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S Michaelis

Publications and source records attributed to S Michaelis.

At least 37 records · Page 2Linked to original sources
PubMed1998

Functional assays for analysis of yeast ste6 mutants.

As a member of the ABC superfamily, STE6 is unique in that it has a well-characterized substrate, a-factor, and can be easily manipulated in the yeast system. Functional assays have been extensively used, and methods to examine trafficking and stability of STE6 are well established. In addition, STE6 chimeras and ste6 deletion strains are useful for the analysis of many nonyeast ABC proteins. Continuing studies of STE6 are expected to aid in the identification of novel cellular components involved in the trafficking and functioning of not only STE6, but of other members of the ABC superfamily as well.

ATP-Binding Cassette Transporters↗
PubMed1998

Ste6p mutants defective in exit from the endoplasmic reticulum (ER) reveal aspects of an ER quality control pathway in Saccharomyces cerevisiae.

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6-166p, is highly unstable. We show that its degradation involves the ubiquitin-proteasome system, as indicated by its in vivo stabilization in certain ubiquitin-proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6-13p and Ste6-90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.

ATP-Binding Cassette Transporters↗
PubMed1998

The Saccharomyces cerevisiae prenylcysteine carboxyl methyltransferase Ste14p is in the endoplasmic reticulum membrane.

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14 gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Deltaste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

Amino Acid Sequence↗
PubMed1998

Role for the ubiquitin-proteasome system in the vacuolar degradation of Ste6p, the a-factor transporter in Saccharomyces cerevisiae.

Ste6p, the a-factor transporter in Saccharomyces cerevisiae, is a multispanning membrane protein with 12 transmembrane spans and two cytosolic ATP binding domains. Ste6p belongs to the ATP binding cassette (ABC) superfamily and provides an excellent model for examining the intracellular trafficking of a complex polytopic membrane protein in yeast. Previous studies have shown that Ste6p undergoes constitutive endocytosis from the plasma membrane, followed by delivery to the vacuole, where it is degraded in a Pep4p-dependent manner, even though only a small portion of Ste6p is exposed to the vacuolar lumen where the Pep4p-dependent proteases reside. Ste6p is known to be ubiquitinated, a modification that may facilitate its endocytosis. In the present study, we further investigated the intracellular trafficking of Ste6p, focusing on the role of the ubiquitin-proteasome machinery in the metabolic degradation of Ste6p. We demonstrate by pulse-chase analysis that the degradation of Ste6p is impaired in mutants that exhibit defects in the activity of the proteasome (doa4 and pre1,2). Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in the doa4 mutant, as it does in the vacuolar protease-deficient pep4 mutant. One model consistent with our results is that the degradation of Ste6p, the bulk of which is exposed to the cytosol, requires the activity of both the cytosolic proteasomal degradative machinery and the vacuolar lumenal proteases, acting in a synergistic fashion. Alternatively, we discuss a second model whereby the ubiquitin-proteasome system may indirectly influence the Pep4p-dependent vacuolar degradation of Ste6p. This study establishes that Ste6p is distinctive in that two independent degradative systems (the vacuolar Pep4p-dependent proteases and the cytosolic proteasome) are both involved, either directly or indirectly, in the metabolic degradation of a single substrate.

ATP-Binding Cassette Transporters↗
PubMed1997

Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.

The Saccharomyces cerevisiae mating pheromone a-factor is a prenylated and carboxyl methylated extracellular peptide signaling molecule. Biogenesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification. NH2-terminal proteolysis, and a nonclassical export mechanism. In this study, we examine the formation and fate of a-factor biosynthetic intermediates to more precisely define the events that occur during a-factor biogenesis. We have identified four distinct a-factor biosynthetic intermediates (P0, P1, P2, and M) by metabolic labeling, immunoprecipitation, and SDS-PAGE. We determined the biochemical composition of each by defining their NH2-terminal amino acid and COOH-terminal modification status. Unexpectedly, we discovered that not one, but two NH2-terminal cleavage steps occur during the biogenesis of a-factor. In addition, we have shown that COOH-terminal prenylation is required for the NH2-terminal processing of a-factor and that all the prenylated a-factor intermediates (P1, P2, and M) are membrane bound, suggesting that many steps of a-factor biogenesis occur in association with membranes. We also observed that although the biogenesis of a-factor is a rapid process, it is inherently inefficient, perhaps reflecting the potential for regulation. Previous studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH2-terminal processing (Ste24p, Axl1p), and export (Ste6p) of a-factor. The intermediates defined in the present study are discussed in the context of these biogenesis components to formulate an overall model for the pathway of a-factor biogenesis.

Amino Acid Sequence↗
PubMed1997

A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.

