Sec20p-interacting proteins (Tip20p,Ufe1p) in the retrograde secretory pathway of the fungal pathogen<Emphasis Type="Italic"> Candida albicans</Emphasis>

Sec20p is an essential Type-II membrane protein of the human fungal pathogen Candida albicans, which is thought to be involved in mediating retrograde vesicle traffic from the Golgi to the endoplasmic reticulum (ER). Using an epitope-tagged Sec20p we obtained evidence for its localization in ER membranes, which is consistent with its proposed role in an ER-tSNARE complex. Two genes encoding potential interaction partners for Sec20p, Tip20p and Ufe1p, were identified in genomic sequences of C. albicans; these show 18% and 27% identity, respectively, to homologues in Saccharomyces cerevisiae. An interaction between the cytoplasmic domain of Sec20p and Tip20p was demonstrated by two-hybrid analysis; in addition, Tip20p was found to form homodimers. Interaction between Sec20p and Tip20p in vivo was verified by co-immunoprecipation experiments. CaUFE1, which encodes a potential ER-tSNARE, was able to complement a thermosensitive ufe1 mutation in S. cerevisiae, suggesting functional conservation between the two fungal proteins. Thus, although the sequences of some components of the ER-tSNARE complex have diverged considerably during evolution, it appears that they have retained similar functions in C. albicans and S. cerevisiae.     

Overexpression of a dominant-negative allele of SEC4 inhibits growth and protein secretion in Candida albicans

Candida albicans SEC4 was cloned by complementing the Saccharomyces cerevisiae sec4-8 mutation, and its deduced protein product (Sec4p) was 63% identical to S. cerevisiae Sec4p. One chromosomal SEC4 allele in C. albicans CAI4 was readily disrupted by homologous gene targeting, but efforts to disrupt the second allele yielded no viable null mutants. Although this suggested that C. albicans SEC4 was essential, it provided no information about this gene's functions. Therefore, we constructed a mutant sec4 allele encoding an amino acid substitution (Ser-28-->Asn) analogous to the Ser-17-->Asn substitution in a trans-dominant inhibitor of mammalian Ras protein. GAL1-regulated expression plasmids carrying the mutant sec4 allele (pS28N) had minimal effects in glucose-incubated C. albicans transformants, but six of nine transformants tested grew very slowly in galactose. Incubation of pS28N transformants in galactose also inhibited secretion of aspartyl protease (Sap) and caused 90-nm secretory vesicles to accumulate intracellularly, and plasmid curing restored growth and Sap secretion to wild-type levels. These results imply that C. albicans SEC4 is required for growth and protein secretion and that it functions at a later step in the protein secretion pathway than formation of post-Golgi secretory vesicles. They also demonstrate the feasibility of using inducible dominant-negative alleles to define the functions of essential genes in C. albicans.     

Deletion analysis of yeast Sec65p reveals a central domain that is sufficient for function in vivo

The Saccharomyces cerevisiae SEC65 gene encodes a 32 kDa subunit of yeast signal recognition particle that is homologous to human SRP19. Sequence comparisons suggest that the yeast protein comprises three distinct domains. The central domain (residues 98–171) exhibits substantial sequence similarity to the 144 residue SRP19. In contrast, the N-terminal and C-terminal domains (residues 1–97 and 172–273 respectively) share no similarity to SRP19, with the exception of a cluster of positively charged residues at the extreme C-terminus of both proteins. Here, we report the cloning of a Sec65p homologue from the yeast Candida albicans that shares the same extended domain structure as its S. cerevisiae counterpart. This conservation of sequence is reflected at the functional level, as the C. albicans gene can complement the conditional lethal sec65-1 mutation in S. cerevisiae . In order to examine the role of the N- and C- terminal domains in Sec65p function, we have engineered truncation mutants of S. cerevisiae SEC65 and tested these for complementing activity in vivo and for SRP integrity in vitro . These studies indicate that a minimal Sec65p comprising residues 76–209, which includes the entire central SRP19-like domain, is sufficient for SRP function in yeast.     

