believed to play a key role among the factors making this yeast an opportunistic pathogen.This hypothesis is supported by several studies performed in different Candidu species in which the protease is detected in experimental infections or in which non-defined and relatively unstable ACP negative mutants are utilized (Ross et al., 1990;Ruchel et al., 1991).
An approach making use of targeted gene disruption for obtaining ACP negative mutants would have the advantage to generate defined stable mutants, so that the role of ACP could be clearly established.Therefore the isolation of the gene for ACP (ACP) and the development of the genetical tools, i.e. a transformation system allowing the design of gene disruptions, which both were prerequisites for this approach, were undertaken and could be achieved successfully (Togni et al., 1991;Sanglard et al., submitted).
The disruption of ACP was performed by sequential gene disruption since C. tropicalis is a diploid yeast.The resulting acp deletion mutants were completely devoided of any extracellular protease activity and are currently being tested for their virulence in animals.
The acp deletion mutants have been also utilized for the complementation of protease secretion by the C. tropicalis and other Cundidu ACP genes after their insertion in the mulicopy vector pMK16 (Kurtz et al., 1987).The mutant could be complemented not only as expected by the C. tropicalis ACf but also by other Cadidu ACP thus affording a useful tool for the screening of functional ACf genes.The availability of the acp deletion mutant combined with the transformation system of C. tropicalis makes now also possible the understanding of the secretion mechanisms of this protease.11,8724385,1991).YP7'l codes for a small GTP-binding protein that is required for ER+Golgi transport.There is evidence that the suppressor genes (including the conventional suppressor SLYI) are also required for this step in vesicular traffic (Ossig et al., Mol. Cell. Biol., 11,2980-2993, 1991).SLY2 and SLY12 code for proteins which carry a stretch of hydrophobic amino acids at their C-terminal end.Both proteins share some structural features with synaptobrevin, a type II transmembrane protein which was found on synaptic vesicles.By using antibodies specific for Sly2p we could show that Sly2p is in fact an integral membrane protein (Ossig et al., op.cit.).
To study the topology of the Sly2p and Slyl2p proteins we fused the region of the invertase (SUC2) gene which codes for the secreted form of this enzyme to the 3' end of the SLY2 and the SLY12 gene, respectively.The extensive N-glycosylation of the Suc2 portion of these hybrid proteins indicates that the N-terminus of Sly2p and Slyl2p faces the cytoplasmic side of the membrane.This approach was used previously by dEnfert et al. (Mol.Cell.Biol.I I, [5727][5728][5729][5730][5731][5732][5733][5734]1991) to demonstrate that Secl2p is a type I1 transmembrane protein.
When expressing the different hybrid genes in a Suc-(suc2d9) strain, we found that only cells carrying the SLY2-SUC2 fusions can become Sue+.This can be achieved either by overexpressing the fusion gene or by inserting a Kex2 cleavage site between the Sly2 and the Suc2 portion of the hybrid protein.The cells stay Suc-, however, if SLYZ2-SUC2 and SECZ2-SUC2 fusions are overproduced or if a Kex2 cleavage site is inserted into these fusion genes.This suggests that the presence of a Kex2 cleavage site may allow us to differentiate between fusion proteins which reach a late Golgi compartment or those which stay in an early secretory compartment.The selection of Suc+ clones from cells expressing the SECl2 or SLY12 genes fused to SUC2 may allow the selection of mutants that mislocalize these proteins.Starting with a SECl2-SUC2 fusion gene three complementation groups were identified which require a Kex2 cleavage site to give Suc+ phenotype.We are currently examining these mutants to determine whether they mislocalize the fusion protein or they mislocalize the Kex2 protease.(reviewed in ref. 1).What might be the function of a multiUb chain?One idea is that the chain's formation on a targeted substrate produces an addtional binding site (or sites) for components of the proteolytic machine.The resulting increase in affinity, i.e., a decrease in the rate of dissociation of the machine-substrate complex, could be used to facilitate subsequent proteolytic steps.Suppose, for instance, that the rate-limiting step for the first proteolytic cleavage of the machine-bound substrate is a spontaneous unfolding (driven by thermal fluctuations) of a relevant region of the substrate.In this case, an increase in stability of the machine-substrate complex, brought about by the formation of a multiUb chain, would facilitate substrate degradation, because the longer the allowed "waiting" time, the greater the probability of a required unfolding event.One prediction of this model is that the degradation of a substrate whose conformation presents less of a kinetic impediment to the proteolytic machine should be less dependent on Ub and ubiquitination than the degradation of an otherwise similar but less stably folded substrate.