Many secreted bioactive signaling molecules, including the yeast mating pheromones a-factor and alpha-factor, are initially synthesized as precursors requiring multiple intracellular processing enzymes to generate their mature forms. To identify new gene products involved in the biogenesis of a-factor in Saccharomyces cerevisiae, we carried out a screen for MA Ta-specific, mating-defective mutants. We have identified a new mutant, ste24, in addition to previously known sterile mutants. During its biogenesis in a wild-type strain, the a-factor precursor undergoes a series of COOH-terminal CAAX modifications, two sequential NH2-terminal cleavage events, and export from the cell. Identification of the a-factor biosynthetic intermediate that accumulates in the ste24 mutant revealed that STE24 is required for the first NH2-terminal proteolytic processing event within the a-factor precursor, which takes place after COOH-terminal CAAX modification is complete. The STE24 gene product contains multiple predicted membrane spans, a zinc metalloprotease motif (HEXXH), and a COOH-terminal ER retrieval signal (KKXX). The HEXXH protease motif is critical for STE24 activity, since STE24 fails to function when conserved residues within this motif are mutated. The identification of Ste24p homologues in a diverse group of organisms, including Escherichia coli, Schizosaccharomyces pombe, Haemophilus influenzae, and Homo sapiens, indicates that Ste24p has been highly conserved throughout evolution. Ste24p and the proteins related to it define a new subfamily of proteins that are likely to function as intracellular, membrane-associated zinc metalloproteases.

Amino Acid Sequence↗
PubMed1997

Recombination-mediated PCR-directed plasmid construction in vivo in yeast.

We have extended the technique of PCR-directed recombination in Saccharomyces cerevisiae to develop a simple method for plasmid or gene construction in the absence of suitable restriction sites. The DNA to be cloned is PCR-amplified with 30-40 bp of homology to a linearized yeast plasmid. Co-transformation into yeast results in homologous recombination at a position directed by the PCR oligonucleotides.

Cloning, Molecular↗
PubMed1997

Identification of novel 'expressed sequence tags' within the FHIT gene locus in human chromosome region 3p14.2.

Losses of genetic material within human chromosome regions (HCR) 3p12-p14 and 3p21-p22 are observed in various neoplasias, suggesting tumor suppressor gene (TSG) loci within these regions. HCR 3p14 is particularly interesting as it contains the t(3;8) translocation breakpoint of a hereditary renal cell carcinoma, the FRA3B fragile site, and DNA markers deleted in several types of human cancer. We here report on the identification of five novel 'expressed sequence tags' (ESTs) within 3p14.2 which map proximal to exon 9 of the candidate TSG, FHIT. These ESTs may be valuable for elucidation of the supposed TSG content in 3p14.2.

Acid Anhydride Hydrolases↗
PubMed1997

A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism.

Many secreted signaling molecules are synthesized as precursors that undergo multiple maturation steps to generate their mature forms. The Saccharomyces cerevisiae mating pheromone a-factor is a C-terminally isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids. We have previously shown that the maturation of a-factor occurs by an ordered biogenesis pathway involving 1) three C-terminal modification steps, 2) two N-terminal proteolytic processing events, and 3) a nonclassical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p. In the present study, we demonstrate that an unexpected and abundant a-factor-related peptide (AFRP) exists in the culture fluid of MATa cells and that its biogenesis is integrally related to that of mature a-factor itself. We show by purification followed by mass spectrometry that AFRP corresponds to the C-terminal 7 amino acids (VFWDPAC) of mature a-factor (YIIKGVFWDPAC), including both the farnesyl- and carboxylmethylcysteine modifications. The formation and export of AFRP displays three striking features. First, we show that AFRP is produced intracellularly and that mutants (ste24 and axl1) that cannot produce mature a-factor due to an N-terminal processing defect are nevertheless normal for AFRP production. Thus, AFRP is not derived from mature a-factor but, instead, from the P1 form of the a-factor precursor. Second, fusion constructs with foreign amino acids substituted for authentic a-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds to the altered residues instead of to AFRP residues. Thus, AFRP may be generated by a sequence-dependent but length-specific proteolytic activity. Third, a-factor and AFRP use distinct cellular machinery for their secretion. Whereas a-factor export is Ste6p-dependent, AFRP is secreted normally even in a ste6 deletion mutant. Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a different type of transporter altogether, or possibly by diffusion. Taken together, these studies indicate that the biogenesis of AFRP involves novel mechanisms and machinery, distinct from those used to generate mature a-factor. Because AFRP neither stimulates nor inhibits mating or a-factor halo activity, its function remains an intriguing question.

ATP-Binding Cassette Transporters↗
PubMed1996

Functional and physical interactions between partial molecules of STE6, a yeast ATP-binding cassette protein.

