It has become increasingly clear that in Escherichia coli, most exported proteins are translocated either posttranslationally or late in their synthesis, and that a component of the export apparatus, SecB, facilitates the export of a subset of the secreted proteins by maintaining them in an export-competent, unfolded form. In an effort to understand how SecB functions as an antifolding factor, we mapped and characterized the sites of SecB interaction in the outer membrane protein LamB. We found that the interaction of SecB with LamB was dependent on the LamB signal sequence as well as on a region in the mature LamB protein. The simplest interpretation of these findings is that SecB binds to both the LamB signal sequence and a mature region in LamB, and that this interaction promotes the antifolding activity of SecB.
\r\n\r\nGiven the fact that several heat-shock proteins have also been shown to function as antifolding factors, we wanted to investigate whether heat-shock proteins might act in a manner analogous to SecB in facilitating the export process. We found that induction of the heat-shock response could substitute for SecB function (SecB is not a heat-shock protein), and that a basal level of heat-shock proteins was necessary for the cell to survive in the absence of SecB protein. These results suggested that heat-shock proteins might indeed be involved in the secretory process and function in a manner similar to that of SecB.
\r\n\r\nIn an attempt to identify these proteins, suppressors of a secB null mutation were isolated and characterized. Not unexpectedly, most of these suppressors mapped to the rpoH locus. Since rpoH encodes \u03c3\u00b3\u00b2, the heat-shock transcription factor, it is likely that these suppressors affect the synthesis levels of heat-shock proteins, which can substitute for SecB function. The remaining suppressors did not map to any known heat-shock or export genes, and potentially represent unidentified heat-shock proteins or export factors that act in a manner similar to SecB in facilitating the export process in E. coli.
", "doi": "10.7907/tq2t-3k47", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2656", "collection": "thesis", "collection_id": "2656", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06202007-083735", "primary_object_url": { "basename": "Herman_pk_1991.pdf", "content": "final", "filesize": 13913514, "license": "other", "mime_type": "application/pdf", "url": "/2656/1/Herman_pk_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Protein Sorting in the Eukaryotic Secretory Pathway: An Essential Role for a Novel Yeast Protein Kinase", "author": [ { "family_name": "Herman", "given_name": "Paul Kenneth", "clpid": "Herman-Paul-Kenneth" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Dunphy", "given_name": "William G.", "orcid": "0000-0001-7598-8939", "clpid": "Dunphy-W-G" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The yeast vps mutants are defective for the intracellular sorting of proteins to the vacuolar compartment. Mutants from two particular vps complementation groups, vpsl5 and vps34, share a common set of phenotypes that suggested that the VPS15 and VPS34 gene products might be functioning at a similar step of the vacuolar protein sorting pathway. vpsl5 and vps34 mutants exhibit specific defects in the sorting of soluble hydrolases to the vacuolar compartment. Whereas soluble hydrolases such as carboxypeptidase Y are almost quantitatively mislocalized to the cell surface, vacuolar membrane proteins appear to be properly localized to the vacuole.\r\n\r\nThe wild-type VPS15 and VPS34 genes were both cloned from yeast genomic DNA libraries by complementation of temperature-sensitive growth defects associated with mutations in these genes. Haploid yeast strains carrying a disruption of either locus were viable but exhibited a severe ts growth defect indicating that both genes are essential for vegetative growth at elevated temperatures. The vps34 null mutant was also found to exhibit a defect in the segregation of the vacuolar compartment upon cell division.