The transmembrane domain is sufficient for Sbh1p function, its association with the Sec61 complex, and interaction with Rtn1p.

The Sec61 protein translocation complex in the endoplasmic reticulum (ER) membrane is composed of three subunits. The alpha-subunit, called Sec61p in yeast, is a multispanning membrane protein that forms the protein conducting channel. The functions of the smaller, carboxyl-terminally tail-anchored beta subunit Sbh1p, its close homologue Sbh2p, and the gamma subunit Sss1p are not well understood. Here we show that co-translational protein translocation into the ER is reduced in sbh1Delta sbh2Delta cells, whereas there is a limited reduction of post-translational translocation and no effect on export of a mutant form of alpha-factor precursor for ER-associated degradation in the cytosol. The translocation defect and the temperature-sensitive growth phenotype of sbh1Delta sbh2Delta cells were rescued by expression of the transmembrane domain of Sbh1p alone, and the Sbh1p transmembrane domain was sufficient for coimmunoprecipitation with Sec61p and Sss1p. Furthermore, we show that Sbh1p co-precipitates with the ER transmembrane protein Rtn1p. Sbh1p-Rtn1p complexes do not appear to contain Sss1p and Sec61p. Our results define the transmembrane domain as the minimal functional domain of the Sec61beta homologue Sbh1p in ER translocation, identify a novel interaction partner for Shb1p, and imply that Sbh1p has additional functions that are not directly linked to protein translocation in association with the Sec61 complex.

A cytosolic splice variant of Cab45 interacts with Munc18b and impacts on amylase secretion by pancreatic acini.

We identified in a yeast two-hybrid screen the EF-hand Ca(2+)-binding protein Cab45 as an interaction partner of Munc18b. Although the full-length Cab45 resides in Golgi lumen, we characterize a cytosolic splice variant, Cab45b, expressed in pancreatic acini. Cab45b is shown to bind (45)Ca(2+), and, of its three EF-hand motifs, EF-hand 2 is demonstrated to be crucial for the ion binding. Cab45b is shown to interact with Munc18b in an in vitro assay, and this interaction is enhanced in the presence of Ca(2+). In this assay, Cab45b also binds the Munc18a isoform in a Ca(2+)-dependent manner. The endogenous Cab45b in rat acini coimmunoprecipitates with Munc18b, syntaxin 2, and syntaxin 3, soluble N-ethylmaleimide-sensitive factor attachment protein receptors with key roles in the Ca(2+)-triggered zymogen secretion. Furthermore, we show that Munc18b bound to syntaxin 3 recruits Cab45b onto the plasma membrane. Importantly, antibodies against Cab45b are shown to inhibit in a specific and dose-dependent manner the Ca(2+)-induced amylase release from streptolysin-O-permeabilized acini. The present study identifies Cab45b as a novel protein factor involved in the exocytosis of zymogens by pancreatic acini.

Identification of mutations causing temperature-sensitive defects in Semliki Forest virus RNA synthesis.

We have sequenced the nonstructural protein coding region of Semliki Forest virus temperature-sensitive (ts) mutant strains ts1, ts6, ts9, ts10, ts11, ts13, and ts14. In each case, the individual amino acid changes uncovered were transferred to the prototype strain background and thereby identified as the underlying cause of the altered RNA synthesis phenotype. All mutations mapping to the protease domain of nonstructural protein nsP2 caused defects in nonstructural polyprotein processing and subgenomic RNA synthesis, and all mutations in the helicase domain of nsP2 affected subgenomic RNA production. These types of defects were not associated with mutations in other nonstructural proteins.

Characterization of GPI14/YJR013w mutation that induces the cell wall integrity signalling pathway and results in increased protein production in Saccharomyces cerevisiae.

We report here identification and characterization of a mutation in the GPI14 gene, the yeast homologue of the mammalian PIG-M that functions in the synthesis of the GPI moiety anchoring proteins to the plasma membrane. We show that the first putative transmembrane domain of Gpi14p is not essential for its function. Downregulation of GPI14 expression/reduced protein function due to an amino terminal deletion resulted in increased transcription and production of an endogenous and a heterologous secreted protein expressed from HSP150 and ADH1 promoter, respectively. In these cells, unfolded protein response was induced but was not responsible for the enhanced production of these proteins. A cell wall defect in the gpi14 mutant cells was suggested by cell aggregation phenotype, increased sensitivity to Calcofluor white, an increased release of Gas1p and total protein into the culture medium. In the gpi14 mutant cells, transcription of RLM1, a transcription factor participating in the cell wall integrity signalling pathway, was increased, and deletion of RLM1 resulted in a synthetic lethal phenotype with the gpi14 mutation. These results suggest that partial inactivation of Gpi14p causes defects in the cell wall structure and suggest that compromised GPI anchor synthesis results in enhanced protein production via the cell wall integrity signalling pathway.

Molecular interactions position Mso1p, a novel PTB domain homologue, in the interface of the exocyst complex and the exocytic SNARE machinery in yeast.