The Saccharomyces cerevisiae a-factor transporter, STE6, is a member of the ATP binding cassette (ABC) transporter superfamily. ABC proteins consist of four modular units that comprise two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). Like many ABC proteins, STE6 contains these four domains in a single polypeptide; certain other ABC proteins are encoded as pairs of "half-molecules" or are further subdivided. Our previous studies demonstrated that STE6 can be expressed as two half-molecules that are functional when co-expressed. Here we dissect the interactions between modules of STE6 in greater detail. We show by co-immunoprecipitation that STE6 half-molecules interact physically, supporting the view that they co-assemble in vivo to form a functional transporter. We also demonstrate a physical interaction between a STE6 half-molecule and full-length STE6; such complexes appear to be functional, based on the striking finding that the defective activity of full-length STE6 mutated in one of its NBDs can be corrected by co-expression of the corresponding "wild-type" half-molecule. We also show that a quarter-molecule consisting solely of the N-terminal MSD of STE6 can interact physically and functionally with a C-terminal three-quarter molecule of STE6, indicating that information directing the assembly of STE6 from partial molecules is contained, at least in part, within its membrane spans.

ATP-Binding Cassette Transporters↗
PubMed1996

Comparative topology studies in Saccharomyces cerevisiae and in Escherichia coli. The N-terminal half of the yeast ABC protein Ste6.

Gene fusions have provided a strategy for determining the topology of polytopic membrane proteins in Escherichia coli. To evaluate whether this highly effective approach is applicable to heterologously expressed eukaryotic integral membrane proteins, we have carried out a comparative topological study of the eukaryotic membrane protein Ste6 both in bacteria and in yeast. Ste6, is an ATP binding cassette (ABC) protein, essential for export of the a-factor mating pheromone in Saccharomyces cerevisiae. The topogenic reporters, invertase in S. cerevisiae and alkaline phosphatase in E. coli, were fused to Ste6 at identical sites and the fusions were expressed in yeast and bacteria, respectively. The results obtained in both systems are similar, although more definitive in E. coli, and support the predicted six-transmembrane spans organization of the N-terminal half of Ste6. Thus, the topological determinants for membrane insertion of polytopic proteins in prokaryotic and in eukaryotic systems appear to be highly similar. In this study we also demonstrate that Ste6 does not contain a cleaved signal sequence.

ATP-Binding Cassette Transporters↗
PubMed1996

Analysis of the localization of STE6/CFTR chimeras in a Saccharomyces cerevisiae model for the cystic fibrosis defect CFTR delta F508.

The use of yeast as a model system to study mammalian proteins is attractive, because yeast genetic tools can be utilized if a suitable phenotype is created. STE6, the Saccharomyces cerevisiae a-factor mating pheromone transporter, and CFTR, the mammalian cystic fibrosis transmembrane conductance regulator, are both members of the ATP binding cassette (ABC) superfamily. Teem et al. (1993) described a yeast model for studying a mutant form of the cystic fibrosis protein, CFTR delta F508. The model involved expression of a chimeric molecule in which a portion of yeast STE6 was replaced with the corresponding region from mammalian CFTR. The STE6/CFTR chimera complemented a ste6 mutant strain for mating, indicating that it could export a-factor. However, mating efficiency was dramatically reduced upon introduction of delta F508, providing a yeast phenotype for this mutation. In human cells, the delta F508 mutation results in retention of CFTR in the endoplasmic reticulum (ER), and possibly in reduction of its chloride-channel activity. Here we examine the basis for the differences in STE6 activity promoted by the wild-type and mutant STE6/CFTR chimeras. By analysis of protein stability and subcellular localization, we find that the mutant chimera is not ER-retained in yeast. We conclude that the molecular basis for the reduced mating of the STE6/CFTR delta F508 chimera must reflect a reduction in its capacity to transport a-factor, rather than mistrafficking. Thus, STE6/CFTR delta F508 in yeast appears to be a good genetic model to probe certain aspects of protein function, but not to study protein localization.

ATP-Binding Cassette Transporters↗
PubMed1996

Genetic instability of 3p12-p21-specific microsatellite sequences in renal cell carcinoma.