The genes of two G-proteins involved in protein transport in Pichia pastoris

Members of the Rab protein family play essential roles in vesicle fusion during protein secretion and represent highly conserved GTP binding proteins. The Saccharomyces cerevisiae Sec4p and Ypt1p, promoting vesicle fusion at the plasma membrane and in ER-Golgi transport, respectively, are among the best characterised yeast members. We have here cloned the Pichia pastoris SEC4 homologue using a S. cerevisiae SEC4 probe. In addition we isolated a crosshybridising clone encoding another Rab-/Ypt-like protein. The deduced full-length PpSec4p comprises 204 amino acid residues with an over all identity of 64% to the Sec4p from S. cerevisiae and 72% to the Candida albicans Sec4p. The YPT-like gene encodes a 216 amino acid residue protein showing highest similarity to the S. cerevisiae Ypt10p and Ypt53p. Both PpSec4p and the Ypt-like protein carry a -Cys-Cys C-terminus, indicating that these proteins are targets for geranyl-geranylation by a type II prenyltransferase.     

The TIP1 gene of Saccharomyces cerevisiae encodes an 80 kDa cytoplasmic protein that interacts with the cytoplasmic domain of Sec20p.

The SEC20 gene of Saccharomyces cerevisiae encodes a 50 kDa type II integral membrane glycoprotein that is required for endoplasmic reticulum (ER) to Golgi transport. Here, we have used a genetic screen, based on the lethal effect of overexpressing the cytoplasmic domain of Sec20p, to identify a novel cytosolic factor that interacts with SEC20. This factor is an 80 kDa cytoplasmic protein encoded by the TIP1 (SEC twenty interacting protein) gene. Coimmunoprecipitation and immunofluorescence using Tip1p and Sec20p or its cytoplasmic domain showed that the two proteins physically interact to form a stable complex. Like SEC20, TIP1 is required for ER to Golgi transport and depletion of Tip1p results in accumulation of an extensive network of ER plus small transport vesicles. We therefore propose that Sec20p and Tip1p act together as a functional unit in the ER to Golgi transport step.     

Sec8p and Sec15p are components of a plasma membrane-associated 19.5S particle that may function downstream of Sec4p to control exocytosis

The SEC8 and SEC15 genes are essential for exocytosis in the yeast Saccharomyces cerevisiae and exhibit strong genetic interactions with SEC4, a gene of the ras superfamily. The SEC8 gene encodes a hydrophilic protein of 122 kD, while the temperature-sensitive sec8-9 allele encodes a protein prematurely truncated at 82 kD by an opal stop codon. The Sec8p sequence contains a 202 amino acid region that is 25% identical to the leucine rich domain of yeast adenylate cyclase that has been implicated in ras responsiveness. Fractionation, stability, and cross-linking studies indicate that Sec8p is a component of a 19.5S particle that also contains Sec15p. This particle is found both in the cytosol and peripherally associated with the plasma membrane, but it is not associated with secretory vesicles. Gel filtration studies suggest that a portion of Sec4p is in association with the Sec8p/Sec15p particle. We propose that this particle may function as a downstream effector of Sec4p, serving to direct the fusion of secretory vesicles with the plasma membrane.     

Functional expression of the Candida albicans alpha-factor receptor in Saccharomyces cerevisiae

Candida albicans genes involved in mating have been identified previously by homology to Saccharomyces cerevisiae mating pathway components. The C. albicans genome encodes CaSte2p, a homolog of the S. cerevisiae alpha-mating pheromone receptor Ste2p, and two potential pheromones, alpha-F13 (GFRLTNFGYFEPG) and alpha-F14 (GFRLTNFGYFEPGK). The response of several C. albicans strains to the synthesized peptides was determined. The alpha-F13 was degraded by a C. albicans MTLa strain but not by S. cerevisiae MATa cells. The CaSTE2 gene was cloned and expressed in a ste2-deleted strain of S. cerevisiae. Growth arrest and beta-galactosidase activity induced from a FUS1-lacZ reporter construct increased in a dose-dependent manner upon exposure of transgenic S. cerevisiae to alpha-F13. Mating between the strain expressing CaSTE2 and an opposite mating type was mediated by alpha-F13 and not by the S. cerevisiae alpha-factor. The results indicated that CaSte2p effectively coupled to the S. cerevisiae signal transduction pathway. Functional expression of CaSte2p in S. cerevisiae provides a well-defined system for studying the biochemistry and molecular biology of the C. albicans pheromone and its receptor.     