In a test of this and related ideas, we have converted, using the Ub fusion technique [l], the 102-residue N-terminal domain of h repressor and its derivatives into a series of N-end rule substrates, and are testing them in wild-type and mutant strains of S. cerevisiae.Previous work by Parcell and Sauer [2] produced a series of h(1-102) derivatives in which Leu57 is replaced with smaller residues; the melting temperature of these otherwise identical proteins ranges from 54OC for the wf h(1-102) protein to 5°C in hL57G (Leu 57+Gly57).The advantage of using h(1-102) and its derivatives is the ability to correlate the extent of Ub dependence of their in vivo degradation (assayed in yeast) with their conformational stability and other structural features that are accessible due to an extensive crystallographic and physico-chemical characterization of the h(1-102) protein.Friedrich-Miescher-Laboratonum der Max-Planck-Gesellschaft, Spemannstr.37-39 D 7400 Tubingen, Germany In the yeast Saccharomyces cerevisiae the genes SEC61, SEC62 and SEC63 are required for the translocation of proteins across the ER membrane.The three gene products are integral membrane proteins and form a protein complex in the ER membrane.At the nonpermissive temperature, sec61, sec62 and sec63 mutant cells fail to translocate certain proteins across the ER, accumulate unprocessed precursors, and cease to grow.
The ubiquitin system is known to mediate ATP-dependent nonlysosomal protein degradation.This pathway is involved in proteolysis of abnormal proteins and in the turnover of cellular regulators.Known targets include transcriptional regulators, p53 and cyclins.Previously, yeast ubiquitin-conjugating enzymes were shown to be involved in diverse cellular functions, including DNA repair, cell cycle control and the stress response.In addition to a proteolytic role of ubiquitin, a function in reversible protein modification has been suggested.
Recently, we have isolated UBC6 encoding a novel ubiquitin-conjugating enzyme of S. cerevisiae.UBC6 has a C-terminal signal-anchor sequence which localizes the protein to the ER membrane.The enzyme domain of UBC6 faces the cytoplasmic side of the ER membrane.Deletion mutants of UBC6 have no obvious phenotypes.However, UBC6 is a high copy number suppressor of sec61.
UBC6 overexpression in sec61 at the nonpermissive temperature restores viability and growth, however, not to wild-type rates.Importantly, UBC6 overexpression rescues the translocation defect fcr certain proteins including invertase and KAR2.No effect was observed with the secretion of a-Factor.Thus, the membrane-bound uBC6 seem to effect the secretion of proteins differentially.Since the soluble UBC4 enzyme also suppresses sec61, but to a significantly lower level than UBC6, we assume that the suppression is mediated by ubiquitindependent protein degradation, possibly of accumulated precursors.Alternatively, ubiquitin conjugation might directly participate in protein translocation.In the yeast Saccheromyces cerevisiae as well as in a number of other fungi the initial steps of protein 0glycosylation take place at the endoplasmic reticulum: Reaction 1, the synthesis of the lipid intermediate Dd-P-Man, is highly conserved in all eucaryotic organisms.In contrast reaction 2 has only been shown so far for fungal cells.In this step mannose is transfered from Dol-P-Man to protein bound seryl and threonyl residues.The enzyme catalysing this reaction is the Dd-P-D-Man: protein 0-D-mannosyltransferase.
This enzyme was purified to homogenity following the mannosyl transfer activity from Dd-P-Man to a short synthetic peptide (Tyr-Asn-Pro-Thr-Ser-Val) [Strahl-Bdsinger & Tanner, 19911.The pH-optimum for the enzyme reaction is 7.5.M$' ions stimulate the reaction twofold, however EDTA does not inhibit the basal rate.The K va~ue for Doi-P-Man Is 4x104M, for the hexapeptide 3.3x103M.Selective hergent extraction of total yeast membranes, column chromatography on hydraxylapatite and DEAEcellulose fdlowed by gelelectrophoresis under non denaturating conditions lead to a protein band with an apparent mdecular weigM of 92 kD.This protein correlates well with the enzyme activity.Treatment with Endoglycosidase F decreases the mdecular weigM from 92 kD to 84 kD.indicating that the mannosyltransferase is a glycoprotein with four N-linked carbohydrate chains of the "high mannose' type.
A pdydonal antibody raised against the 92 kD protein precipitates the mannosyltransferase activii.