\r\n\r\nThe predicted sequence of the VPS15 gene product exhibits significant similarity to the catalytic domains of the serine/threonine family of protein kinases. Point mutations altering specific amino acid residues of Vpsl5p that are highly conserved in all protein kinases result in the biological inactivation of Vpsl5p. The kinase domain mutants exhibit severe vacuolar protein sorting and ts growth defects. In addition, Vpsl5p is phosphorylated in vivo in a reaction that requires a wild-type Vpsl5p kinase domain. Subcellular fractionation experiments indicate that Vpsl5p is peripherally associated with the cytoplasmic face of a late Golgi or vesicle compartment. A vpsl5 mutant that encodes a protein lacking 30 carboxy-terminal amino acids exhibits a severe ts defect in vacuolar protein delivery. At the restrictive temperature, carboxpeptidase Y accumulates in a specific intracellular compartment that may represent a normal transport intermediate between the Golgi and vacuolar compartments. The vacuolar delivery defect in this mutant has an extremely rapid rate of onset suggesting that Vps15p is directly involved in the sorting of soluble proteins to the vacuole. Altogether, these data suggest that Vpsl5p regulates specific protein phosphorylation reactions in vivo that are required for the delivery of soluble hydrolases to the vacuole.", "doi": "10.7907/38ba-0w26", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:6290", "collection": "thesis", "collection_id": "6290", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04112011-112805564", "type": "thesis", "title": "Genetic, Molecular and Biochemical Studies of Vacuole Biogenesis and Maintenance in the Yeast Saccharomyces cerevisiae", "author": [ { "family_name": "Robinson", "given_name": "Jane Suzanna", "clpid": "Robinson-Jane-Suzanna" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Using a selection for spontaneous mutants that mislocalize a vacuolar CPY-Inv\r\nfusion protein to the cell surface, 505 Saccharomyces cerevisiae mutants with defects in\r\nprotein sorting were identified. Seventeen of these mutants were dominant; the others\r\ndefined 25 new vps (for vacuolar protein sorting) complementation groups. Alleles of\r\neach vps complementation group exhibit defects in the targeting and final processing of\r\nsoluble vacuolar enzymes (CPY, PrA and PrB). Two of the genes, VPS17 and VPS15,\r\nmap to ChXV and ChII, respectively. The vpsll, vps16, vps18 and vps33 mutants\r\nexhibit morphological defects, their cells containing debris of a membranous nature and\r\nhighly abnormal vacuole remnants. Intercrosses with other vacuole function-defective\r\nmutants (vpl, pep, sip and end), revealed genetic overlaps. It is evident that more than\r\n50 gene products are involved in biogenesis and maintenance of the yeast vacuole.\r\nAlleles of 7 of the vps complementation groups are temperature-sensitive for vegetative\r\ncell growth at 37\u00b0C, and this recessive, Ts phenotype cosegregated with the vps defect\r\nin each case. This easily complemented phenotype has facilitated cloning of six of the\r\ngenes.\r\nThe VPS18 gene was chosen for further studies. A plasmid complementing the\r\nTs growth defect of vps18-1 was isolated and shown by integrative mapping to carry\r\nDNA from the VPS18 locus. Yeast strains with a deletion of the entire VPS18 coding\r\nregion (\u0394vps18), are viable and exhibit the same phenotypes as vps18-1. \u0394vps18, \u03b1 strains have smaller \u03b1-factor halos on sst2,a lawns than do VPS18, \u03b1 strains.\r\nImmunoprecipitation of \u03b1-factor indicated that it is secreted from the \u0394vps18 mutant in\r\nprecursor form. Several of the other severely defective vps mutants also show this \u03b1-factor\r\nprocessing defect. DNA sequencing of VPs18 showed an open reading frame\r\nencoding a 918aa protein, hydrophilic in nature. The protein sequence revealed a zinc finger like, cysteine-rich motif in its C-terminal region. A synthetic mutant with a\r\ncysteine to serine alteration has a temperature-conditional CPY sorting defect with very\r\nrapid onset. Therefore, Vps18p may be a zinc-binding protein, directly necessary for\r\ncorrect vacuolar protein sorting and proper functioning of the Golgi compartment that\r\ncontains Kex2p.\r\n", "doi": "10.7907/zfzc-0035", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2549", "collection": "thesis", "collection_id": "2549", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06112007-104332", "type": "thesis", "title": "Vacuolar Protein Sorting in Yeast: Characterization of Mutants and Identification of a Protein Required for Vacuole Biogenesis", "author": [ { "family_name": "Banta", "given_name": "Lois Margaret", "clpid": "Banta-Lois-Margaret" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The lysosome-like vacuole of the yeast Saccharomyces cerevisiae is an acidic compartment containing a number of hydrolytic glycoproteins including carboxypeptidase