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S Michaelis

Publications and source records attributed to S Michaelis.

At least 55 records · Page 3Linked to original sources
PubMed1994

Metabolic instability and constitutive endocytosis of STE6, the a-factor transporter of Saccharomyces cerevisiae.

STE6, a member of the ATP binding cassette (ABC) transporter superfamily, is a membrane protein required for the export of the a-factor mating pheromone in Saccharomyces cerevisiae. To initiate a study of the intracellular trafficking of STE6, we have examined its half-life and localization. We report here that STE6 is metabolically unstable in a wild-type strain, and that this instability is blocked in a pep4 mutant, suggesting that degradation of STE6 occurs in the vacuole and is dependent upon vacuolar proteases. In agreement with a model whereby STE6 is routed to the vacuole via endocytosis from the plasma membrane, we show that degradation of STE6 is substantially reduced at nonpermissive temperature in mutants defective in delivery of proteins to the plasma membrane (sec6) or in endocytosis (end3 and end4). Whereas STE6 appears to undergo constitutive internalization from the plasma membrane, as do the pheromone receptors STE2 and STE3, we show that two other proteins, the plasma membrane ATPase (PMA1) and the general amino acid permease (GAP1), are significantly more stable than STE6, indicating that rapid turnover in the vacuole is not a fate common to all plasma membrane proteins in yeast. Investigation of STE6 partial molecules (half- and quarter-molecules) indicates that both halves of STE6 contain sufficient information to mediate internalization. Examination of STE6 localization by indirect immunofluorescence indicates that STE6 is found in a punctate, possibly vesicular, intracellular pattern, distinct from the rim-staining pattern characteristic of PMA1. The punctate pattern is consistent with the view that most of the STE6 molecules present in a cell at any given moment could be en route either to or from the plasma membrane. In a pep4 mutant, STE6 is concentrated in the vacuole, providing further evidence that the vacuole is the site of STE6 degradation, while in an end4 mutant STE6 exhibits rim-staining, indicating that it can accumulate in the plasma membrane when internalization is blocked. Taken together, the results presented here suggest that STE6 first travels to the plasma membrane and subsequently undergoes endocytosis and degradation in the vacuole, with perhaps only a transient residence at the plasma membrane; an alternative model, in which STE6 circumvents the plasma membrane, is also discussed.

ATP-Binding Cassette Transporters↗
PubMed1994

Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.

Eukaryotic proteins initially synthesized with a C-terminal CAAX motif (C is Cys, A is aliphatic, and X can be one of several amino acids) undergo a series of modifications involving isoprenylation of the Cys residue, proteolysis of AAX, and alpha-carboxyl methyl esterification of the newly formed isoprenyl cysteine. We have previously demonstrated that STE14 encodes the enzyme which mediates carboxyl methylation of the Saccharomyces cerevisiae CAAX proteins a-factor, RAS1, and RAS2. Here we report the nucleotide sequence of STE14, which indicates that STE14 encodes a protein of 239 amino acids, predicted to contain multiple membrane-spanning segments. Mapping data indicate that STE14 resides on chromosome IV, tightly linked to ADE8. By analysis of ste14 null alleles, we demonstrated that MATa ste14 mutants are unable to mate but are viable and exhibit no apparent growth defects. Additional analysis of ste14 ras 1 and ste14 ras2 double mutants, which grow normally, reinforces our previous conclusion that RAS function is not significantly influenced by its methylation status. We examine a-factor biogenesis in a ste14 null mutant by metabolic labeling and immunoprecipitation and demonstrate that although proteolytic processing and membrane localization of a-factor are normal, the ste14 null mutant exhibits a profound block in a-factor export. This observation suggests that the methyl group is likely to be a critical recognition determinant for the a-factor transporter, STE6, thus providing insight into the substrate specificity of STE6 and also supporting the hypothesis that carboxyl methylation can have a dramatic impact on protein-protein interactions.

Amino Acid Sequence↗
PubMed1993

STE6, the yeast a-factor transporter.

In eukaryotic cells, most extracellular proteins exit the cell via the classical secretory pathway (ER-->Golgi-->secretory vesicles). A notable exception to this pattern is the Saccharomyces cerevisiae mating pheromone alpha-factor, an isoprenylated, methylated, oligopeptide signaling molecule which uses a distinctly non-classical mechanism for secretion. Export of alpha-factor from the yeast cell is mediated by STE6, a member of the ABC protein superfamily. STE6 is one of the few eukaryotic ABC proteins for which a true physiological substrate is known. The ability to carry out molecular manipulations with ease in yeast, together with the possibility of probing substrate-transporter interactions via genetic analysis, affords an excellent opportunity to rigorously dissect the workings of this ABC family member.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins.