Candida albicans VPS1 contributes to protease secretion, filamentation, and biofilm formation

To investigate the pre-vacuolar secretory pathway in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS1. C. albicans VPS1 encodes a predicted 694-aa dynamin-like GTPase that is 73.3% similar to S. cerevisiae Vps1p. Plasmids bearing C. albicans VPS1 complemented the temperature-sensitive growth, abnormal class F vacuolar morphology, and carboxypeptidase missorting of a S. cerevisiae vps1 null mutant. To study VPS1 function in C. albicans, a conditional mutant strain (tetR-VPS1) was generated by deleting the first allele of VPS1 and placing the second allele under control of a tetracycline-regulatable promoter. With doxycycline, the tetR-VPS1 mutant was hyper-susceptible to sub-inhibitory concentrations of fluconazole, but not amphotericin B, 5-fluorocytosine, or non-specific osmotic stresses. The repressed tetR-VPS1 mutant was defective in filamentation and secreted less extracellular protease activity. Biofilm production and filamentation within the biofilm were markedly reduced. These results suggest that C. albicans VPS1 has a key role in several important virulence-related phenotypes.     

The budding yeast Pichia pastoris has a novel Sec23p homolog

In Pichia pastoris, coat protein complex II (COPII) vesicles form at discrete transitional ER (tER) sites. Analyzing COPII coat proteins in this yeast will help to reveal the mechanisms of tER organization. Here, we show that like Saccharomyces cerevisiae, P. pastoris contains essential SEC23 and SEC24 genes, as well as the non-essential SEC24 homolog LST1. In addition, P. pastoris contains a novel non-essential SEC23 homolog that we have designated SHL23. The products of all four genes are concentrated at tER sites. Deletion of SHL23 does not disrupt tER morphology. As judged by two-hybrid analysis, Sec23p associates with both Sec24p and Lst1p, whereas Shl23p associates selectively with Lst1p. These results suggest that P. pastoris COPII vesicles contain an Shl23p/Lst1p complex that is absent in S. cerevisiae.     

Distinct and redundant roles of the two protein kinase A isoforms Tpk1p and Tpk2p in morphogenesis and growth of Candida albicans

TPK1 and TPK2 encode both isoforms of protein kinase A (PKA) catalytic subunits in Candida albicans. Mutants lacking both TPK1 alleles showed defective hyphal morphogenesis on solid inducing media, whereas in liquid hypha, formation was affected slightly. In contrast, tpk2 mutants were only partially morphogenesis defective on solid media, whereas a strong block was observed in liquid. In addition, the yeast forms of tpk2-- but not tpk1-- mutants were completely deficient in invading agar. Because Tpk1p and Tpk2p differ in their N-terminal domains of approximately 80--90 amino acids, while the catalytic portions are highly homologous, the functions of hybrid Tpk proteins with exchanged N-terminal domains were tested. The results demonstrate that the catalytic portions mediate Tpk protein specificities with regard to filamentation, whereas agar invasion is mediated by the N-terminal domain of Tpk2p. Homozygous tpk1 and tpk2 mutants grew normally; however, a tpk2 mutant strain containing a single regulatable TPK1 allele (PCK1p-TPK1) at low expression levels was severely growth defective. It was completely blocked in hyphal morphogenesis and was stress resistant to high osmolarities or temperatures. Thus, both Tpk isoforms in C. albicans share growth functions but, unlike Saccharomyces cerevisiae isoforms, they have positive, specific roles in filament formation in different environments.     

Analysis of heterologous expression of Candida albicans SEC61 gene reveals differences in Sec61p homologues related to species-specific functionality

The protein secretory pathway has not been studied in depth in Candida albicans despite its essential role in the secretion of enzymes and cell surface components related to the ability of the fungus to colonize the human host. To gain further insight into the elements that participate in the first stages of the secretory process in this fungal pathogen we have isolated and characterized the C. albicans ortholog of SEC61. In other species SEC61 has been shown to encode the core element of the protein translocation apparatus within the ER membrane. The cloned gene appears to be essential for cell viability and encodes a highly conserved protein, very similar to the Sec61p from other yeast species both in sequence and hydropathy profile. However, CaSec61p is not able to complement the thermosensitive-growth phenotype of a Saccharomyces cerevisiae sec61 mutant, even though it is expressed and correctly incorporated into the ER membrane of the transformant cells. We report results indicating that the lack of functional complementation could be related to differences in the primary structure of the cytosolic domain located between the fourth and fifth transmembrane domains of the accepted topological model of Sec61p.     

Structural and functional dissection of a membrane glycoprotein required for vesicle budding from the endoplasmic reticulum.