OBJECTIVE: To determine the role of structural alterations of human chromosome region 3p12-p21 in the possible inactivation of one or more tumour-suppressor genes in the pathogenesis of renal cell carcinoma (RCC), lung cancer and other neoplasms. MATERIALS AND METHODS: As microsatellite instability (MI), in particular MI with loss of heterozygosity (LOH), may indicate putative tumour-suppressor gene loci, 20 kidney tumours, including 14 clear cell carcinomas and six non-clear cell neoplasms, were investigated with 10 polymorphic simple sequence-repeat markers spanning 3p12-p21. Six of these markers map to the region of deletion flanked by markers D3S1285 and D3S1295 bracketing the t(3;8) translocation break-point in 3p14.2 of hereditary RCC. RESULTS: Twelve of 14 clear cell RCCs displayed MI for at least one locus, as opposed to none of the non-clear cell tumours (P = 0.001). Locus D3S1274 in 3p13 located in the region deleted in lung cancer line U2020 and loci D3S1313 and D3S1300 in 3p14.3 characterized common regions of instability and LOH. Two patients with RCC who also had lung cancer and colon cancer, respectively, showed LOH at D3S1313 or D3S1300 as the only alterations of their kidney tumours. CONCLUSIONS: These results suggest that human chromosome region 3p14.3 distal to the hereditary t(3;8) translocation breakpoint and the region deleted in the U2020 lung cancer cell line might be involved in the tumorigenesis or progression of clear cell RCC.

Adult↗
PubMed1996

Cell fusion during yeast mating requires high levels of a-factor mating pheromone.

During conjugation, two yeast cells fuse to form a single zygote. Cell fusion requires extensive remodeling of the cell wall, both to form a seal between the two cells and to remove the intervening material. The two plasma membranes then fuse to produce a continuous cytoplasm. We report the characterization of two cell fusion defective (Fus-) mutants, fus5 and fus8, isolated previously in our laboratory. Fluorescence and electron microscopy demonstrated that the fus5 and fus8 mutant zygotes were defective for cell wall remodeling/removal but not plasma membrane fusion. Strikingly, fus5 and fus8 were a specific; both mutations caused the mutant phenotype when present in the MATa parent but not in the MAT alpha parent. Consistent with an a-specific defect, the fus5 and fus8 mutants produced less a-factor than the isogenic wild-type strain. FUS5 and FUS8 were determined to be allelic to AXL1 and RAM1, respectively, two genes known to be required for biogenesis of a-factor. Several experiments demonstrated that the partial defect in a-factor production resulted in the Fus- phenotype. First, overexpression of a-factor in the fus mutants suppressed the Fus- defect. Second, matings to an MAT alpha partner supersensitive to mating pheromone (sst2 delta) suppressed the Fus- defect in trans. Finally, the gene encoding a-factor, MFA1, was placed under the control of a repressible promoter; reduced levels of wild-type a-factor caused an identical cell fusion defect during mating. We conclude that high levels of pheromone are required as one component of the signal for prezygotes to initiate cell fusion.

Alleles↗
PubMed1996

Construction of a consistent YAC contig for human chromosome region 3p14.1.

Chromosomal deletions and translocations of human chromosome region 3p14 are observed in various human malignancies and suggest the existence of a tumor suppressor gene locus within this region. Tumors most frequently affected by these aberrations are small-cell lung cancer and renal-cell carcinoma. In continuation of our previously published YAC contig of chromosome region 3p14.2-p14.3, we report here on the construction of a YAC contig of at least 11 Mb that consisted of 171 YACs and covers the entire subregion 3p14.1. This contig includes the t(3;8) breakpoint of a hereditary renal-cell carcinoma localized in 3p14.2 and extends into human chromosome region 3p12-p13. It defines the order of 34 DNA probes in relation to reference markers D3S6 and D3S30 as well as the human protein tyrosine phosphatase-gamma gene. For 31 DNA probes we identified nonchimeric YACs by fluorescence in situ hybridization. The minimal tilling pathway consists of 16 yeast artificial chromosomes. As a prerequisite for identification of a putative tumor suppressor gene within this region, this contig renders human chromosome region 3p14.1 accessible to gene isolation.

Blotting, Southern↗
PubMed1994

Mapping and sequencing of two yeast genes belonging to the ATP-binding cassette superfamily.

ATP-binding cassette (ABC) transporters share significant sequence identity within their ATP-binding domains. Degenerate oligonucleotides based on highly conserved portions of the ATP-binding domain genes were used to clone portions of two members of the ABC gene superfamily from Saccharomyces cerevisiae DNA. These genes were designated MDL1 and MDL2 (for multidrug resistance-like). Each MDL gene is predicted to encode a single set of transmembrane domains and a single ATP-binding domain, thus the MDL gene products are 'half-molecule' ABC proteins. The two genes were mapped to precise regions on chromosomes XII and XVI and show a considerable similarity to the mammalian P-glycoprotein/multidrug resistance (MDR) and peptide transporter (TAP) genes. Preliminary analysis of null mutants constructed by gene replacement has indicated that the MDL genes are not essential for viability of yeast. The sequences have been deposited in the GenBank data library under Accession Numbers L16958 (Locus YSCBCSA) and L16959 (Locus YSCBCSB).

ATP Binding Cassette Transporter, Subfamily B, Mem↗