Using this antibody about 10-20 bg of the 92 kD protein were purified to generate peptides.Llquid phase sequencing yielded three non overlapping peptkies.Chemically synthesized digodeoxynudeotides corresponding to the peptide sequences were used to screen a plasmid gene bank of yeast genomic DNA.A positive done reacting with all three digonudeotides was identified.Evidence will be presented showing that indeed the mannosyltransferase gene has been cloned.Temperature s e n s i t i v e c e l l l y s i s mutants were i n i t i a l l y s e l e c t e d f o r t h e i r a b i l i t y t o give p o s i t i v e s t a i n i n g f o r a l k a l i n e phosphatase a f t e r c u l t i v a t i o n a t 37OC.A more d e t a i l e d study of c e l l l y s i s revealed t h a t some of t h e mutants did not l y s e suggesting an enhanced export of p r o t e i n s a t 37OC r a t h e r t h a n c e l l l y s i s .Here, t h e r e s u l t s with 1-4B mutant a r e presented.

Strahl
C u l t i v a t i o n a t 37OC i n YPD medium supplemented with 10% of s o r b i t o l l e a d s t o export of 60 ug and 5 ug of p r o t e i n from lo8 c e l l s of t h e 1-4B mutant and S288C p a r e n t a l s t r a i n s , r e s p e c t i v e l y .This accounts f o r 25% and 2% of t h e c e l l u l a r p r o t e i n of t h e two s t r a i n s .P r o t e i n export a t 23OC was n e g l i g i b l e ( l e s s than 3 ug p r o t e i n / l O s t r a i n s .Export of p r o t e i n s cosegregates with t h e phenotype i n progeny of 1-4B x wild type c r o s s e s and is due t o a s i n g l e n u c l e a r mutation.The export of p r o t e i n s a t 37OC s t a r t s when 1-4B c e l l s e n t e r t h e s t a t i o n a r y phase and continues approximately 20 hours.P r o t e i n export does not occur i f p r o t e i n s y n t h e s i s i s i n h i b i t e d o r energy supply i s blocked suggesting a n a c t i v e process.SDS-PAGE a n a l y s i s shows d i f f e r e n t p a t t e r n of p r o t e i n s exported i n t o t h e medium of 1-4B a t 37OC compared t o t h a r of wild type p a r e n t a l c e l l s .The range of exported p r o t e i n s i s from 1 2 KD t o more than 200 KD.When measuring t h e export of periplasmic and i n t e r n a l p r o t e i n s we found t h a t t h e i n d u c i b l e a c i d phosphatase and i n v e r t a s e were exported a t e l e v a t e d l e v e l s from 1-4B c e l l s a t 37OC while two i n t e r n a l p r o t e i n s behaved d i f f e r e n t l y : a l k a l i n e phosphatase was exported but a r g i n a s e was n o t .It might be concluded t h a t i n a d d i t i o n t o an enhanced export of periplasmic p r o t e i n s , t h e 1-4B mutant c e l l s become leaky a t 37OC f o r some i n t e r n a l p r o t e i n s . Although t h e d a t a obtained suggest a n a c t i v e process i n a l i v e c e l l s , we c a r r i e d out experiments t o exclude c e l l l y s i s of a small percentage of s t a t i o n a r y c e l l s a s a reason f o r t h e p r o t e i n export observed: i ) no r e l e a s e of previously r a d i o a c t i v e l y labeled RNA was found i n c u l t u r e s of s t a t i o n a r y 1-4B c e l l s a c t i v e 1 8 exporting p r o t e i n s , and i i ) p r o t e i n export was observed 24 hours a f t e r t h e s h i f t t o 37 C from 1-4B c e l l s growing e x p o n e n t i a l l y i n chemostat.
The d a t a obtained a r e c o n s i s t a n t with t h e idea t h a t t h e mutation i n 1-4B c e l l s i s i n a gene whose product i s important f o r t h e maintenance of t h e permeability b a r r i e r of plasma membrane i n S. c e r e v i s i a e .In the yeast Saccharomyces cerevisiae, the uptake of uracil is mediated by a specific permease encoded by the FUR4 gene.This uracil permease is a multispanning membrane protein which follows the secretory pathway to the plasma membrane.It has a long hydrophilic N-terminal region, followed by 10 to 12 putative transmembrane segments, and an hydrophilic C-terminus.Its N-terminus is oriented towards the cytosplasm and the first a-helical hydrophobic segment of the permease was found to act as a signal anchor sequence involved in the insertion of uracil permease in the endoplasmic reticulum membrane (1).This insertion was prevented in a sec6l but not in a sec62 secretory mutant in conditions leading to a complete block in the translocation of soluble proteins.