Sec12p is a membrane glycoprotein required for the formation of a vesicular intermediate in protein transport from the endoplasmic reticulum to the Golgi apparatus in Saccharomyces cerevisiae. Comparison of the N-linked glycosylation of Sec12p, a Sec12p-invertase hybrid protein, and a derivative of Sec12p lacking 71 carboxy-terminal amino acids showed that Sec12p is a type II membrane protein. Analysis of two truncated forms of Sec12p and of a temperature-sensitive mutant indicated that the C-terminal domain of Sec12p is not essential for protein transport, whereas the integrity and membrane attachment of the cytoplasmic N-terminal domain are essential. Expression of a soluble cytoplasmic domain dramatically inhibited the growth of a sec12 temperature-sensitive strain by increasing the transport defect at a normally permissive temperature. This growth inhibition as well as the sec12 temperature-sensitive defect were suppressed by the overproduction of Sar1p, a small GTP-binding protein that participates in protein transport. Sar1p membrane association was enhanced by elevated levels of Sec12p. These results suggest that the cytoplasmic domain of Sec12p interacts with Sar1p and that the complex may function to promote vesicle formation.     

Structural and functional dissection of Sec62p, a membrane-bound component of the yeast endoplasmic reticulum protein import machinery.

SEC62 is required for the import of secretory protein precursors into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae. The DNA sequence of SEC62 predicts a 32-kDa polypeptide with two potential membrane-spanning segments. Two antisera directed against different portions of the SEC62 coding region specifically detected a 30-kDa polypeptide in cell extracts. A combination of subcellular fractionation, detergent and alkali extraction, and indirect immunofluorescence studies indicated that Sec62p is intimately associated with the ER membrane. Protease digestion of intact microsomes and analysis of the oligosaccharide content of a set of Sec62p-invertase hybrid proteins suggested that Sec62p spans the ER membrane twice, displaying hydrophilic amino- and carboxy-terminal domains towards the cytosol. Sec62p-invertase hybrid proteins that lack the Sec62p C terminus failed to complement the sec62-l mutation and dramatically inhibited the growth of sec62-l cells at a normally permissive temperature. The inhibitory action of toxic Sec62p-invertase hybrids was partially counteracted by the overexpression of Sec63p. Taken together, these data suggest that the C-terminal domain of Sec62p performs an essential function and that the N-terminal domain associates with other components of the translocation machinery, including Sec63p.     

The engagement of Sec61p in the ER dislocation process

Sec61p comprises the endoplasmic reticulum (ER) channel through which nascent polypeptides are imported and from which malfolded proteins have been suggested to be exported, or dislocated, back to the cytoplasm. We have devised a genetic screen for dislocation-specific mutant alleles of SEC61 from S. cerevisiae by employing the unfolded protein response to report on the accumulation of misfolded proteins in the ER. Three of the isolated sec61 alleles are fully proficient in protein translocation into the ER, but defective in the elimination of a misfolded ER luminal substrate and a short-lived ER membrane-spanning model protein, which are otherwise rapidly degraded by cytoplasmic proteolysis in wild-type cells. Our results point to the fourth luminal loop and third transmembrane domain of Sec61p that markedly influence dislocation. We suggest that distinct features of the Sec61-translocon direct the two-way translocation processes.     

Identification of Sec36p,Sec37p,and Sec38p: components of yeast complex that contains Sec34p and Sec35p

The Saccharomyces cerevisiae proteins Sec34p and Sec35p are components of a large cytosolic complex involved in protein transport through the secretory pathway. Characterization of a new secretion mutant led us to identify SEC36, which encodes a new component of this complex. Sec36p binds to Sec34p and Sec35p, and mutation of SEC36 disrupts the complex, as determined by gel filtration. Missense mutations of SEC36 are lethal with mutations in COPI subunits, indicating a functional connection between the Sec34p/sec35p complex and the COPI vesicle coat. Affinity purification of proteins that bind to Sec35p-myc allowed identification of two additional proteins in the complex. We call these two conserved proteins Sec37p and Sec38p. Disruption of either SEC37 or SEC38 affects the size of the complex that contains Sec34p and Sec35p. We also examined COD4, COD5, and DOR1, three genes recently reported to encode proteins that bind to Sec35p. Each of the eight genes that encode components of the Sec34p/sec35p complex was tested for its contribution to cell growth, protein transport, and the integrity of the complex. These tests indicate two general types of subunits: Sec34p, Sec35p, Sec36p, and Sec38p seem to form the essential core of a complex to which Sec37p, Cod4p, Cod5p, and Dor1p seem to be peripherally attached.     