In the yeast Saccharomyces cerevisiae, mutations in either of two unlinked genes, RAM1 or RAM2, abolish the farnesyltransferase activity responsible for prenylation of Ras proteins and the a-factor mating pheromone. Here we report that the function of RAM1 and RAM2 genes is required for the membrane localization of Ras proteins and a-factor. The RAM2 gene was sequenced and can encode a 38-kDa protein. We examined the functional interaction of RAM2 and RAM1 by expressing the genes in Escherichia coli. Extracts derived from an E. coli strain that coexpressed RAM1 and RAM2 efficiently farnesylated a-factor peptide and Ras protein substrates. In contrast, extracts derived from E. coli strains that expressed either RAM gene alone were devoid of activity; however, when the latter extracts were mixed, protein farnesyltransferase activity was reconstituted. These results indicate that the yeast farnesyl-protein transferase is comprised of Ram1 and Ram2 polypeptides. Although Ram1 is a component of the enzyme, disruption of the RAM1 gene in yeast was not lethal, indicating that the Ram1-Ram2 farnesyltransferase is not essential for viability. In contrast, disruption of RAM2 was lethal, suggesting that Ram2 has an essential function in addition to its role with Ram1 in protein farnesylation.

Alkyl and Aryl Transferases↗
PubMed1991

Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily.

STE6, the yeast a-factor transporter, is a member of the ATP binding cassette protein superfamily, which also includes the mammalian multidrug resistance protein and the cystic fibrosis gene product. These proteins contain two homologous halves, each with six membrane spanning segments and a predicted ATP nucleotide binding domain. To assess the importance of the two halves of STE6, and to examine the functional significance of residues conserved among members of the ATP binding cassette superfamily, we introduced mutations into the nucleotide binding domains of STE6. Our analysis demonstrates that both halves of STE6 are critical for function and that some, but not all, mutations analogous to those known to result in cystic fibrosis impair STE6 activity. To examine further the functional contribution of each half of the STE6 protein, we severed the STE6 coding sequence and expressed the two halves of the transporter as separate polypeptides. Whereas 'half-molecules' are unable to provide transport function individually, co-expression of both half-molecules in the same cell leads to functional reconstitution of STE6-mediated a-factor transport.

ATP-Binding Cassette Transporters↗
PubMed1991

The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and RAS proteins.

Post-translational processing of a distinct group of proteins and polypeptides, including the a-factor mating pheromone and RAS proteins of Saccharomyces cerevisiae, results in the formation of a modified C-terminal cysteine that is S-isoprenylated and alpha-methyl esterified. We have shown previously that a membrane-associated enzymatic activity in yeast can mediate in vitro methylation of an isoprenylated peptide substrate and that this methyltransferase activity is absent in ste14 mutants. We demonstrate here that STE14 is the structural gene for this enzyme by expression of its product as a fusion protein in Escherichia coli, an organism in which this activity is lacking. We also show that a-factor, RAS1 and RAS2 are physiological methyl-accepting substrates for this enzyme by demonstrating that these proteins are not methylated in a ste14 null mutant. It is notable that cells lacking STE14 methyltransferase activity exhibit no detectable impairment of RAS function or cell viability. However, we did observe a kinetic delay in the rate of RAS2 maturation and a slight decrease in the amount of membrane localized RAS2. Thus, methylation does not appear to be essential for RAS2 maturation or localization, but the lack of methylation can have subtle effects on the efficiency of these processes.

Amino Acid Sequence↗
PubMed1988

The a-factor pheromone of Saccharomyces cerevisiae is essential for mating.

The Saccharomyces cerevisiae pheromone a-factor is produced by a cells and interacts with alpha cells to cause cell cycle arrest and other physiological responses associated with mating. Two a-factor structural genes, MFA1 and MFA2, have been previously cloned with synthetic probes based on the a-factor amino acid sequence (A. Brake, C. Brenner, R. Najarian, P. Laybourn, and J. Merryweather, cited in M.-J. Gething [ed.], Protein transport and secretion, 1985). We have examined the function of these genes in a-factor production and mating by construction and analysis of chromosomal null mutations. mfa1 and mfa2 single mutants each exhibited approximately half the wild-type level of a-factor activity and were proficient in mating, whereas the mfa1 mfa2 double mutant produced no a-factor and was unable to mate. These results demonstrate that both genes are functional, that each gene makes an equivalent contribution to the a-factor activity and mating capacity of a cells, and that a-factor plays an essential role in mating. Strikingly, exogenous a-factor did not alleviate the mating defect of the double mutant, suggesting that an a cell must be producing a-factor to be an effective mating partner.

Amino Acid Sequence↗
PubMed1987

secD, a new gene involved in protein export in Escherichia coli.