We have shown by in vivo pulse labeling and immunoprecipitation that the uracil permease is phosphorylated.Phosphoamino acid analysis indicates that the phosphorylation occurs on seryl residue(s).Experiments with temperature sensitive secretory mutants have established that the phosphorylation of the permease takes place at the plasma membrane.Potential recognition sites for the cAMP protein kinase A and for kinase C are present in the hydrophilic N-terminus of this protein and in a loop predicted to be cytoplasmic.However, uracil permease is phosphorylated in cdc25 mutant cells at non permissive temperature suggesting that at least part of the phosphorylation is cAMP independent.ln vivo and in vitro experiments are in progress to define the phosphorylation site@) of the uracil permease as a first step to elucidate the functional significance of its phosphorylation.Strategy of isolation and main characteristics of strain oversecreting calf chymosin: Protoplasts of S.cerevisiae GRF18 (a, his3, led) were transformed with recombinant plasmid pJDBaCHE containing ori and amp' from pBR322 (E.coli), LEU;! and 2 p ARS from S.cerevisiae and calf-prochymosin cDNA sandwiched between promoter-preproa and terminator of MFa (S.cerevisiae).Accumulation of spontaneous mutants oversecreting chymosin was based on the assumption that these clones will grow faster due to the lower concentration of toxic chymosin proteins inside of cells.After repeated cultivation of S.cerevisiae GRFlS(pJDBaCHE) the oversecreting strain WSl(pJDBaCHE1 was selected using a modified milk-clotting assay.More then 10 fold higher maximal concentration of secreted chymosin proteins in the supplemented minimal medium (pH 3.7) w a s observed after cultivation of GOSl(pJDBaCHE) in comparison with GRFl8(pJDBaCHE) under the same conditions.Accumulation of chymosin proteins was faster, maximal level of activity was reached in early stationary phase of growth and in contrary to GRFlS (pJDBaCHE) no rapid decrease of activity was observed.Construction of killer toxin K1 overproducing strains: The strain GOSl was obtained by isolation of clone spontaneously cured of the pJDBaCHE.Both, hybrids (WSMH) and cybrids (GOSMC) containing nuclei only from GOSl were constructed by induced protopl_ast fusion of GOSl with killer strain S.cerevisiae M3 (a, ade , ilv , trp-, K l ' , R l ' ) .Analogically, hybrids (GMH) and cybrids (GMC) of G W 1 8 with M3 were also constructed for comparison."Killer plaque technique" and "killer stepwise procedure" (Vondrejs et al., Biotechnology -Current Progress 1: 227, 1991) were used for selecting these clones.About 30 fold increase in toxin-activity secreted for MSMH or GOSMC in comparison with GMH or GMC was demonstrated.
It follows from experiments mentioned above, that mutation causing oversecretion is very likely dominant and is not specifically connected with chymosin production only.
Time course of growth and accumulation of chymosin or K1 toxinactivity by oversecreting strains was determined under various conditions.It was found that proteins secretion continues very intensively after transfer of stationary phase culture to the fresh medium namely if cell concentration takes place as well.
It has been shown that the exploitation of killer overproducing strains is advantageous namely in the case of its application in vari-

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INT. CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY S461 10-11B TARGETING AND FUNCTIONAL FOLDING OF THE INTEGRAL MEMBRANE PROTEIN BACTERIORHODOPSIN (BR) INTO THE MITOCHONDRIAL INNER MEMBRANE OF THE FISSION YEAST SCHIZOSACCHAROMYCES POMBE -AN IN VIVO APPROACH A. Hoffmann, V. Hildebrandt and G. Buldt FB Physik, Abt.Biophysik, Freie Universitat Berlin, Arnimallee 14, W-1000 Berlin 3 3 , Germany A system was developed to study structural aspects of transport and functional incorporation of an integral membrane protein into a biological membrane of an eucaryotic cell.Bacteriorhodopsin a light driven proton pump from the archaebacteria Halobacterium halobium was expressed in S. pombe.To target BR to the mitochondrial inner membrane fusion-genes were made.N-terminal signalpeptide-sequences of proteins naturally located in the mitochondrial inner membrane (e.g.Cytochrom C-Oxidase, subunit IV) were fused to the N-terminus of BR.Western-Blot-Analyses of purified mitochondria indicated that BR was transported into these organelles, where it was processed releasing the signalpeptid-sequence.Isolation of mitochondrial outer and inner membrane and specific protease digestion gave evidence that BR was incorporated into the mitochondrial inner membrane with the processed N-terminus exposed to the intermembrane-space and the C-terminus towords the matrix.The addition of Retinal to intact cells indicated that BR was regenerated to form a functional proton pump in the mitochondria of S. vombe.0749-503X/92/Spe~.ISS.0461 -01 $05.50 0 1992 by John Wiley & Sons Ltd.S462 16TH INT.CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY 10-12B AN ESSENTlAL GENE FOR VACUOLE BIOSYNTHESIS IN YEAST T. Schlacher, G. Zisser, A. Ebeling and G. H6oenauec lnstitut firr Mikrobiologie, Universwt Graz, Universwtsplatz 2, A-8010 Graz, Austria 0749--503X/92/sps.Iss.0462-01 $05.50 0 1992 bY John W i l C Y 6 ?Sons -' I 16TH INT.CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY S463 10-13B WS5 ENCODES A PHOSPHOPROTEIN REQUIRED FOR EFFICIENT VACUOLAR PROTEIN SORTING AND VACUOLE ASSEMBLY IN YEAST.--Bruce F. Horazdovsky and Scott D. Emr.UCSD School of Medicine, Division of Cellular and Molecular Medicine & Howard Hughes Medical Institute, La Jolla, CA 92093-0668.