Sec62p, a component of the endoplasmic reticulum protein translocation machinery, contains multiple binding sites for the Sec-complex

SEC62 encodes an essential component of the Sec-complex that is responsible for posttranslational protein translocation across the membrane of the endoplasmic reticulum in Saccharomyces cerevisiae. The specific role of Sec62p in translocation was not known and difficult to identify because it is part of an oligomeric protein complex in the endoplasmic reticulum membrane. An in vivo competition assay allowed us to characterize and dissect physical and functional interactions between Sec62p and components of the Sec-complex. We could show that Sec62p binds via its cytosolic N- and C-terminal domains to the Sec-complex. The N-terminal domain, which harbors the major interaction site, binds directly to the last 14 residues of Sec63p. The C-terminal binding site of Sec62p is less important for complex stability, but adjoins the region in Sec62p that might be involved in signal sequence recognition.     

SED4 encodes a yeast endoplasmic reticulum protein that binds Sec16p and participates in vesicle formation

SEC16 is required for transport vesicle budding from the ER in Saccharomyces cerevisiae, and encodes a large hydrophilic protein found on the ER membrane and as part of the coat of transport vesicles. In a screen to find functionally related genes, we isolated SED4 as a dosage- dependent suppressor of temperature-sensitive SEC16 mutations. Sed4p is an integral ER membrane protein whose cytosolic domain binds to the COOH-terminal domain of Sec16p as shown by two-hybrid assay and coprecipitation. The interaction between Sed4p and Sec16p probably occurs before budding is complete, because Sed4p is not found in budded vesicles. Deletion of SED4 decreases the rate of ER to Golgi transport, and exacerbates mutations defective in vesicle formation, but not those that affect later steps in the secretory pathway. Thus, Sed4p is important, but not necessary, for vesicle formation at the ER. Sec12p, a close homologue of Sed4p, also acts early in the assembly of transport vesicles. However, SEC12 performs a different function than SED4 since Sec12p does not bind Sec16p, and genetic tests show that SEC12 and SED4 are not functionally interchangeable. The importance of Sed4p for vesicle formation is underlined by the isolation of a phenotypically silent mutation, sar1-5, that produces a strong ER to Golgi transport defect when combined with sed4 mutations. Extensive genetic interactions between SAR1, SED4, and SEC16 show close functional links between these proteins and imply that they might function together as a multisubunit complex on the ER membrane.     

A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation.

SEC61p is essential for protein translocation across the endoplasmic reticulum membrane of S. cerevisiae. We have found a mammalian homolog that shows more than 50% sequence identity with the yeast protein. Moreover, several regions of SEC61p have significant similarities with corresponding ones of SecYp of bacteria, indicating a strong evolutionary conservation of the mechanism of protein translocation. Mammalian Sec61p, like the yeast protein, is located in the immediate vicinity of nascent polypeptides during their membrane passage. It is tightly associated with membrane-bound ribosomes, suggesting that the nascent chain passes directly from the ribosome into a protein-conducting channel. These results define Sec61p as a ubiquitous key component of the protein translocation apparatus.     

Sec24p and Iss1p function interchangeably in transport vesicle formation from the endoplasmic reticulum in Saccharomyces cerevisiae

The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle transport from the endoplasmic reticulum. Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a close homologue, ISS1 (YNL049C), which is 55% identical to SEC24. SEC24 is essential for vesicular transport in vivo because depletion of Sec24p is lethal, causing exaggeration of the endoplasmic reticulum and a block in the maturation of carboxypeptidase Y. Overproduction of Sec24p suppressed the temperature sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed the temperature sensitivity of sec16-2. SEC24 gene disruption could be complemented by overexpression of ISS1, indicating functional redundancy between the two homologous proteins. Deletion of ISS1 had no significant effect on growth or secretion; however, iss1Delta mutants were found to be synthetically lethal with mutations in the v-SNARE genes SEC22 and BET1. Moreover, overexpression of ISS1 could suppress mutations in SEC22. These genetic interactions suggest that Iss1p may be specialized for the packaging or the function of COPII v-SNAREs. Iss1p tagged with His(6) at its C terminus copurified with Sec23p. Pure Sec23p/Iss1p could replace Sec23p/Sec24p in the packaging of a soluble cargo molecule (alpha-factor) and v-SNAREs (Sec22p and Bet1p) into COPII vesicles. Abundant proteins in the purified vesicles produced with Sec23p/Iss1p were indistinguishable from those in the regular COPII vesicles produced with Sec23p/Sec24p.