New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.

Bacterial Proteins↗
PubMed1986

RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor.

We have identified a gene (SUPH) of S. cerevisiae that is required for both RAS function and mating by cells of a mating type. supH is allelic to ste16, a gene required for the production of the mating pheromone a-factor. Both RAS and a-factor coding sequences terminate with the potential acyltransferase recognition sequence Cys-A-A-X, where A is an aliphatic amino acid. Mutations in SUPH-STE16 prevent the membrane localization and maturation of RAS protein, as well as the fatty acid acylation of it and other membrane proteins. We propose the designation RAM (RAS protein and a-factor maturation function) for SUPH and STE16. RAM may encode an enzyme responsible for the modification and membrane localization of proteins with this C-terminal sequence.

Acylation↗
PubMed1986

Effects of signal sequence mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coli.

We isolated a collection of mutants defective in the export of alkaline phosphatase to the periplasm. Two classes of mutants were obtained: one class with lesions unlinked to the phoA gene and a second class harboring linked mutations. Among the former class, one mutant is cold sensitive for growth and may be defective in a component of the Escherichia coli secretory apparatus. Included in the latter class are 47 mutants which are characterized in detail in this report. To facilitate DNA sequence analysis of these mutants, we devised a convenient method that relies on homologous recombination in vivo to transfer phoA mutations from the bacterial chromosome directly onto the genome of a single-stranded M13 phage vector. DNA sequence analysis revealed that our collection of mutants comprises six unique mutations, all of which reside in the phoA signal sequence coding region and lend further support to the notion that the length of the hydrophobic core of the signal sequence is crucial for its function in protein export. Kinetic studies showed that in these mutants, the small fraction of alkaline phosphatase which succeeds in reaching a periplasmic location, despite a defective signal sequence, is translocated across the membrane in a slow, posttranslational fashion.

Alkaline Phosphatase↗
PubMed1985

Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae.

Some strains of Klebsiella pneumonia secrete pullulanase, a debranching enzyme which produces linear molecules (maltodextrins, amylose) from amylopectin and glycogen. pulA, the structural gene for pullulanase, was introduced into Escherichia coli, either on a multiple-copy-number plasmid or as a single copy in the chromosome. When in E. coli, pulA was controlled by malT, the positive regulatory gene of the maltose regulon. Indeed, pulA expression was undetectable in a malT-negative mutant and constitutive in a malTc strain. Furthermore, the plasmid carrying pulA titrated the MalT protein. When produced in E. coli, pullulanase was not localized in the same way as in K. pneumoniae. In the latter case it was first exported to the outer membrane, with which it remained loosely associated, and was then released into the growth medium. In E. coli the enzyme was distributed both in the inner and the outer membranes and was never released into the growth medium.

Cell Membrane↗
PubMed1983

In vitro construction and characterization of phoA-lacZ gene fusions in Escherichia coli.

Using recombinant DNA techniques, we have constructed phoA-lacZ gene fusions. Two of the fusions encode hybrid proteins containing approximately half of alkaline phosphatase at the amino terminus joined to beta-galactosidase. For the one fusion strain analyzed in detail, it was shown that the hybrid protein is found in the membrane fraction of cells. In its membrane location, the beta-galactosidase activity of the hybrid is not sufficient to support cell growth on lactose. Unexpectedly, fusions containing phoA and lacZ joined in the wrong translational reading frame were also obtained. These fusions direct the phosphate-regulated synthesis of beta-galactosidase, apparently via a translation restart mechanism. Thus, when gene fusions are constructed, the presence of properly regulated beta-galactosidase activity does not necessarily indicate that a hybrid protein is being produced.

Alkaline Phosphatase↗
PubMed1983

Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli.

A phoA-lacZ gene fusion was used to isolate mutants altered in the alkaline phosphatase signal sequence. This was done by selecting Lac+ mutants from a phoA-lacZ fusion strain that produces a membrane-bound hybrid protein and is unable to grow on lactose. Two such mutant derivatives were characterized. The mutations lie within the phoA portion of the fused gene and cause internalization of the hybrid protein. When the mutations were genetically recombined into an otherwise wild-type phoA gene, they interfered with export of alkaline phosphatase to the periplasm. The mutant alkaline phosphatase protein was found instead in the cytoplasm in precursor form. DNA sequence analysis demonstrated that both mutations lead to amino acid alterations in the signal sequence of alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1981

Deletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA.

Lambda transducing phages containing portions of the phoA gene have been isolated and used to construct a deletion map of the phoA gene. The isolation of a plaque-forming lambda transducing phage carrying the entire phoA gene is also described. Two new methods for screening or selection of mutants that have altered levels of alkaline phosphatase activity are reported.

Alkaline Phosphatase↗