16TH 0749 -
INT. CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY S471 10-21B CODON DISTRIBUTION I N mRNA OF YEAST SECRETORY PROTEINS AND C O D I N G OF P R E C U R S O R P R O C E S S I N G AND SIGNAL PEPTIDASE CLEAVAGE SITES -Komar A.A., A d z h u b e i I .A . , K r a s h e n i n n i k o v I .A .Moscow S t a t e U n i v e r s i t y , F a c u l t y o f B i o l o g y , D e p a r t m e n t o f M o l e c u l a r B i o l o g y , 1 1 9 8 9 9 M O S C O W , R u s s i a .S i g n a l p e p t i d a s e r e c o g n i t i o n a n d p r o t e i n p r e c u r s o r s p r o c e s s i n g s i t e s i n s e c r e t o r y p r o t e i n s f o r m s u c h e l e m e n t s o f t h e s e c o n d a r y s t r u c t u r e a s t u r n s a n d b e n d s [ P e r l m a n & H a l v o r s o n , J .M o l .B i o l ., 167, 3 9 1 -3 9 7 , 19831.R e c e n t l y we h a v e shown f o r s e v e r a l p r o t e i n s w i t h known s p a t i a l s t r u c t u r e s t h a t p o l y p e p t i d e c h a i n r e g i o n s t h a t a r e c r u c i a l f o r p r o t e i n f o l d i n g ( s u c h a s t u r n s , b e n d s , e t c ) a r e m a i n l y c o d e d b y g r o u p e s o f c o d o n s w i t h l o w u s a g e f r e q u e n c i e s .A v i e w was s u p p o r t e d t h a t g r o u p e s o f r a r e c o d o n s f o r t h e g i v e n mRNA s e q u e n c e may c a u s e t h e t r a n s l a t i o n a l p a u s e s a n d t h a t t h e mRNA t r a n s l a t i o n k i n e t i c s may f o l l o w t h e c o d o n f r e q u e n c y o c c u r e n c e p r o f i l e [ K r a s h e n i n n i k o v e t a l , J .P r o t .Chem., 10, 4 4 5 -453, 19913.I t was assumed t h a t n o n r a n d o m d i s t r i b u t i o n o f c o d o n s i n mRNA may s e r v e a s a p r o g r a m , d r i v i n g t h e s p a t i a l p a c k i n g o f t h e s y n t h e s i z e d p o l y p e p t i d e c h a i n [ K r a s h e n i n n i k o v e t a l , J .P r o t .Chem., 10, 4 4 5 -4 5 3 , 19911.I n t h e p r e s e n t n o t e we h a v e a p p l i e d a n a p p r o a c h o f c o d o n d i s t r i b u t i o n a n a l y s i s d e v e l o p e d e a r l i e r [ K r a s h e n i n n i k o v e t a l , D o k l .Akad.Nauk SSSR ( P r o c .N a t l .Acad. S c i .USSR) 303, 9 9 5 -9 9 9 , 1 9 8 8 1 f o r t h e i n v e s t i g a t i o n o f s p e s i f i c n a t u r e o f c o d o n d i s t r i b u t i o n a l o n g c o d i n g r e g i o n s o f y e a s t s e c r e t o r y p r o t e i n s .I t was shown f o r s e v e r a l p r o t e i n s (SUC2, MFALPHAI, STE3 a n d o t h e r g e n e p r o d u c t s ) t h a t s i g n a l p e p t i d a s e a n d p r e c u r s o r p r o c e s s i n g s i t e s a r e c o d e d b y g r o u p e s o f c o d o n s w i t h l o w u s a g e f r e q u e n c i e s .One c a n s u g g e s t t h a t t h e s e p o l y p e p t i d e c h a i n r e g i o n s c o u l d b e t r a n s l a t e d s l o w l y t h a n o t h e r s a n d t h u s m i g h t b e c r u c i a l f o r p r o p e r c o t r a n s l a t i o n a l f o l d i n g o f t h e s e p r o t e i n s , a s s u m i n g t h e c o t r a n s l a t i o n a l p r o t e i n f o l d i n g m o d e l s s u g g e s t e d p r e v i o u s l y [ P u r v i s e t a l , J .M o l .B i o l , 193, 4 1 3 -4 1 7 , 1987; K r a s h e n i n n i k o v e t a l , J .P r o t .Chem., 10, 4 503X/92/Sp~.ISS.0471 -01 $05.50 0 1992 by John Wiley & Sons Ltd.S472 16TH INT.CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY 10-22B A GENE11 C ANfil-\'SIS OF AN ALF'I-IA-.-RMYLASESUPER-SECRETOR 1.N YEAST Zbiyniew K o t y l a k Insti.ti.ita! f o r Protection o f Nat.ctra1 Ehvironment 45-0'56 opo1 e , 02 i mska 46a, Pol and Xn t.tie p r e s r ~n t r e p o r t .I d e s c r i b e a gene which i n c r e a s e t h e a c t i v i t y o f ?d alpha-amylase.T h i s gene, which I have c a l l e d SUS, is l o o s e l y I i n k w l t o t t i e s t r u c t t r r a l (jerie STO, f o r alpha-amylase.