Y (CPY), proteinase A (PrA) and proteinase B (PrB). A gene fusion-based selection scheme was utilized to isolate ~600 mutants defective in the localization and processing of vacuolar proteins. These vacuolar protein sorting (vps) mutants define >33 complementation groups and exhibit hybrid protein-independent defects in the sorting of CPY, PrA, and PrB. Light and electron microscopic analyses of the vacuole morphology revealed three distinct classes of vps mutants. The class A mutants (26 complementation groups) contain 1-3 large vacuoles that resemble those of the parental strain. One class A mutant is sensitive to low pH and exhibits a defect in vacuole acidification. Consistent with a role for vacuolar pH in protein sorting, perturbation of vacuole acidification resulted in the missorting and secretion of CPY and PrA in wild-type cells. Mutants in the three class B complementation groups exhibit a fragmented vacuole morphology. The class C vps mutants (four complementation groups) lack any compartment resembling a wild-type vacuole, but accumulate vesicles and other membranous structures. Many class C strains exhibit genetically linked defects including temperature-sensitivity and sensitivity to osmotic stress. Unlike other vps mutants, these mutants secrete up to 50% of a vacuolar membrane marker enzyme. The gene defined by one class C mutant, vps33, has been cloned. The predicted VPS33 gene product is hydrophilic and shares sequence similarity with a family of ATP-binding proteins. Disruption of VPS33 is not lethal but results in temperature-sensitive growth. Vps33p-specific antisera recognize a cytosolic protein of ~75 kD. One temperature-sensitive vps33 mutant carrying a missense mutation contains apparently normal vacuoles at the permissive temperature, but lacks vacuoles specifically in the bud at the nonpermissive temperature. We propose that the abnormalities in vacuole morphology and inheritance in vps33 mutants are a consequence of a primary defect in Golgi-to-vacuole protein delivery. A second VPS gene, VPS28, has also ben cloned. Our data suggest that the VPS28 gene product only indirectly affects vacuole protein sorting, but may function in a late protein modification process.
", "doi": "10.7907/TZHF-3J73", "publication_date": "1990", "thesis_type": "phd", "thesis_year": "1990" }, { "id": "thesis:2312", "collection": "thesis", "collection_id": "2312", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05302007-153631", "type": "thesis", "title": "Characterization of the SEC18 Gene of S. cerevisiae: Identification of a Protein Involved in Yeast Secretion", "author": [ { "family_name": "Eakle", "given_name": "Kurt Andrew", "clpid": "Eakle-Kurt-Andrew" } ], "thesis_advisor": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "thesis_committee": [ { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum and the Golgi complex. We have cloned the SEC18 gene by complementation of the secl8-1 mutation. Deletion/disruption of this gene has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2271 by open reading frame which would code for a protein of 83.9 kd. The predicted protein sequence showed no significant homology to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kd. Antisera raised against a Sec18-?-galactosidase fusion protein, detects two proteins from in vivo 35S labeled yeast cells identical in size to those seen by in vitro translation. Although potential sites for N-linked glycosylation are present in the Sec 18p sequence, the sizes of the in vivo SEC18 gene products are unaffected by the drug tunicamycin. Hydrophobicity analysis indicated that the protein is hydrophilic in nature and lacks any region that would be predicted to serve as a signal sequence or transmembrane anchor. These results suggest that the Secl8p resides in the cell cytoplasm. Pulse-chase experiments indicate that the two forms of Sec 18 protein are not the result of post-translational processing. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec 18 protein do not arise from mRNAs with different 5' ends. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of the Sec 18 protein. While cell fractionation studies show that the Sec 18p are not associated with ER or Golgi compartments, association with a 100,000 x g pellet fraction has been observed suggesting that Sec 18p may bind transiently to small vesicles such as those presumed to participate in ER to Golgi transport.
", "doi": "10.7907/6aqb-xd83", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" } ]