The SUS gene i n i
t s a c t i v e f o r m can be separated b y r e c o m b i n a t i o n f r o m t h e s t r u c t u r a l yerie STfis.Genetic crosses showed t h e pi-esmce o f t h e super-secretor gene i n a c -t i v e and i n a c t i v e form , i n spores w i t h o u t amyl o l i t i c a c t i v i t y .Enzymatic assay shc2wed t h a t SUS gene 15 s p e c i f i c : t h a t i s , i t i n c r e a s e s t h e s e c r e t i c m o f elphi-+-dmylaEie b u t has no i n + l u e n c e on t h e p e r i p l a s m i c enzymes a c i d phosphatase and i n v e r t a s e .The cornparison of s e c r e t i o n o f a c i d phosphatase andi n v e r t a s e between hyper-secr-etor., l o w -s e c r e t o r and t e s t e r s t r a i n s w i l l be an i n t e r e s t i n g phenornerlon f o r f u t u r e study.The t e s t e r s t r a i n s e c r e t e s more a c i d phosptlatase .(thant h e l o w -s e c r e t o r and, i n c o n t r a s t , t h e l o w -s e c r e t o r s e c r e t e s n 1 0 r -e i n v e r t a s e t h a n t h e t e s t e r s t r a i n .The s e c r e t i o n o f a c i d pt1osphataw.i n t h e t e s t e r s t r a i n and o f i n v e r t a s e i n t h e l o w -s e c r e t o r a r e compar-ablt?t o t h e s e c r e t i o n o f t h e s e two enzymes i n hyper-secretor.Pr-el iininar-y g e n e t i c a l and enzymatic d a t a t h a t t h e s e c r e t i o n o f t h e s e enzymes i s p r o b a b l y under t h e c o n t r o l of two s e p a r a t e genes. . . . ..-. 0749-503X/92/Spec.ISS.0472-01 $05.50 0 1992 by John Wiley & Sons Ltd. 16TH INT.CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY S473 10-23B SECl4 DELETED MUTANT OF YARROWlA L f P O L Y T f C A IS ALTERED IN THE SECRETION AND DIFFERENTIATION PROCESSES M.C. w, J.M. Nicaud, and C. Gaillardin.L h o n t o i n de gtnttique nml6culaire et cellulairc.Tnstitut National Apnornique 78850 Thiverval Gngnon.Fmce.The Saccharmnyces cerevisiae SGCl4 geiic is involved in the secretion pathway at the Colgi apparatus.The SEC14 genes of S. cercvisiiie and Kliiyveromyces lads have becn isolated and molecular analysis revealed high homology at the DNA level.SEC14 protein was shown to transfer thc phosphatidylcholine and phosphatidylinositol betwecn intraccllular biological membranes.We cloned and characterized a SEC14 homologous eene of the dimorphic yeast Yurrowiu lipobtica.. Alipnlent of gene sequences mvealed that the N-terminal moiety homologous to tlic S. cerevisine SEC14 gene.C-tenniiial rnoicty has no homolog in data library.The nmlecular mass of traiislntcd prduct inferred from gene sequence is about 58 kD.An h n i u n o r e d v e polypeptide displnying a similar molecular mass is recognized by S. cerevisiue antibodies in the Y. lipolyricu cell c x t ~x l ~.

0749 - 1 .
503X/92/Sp~.ISS.0481 -01 $05.50 0 1992 by John Wiley & Sons Ltd.S482 16TH INT.CONF.ON YEAST GENETICS A N D MOLECULAR BIOLOGY 10-32B NUCLEAR TARGETING AND SUBNUCLEAR LOCATION OF A tRNA BIOSYNTHETIC ENZYME A. M. Rose1, A. K. Hopper'.and N. C. Martin' Department of Biochemistry, University of Louisville School of Medicine, Louisville KY 40292 2. Department of Biological Chemistry, Milton S .Hershey Medical Center, Hershey, PA 17033 N2,N2-dimethylguanosine-specific tRNA methyltransferase (mZGtase) is required for the biosynthesis of m$G in both mitochondria1 and cytoplasmic tRNAs.Previous studies have demonstrated that the m : G modification of cytoplasmic tRNAs occurs in the n u ~l e u s ' * ~~~ and indirect immunofluorescence demonstrates that the majority of the TRMl gene product is nuclear4.

0749 -
503X/92/Spec.ISS.0486-01 $05.50 0 1992 by John Wiley & Sons Ltd. 16TH INT.CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY S487 10-37B ON THE FUNCTION OF MULTIUBIQUITIN CHAIN and A. Varshavsky Division of Biology, California Institute of Technology, Pasadena, California 91125, USA Several apparently distinct degradation signals in short-lived eukaryotic proteins have a common property of directing the formation of a multiubiquitin (multiUb) chain linked to an internal lysine of a substrate 0749-503X1921Spe~.ISS.0488-01 $05.50 0 1992 by John Wiley & Sons Lid.16TH INT.CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY S489 10-39B PROTEIN OGLYCOSYLATION IN SACCHAROMYCES CERNISIAE: PURIFICATION, CHARACTERIZATION AND CLONING OF THE DOLICHYL-PHOSPHATE-D-MANN0SE:PROlElN 0-D-MANNOSYLTRANSFERASE s.Strahl-BdsinpeG T. lmmewdl and W. Tanner Lehrstuhl fiir Zellbidogie und Manzenphysidogie, UnivemiWsstr.31,8400 Regensburg, Germany Two types of protein carbohydrate chains are distinguished.They are either bound to the protein by a Nglycosidic link to asparagine or the carbohydrate part is linked O-glycosidimly to the hydroxylgroup of seryl or threonyl residues.The N-glycosylation of proteins has been studied extensively.It is highly consewed from yeast to man.In contrast, O-glycosylation proceeds differently as far as known in mammalian, plant and fungal cells.
L .s .* .R.S. Full&, and J.W. Nichols* * Department of Physiology, Emory University School of Medicine, Atlanta, Georgia, U.S.A., 30322 # Department of Biochemistry, Stanford University School of Medicine.Stanford, California, U.S.A., 94305 We have used phospholipid analogs bearing the f l -N-4-nitm~2-oxa-l,3-diamle (NBD) reporter group on one acyl chain to p b e the pathway of internalization of plasma memtxane phospholipids in S. cerevkh.We find that there are two pathways for intanalimtion of phosphatidylcholine analogs.(1) These molecules are transported by endocytic lipid traffic to the vacuole.This pathway is temperature and energy dependent.It is also dependent on many secretoty genes, including a a sEc14.a n d m but is not dependent on the m l a t i o n of biochemical analysis with fluoresene mi - Service de Biochimie et de Genetique Moleculaire, Bit.142, CE/Saclay, 91 191 Gif-sur-Yvette Cedex, France Present address : lnstitut Jacques Monod, 2, place Jussieu, 75251 Paris, France The EBB1, and EBE;1 genes.(2) Fluorescent phosphatidylcholine analogs are also gene.It is inhibited by pretreatment of pp.5890-5898) and suggests the existence of a 3749--503X/92/Spec.1%.0466-01 $05.50 0 1992 by John Wiley & Sons Ltd. 16TH INT.CONF.ON YEAST GENETICS AND MOLECULAR BIOLOGY S467 10-17B A NOVEL YEAST GENE FUNCTIONING IN PROTEIN TRANSLOCATION, SSS7, ENCODES A MEMBRANE PROTEIN Y. Esnautt, M.-0.Blondel' and F. K e a

Table 1 .
The sugar composition of sodium carbonate-extracted membrane vesicles.

0749-503X192lSpec. Iss. 0469-01 $05.50 @ 1992 by John Wiley & Sons Ltd. S470 16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY 10-20B
SlE6 protein of S. cerevisae and t h e haemlysin B protein of E. cold, t o establish an alternative route of secretion for heterologous proteins i n yeast, bypassing t h e normal secretory pathway.KZyB hybrid-proteins and analysed them for export of a-factor and DHFR-KZyA fusion proteins i n yeast.One of t h e hybrids is s t i l l able t o secrete a-factor although t o a much reduced extent.
W e investigated whether t h e mating pheromone a-factor which is secreted by t h e STE6 t r a n s p o r t e r has t h e p o t e n t i a l of serving as a s e c r e t i o n s i g n a l i n f u s i o n s with a larger polypeptide.Several f u s i o n s were constructed with parts of t h e pre-a-factor p r o t e i n fused t o t h e Cterminus of mouse DHFR.W e could demonstrate t h a t these fusions l i k e af a c t o r are C-terminally modified by comparing t h e gel mobility of t h e fusion p r o t e i n s from w i l d t y p e and from stel6 (raml) mutant e x t r a c t s .Furthermore t h e s e fusions were s p e c i f i c a l l y farnysylated i n vitro.A large f r a c t i o n of t h e DHER-a-factor fusions (about 50 %) is possibly membrane-associated o r contained within a cell compartment.So f a r we were not able t o detect t h e DHFR-a-factor fusion proteins i n t h e culture s u p e r n a t a n t .It i s p o s s i b l e t h a t t h e s e f u s i o n s are directed t o a location i n t h e cell other than t h e plasmamembrane.W e are currently investigating t h e localization of t h e fusions i n t h e cell.F u r t h e m r e w e are t r y i n g t o use t h e haemolysin system of E.coli t o t r a n s p o r t proteins directly out of t h e yeast cell.The haemolysin B/D translocator of E. coli is able t o secrete a large protein, t h e haemolysin A protein (110 kD).W e have constructed yeast s t r a i n s containing t h e haemolysin B and D functions integrated i n t o t h e yeast genome under t h e control of t h e GALI,10 promoters and we are currently investigating whether these proteins can be used t o secrete haemolysin A fusion proteins i n yeast.I n o r d e r t o map t h e domains t h a t are r e s p o n s i b l e f o r s u b s t r a t e recognition we generated STE6/0749-503X/92/Spec. 11s.0470-01 $05.50 0 1992 by John Wiley & Sons Ltd.

ON YEAST GENETICS AND MOLECULAR BIOLOGY S475 10-25B DUAL REGULATION OF TEE mlSO-GENE ENCODING A SECRETORY GLYCOPROTEIN BY HEAT STRESS AND NUTRIENT STARVATION
Asn mutation at a highly conserved position in the S. cerevisiae RASZ gene yields a novel type of a dominant allele, RASZSn.The effects of this allele include greatly increased resistance of cells to oxidative and thermal stresses, elevated activity of the N-end rule pathway and other proteolytic pathways, changes in the kinetics of entering and leaving the stationary phase, and decreased lifespan of mother cells.These phenotypes of RAS2sn are underlied by overexpression of genes that encode heat stress proteins, the cytosolic and peroxisomal catalases, N-acetylase, ubiquitin-conjugating enzymes and N-recognin.Stress-resistant phenotypes of the previously described mutants in the R A S 2 gene have been shown to result at least in part from low levels of CAMP, which is produced by the Ras-dependent adenylate cyclase.However, the levels of CAMP in the RAS2Sn 0749-503X/92/Spec.ISS.0473-01 $05.50 0 1992 by John Wiley & Sons Ltd. cells are either normal or higher than those in congenic wild-type cells.These and related findings strongly suggest that the R a ~2 ~" protein is perturbed in its interactions with effectors distinct from those of the extensively studied CAMP pathway.Further genetic analysis of RASZSn should help identify these effectors.16TH INT.CONF.

Ltd. S490 16TH INT. CONF. ON YEAST GENETICS AND MOLECULAR BIOLOGY 10-4OB SEQUENCING OF PEPl, A GENE WHOSE PRODUCT IS INVOLVED IN VACUOLAR PROTEIN SORTING.
In the framework of the European Community project for Sequencing the Yeast Genome, we have sequenced the PEPl gene, which is located on the left arm of chromosome 11.The pep1 mutant, togheter with other pep mutants defining 15 complementation groups, was first isolated for its reduced level of carboxypeptidase Y (Jones, 1977).It was found later to exhibit a profound vacuolar protein sorting defect (Rothman et al.,1989).PEPI encodes a 1579 amino acids long protein with an estimated molecular weight of 178 KDa.Search for homology with proteins of the MIPSX data base gave no significant result.PEPl is an integral protein with two transmembrane segments predicted.The first one, at the N-terminal end of the protein, is a secretory signal sequence which targets the peptide to the endoplasmic reticulum.The second one, located at 165 amino acids of the C-terminal end, indicates a 0749-503X/92/Spec.ISS.0489-01 $05.50 G 1992 by John Wiley & Sons