In this study, we have analyzed the association of the Sec1p interacting protein Mso1p with the membrane fusion machinery in yeast. We show that Mso1p is essential for vesicle fusion during prospore membrane formation. Green fluorescent protein-tagged Mso1p localizes to the sites of exocytosis and at the site of prospore membrane formation. In vivo and in vitro experiments identified a short amino-terminal sequence in Mso1p that mediates its interaction with Sec1p and is needed for vesicle fusion. A point mutation, T47A, within the Sec1p-binding domain abolishes Mso1p functionality in vivo, and mso1T47A mutant cells display specific genetic interactions with sec1 mutants. Mso1p coimmunoprecipitates with Sec1p, Sso1/2p, Snc1/2p, Sec9p, and the exocyst complex subunit Sec15p. In sec4-8 and SEC4I133 mutant cells, association of Mso1p with Sso1/2p, Snc1/2p, and Sec9p is affected, whereas interaction with Sec1p persists. Furthermore, in SEC4I133 cells the dominant negative Sec4I133p coimmunoprecipitates with Mso1p-Sec1p complex. Finally, we identify Mso1p as a homologue of the PTB binding domain of the mammalian Sec1p binding Mint proteins. These results position Mso1p in the interface of the exocyst complex, Sec4p, and the SNARE machinery, and reveal a novel layer of molecular conservation in the exocytosis machinery.

Kluyveromyces lactis SSO1 and SEB1 genes are functional in Saccharomyces cerevisiae and enhance production of secreted proteins when overexpressed.

The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae. Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion. Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S. cerevisiae, which suggests that they may be rate-limiting factors in this process. Here we report isolation of Kluyveromyces lactis homologues of these genes. KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature. The encoded Kluyveromyces proteins are up to 70% identical with the S. cerevisiae homologues at the amino acid level and can functionally replace them. Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S. cerevisiae when overexpressed. In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S. cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K. lactis and Pichia pastoris, the latter also in Candida utilis. The GenBank Accession Nos are AF307983 (K. lactis SSO1) and AF318314 (K. lactis SEB1).

Screening for novel essential genes of Saccharomyces cerevisiae involved in protein secretion.

We describe here a screening procedure devised for searching new genes involved in protein secretion in Saccharomyces cerevisiae. The screening procedure takes advantage of yeast strains constructed within the EUROFAN project, in which the promoters of the novel essential genes were replaced by the doxycycline-regulated tetO(7)-CYC1 promoter. This promoter is active in normal growth medium but results in downregulation of the gene in the presence of doxycycline. The yeast cells were grown in the presence or absence of doxycycline, and both the growth and secretion of the heat shock protein, Hsp150p, into the culture medium were determined. In seven strains there was a specific effect on protein secretion. In a strain in which the RPN5 gene was downregulated, the level of secreted Hsp150p was increased compared to the control culture. When RER2 was downregulated, cells secreted Hsp150p that was not of the mature size. In five strains, secretion was more severely reduced than cell growth. One of these downregulated genes, YGL098w, was recently reported to encode an ER-located t-SNARE, USE1. Four of the genes detected, NOG2, NOP15, RRP40 and SDA1, encode proteins involved in ribosome assembly, suggesting a possible new signalling pathway between ribosome biogenesis and production of secreted proteins. The results obtained here indicate that the present screen could be successfully used in larger scale to identify novel secretion-related genes.

Mapping of sporulation-specific functions in the yeast syntaxin gene SSO1.

The yeast Saccharomyces cerevisiae has two closely related plasma membrane syntaxins, Sso1p and Sso2p, which together provide an essential function in vegetative cells. However, Sso1p is also specifically needed during sporulation; and this function cannot be provided by Sso2p. We used fusions between SSO1 and SSO2 to map the sporulation-specific function of SSO1. We found that the two N-terminal alpha-helices Ha and Hb of Sso1p are important for sporulation, since it is reduced 8-fold for fusions where Ha and Hb are derived from Sso2p. In contrast, the C-terminal half of Sso1p does not seem to be specifically required for sporulation. Surprisingly, we further found that the 3' untranslated region (3'UTR) of SSO1 is essential for sporulation. Western blots failed to reveal a preferential expression of Sso1p in sporulating cells, indicating that effects on gene expression are unlikely to explain why the SSO1 3'UTR is needed for sporulation.

Two divergent plasma membrane syntaxin-like SNAREs, nsyn1 and nsyn2, contribute to hyphal tip growth and other developmental processes in Neurospora crassa.

Highly polarized exocytosis of vesicles at hyphal apices is an essential requirement of tip growth. This requirement may be met by the localization and/or activation of an apical SNARE-based machinery. We have cloned nsyn1 and nsyn2, SNAREs predicted to function at the plasma membrane in Neurospora crassa. Transformation of extra copies of nsyn1 into wild-type strains displayed effects consistent with quelling of nsyn1 expression, which was lethal in most transformants. All surviving transformants grew slowly, conidiated poorly, and were male sterile. In addition, antisense nsyn1 strains grew slowly, with abnormal hyphal diameters and polarity and defective conidiation. For nsyn2, several repeat induced point mutation (RIP) crosses produced no, or poorly germinating ascospores. Those that germinated produced slow-growing hyphae with abnormal branching. The defects in nsyn1 and nsyn2 mutants are consistent with differential impaired vesicle fusion in hyphal tips and other developmental stages.

Functional inactivation of the conserved Sem1p in yeast by intrabodies.

Intrabody technology was applied to characterize the function and intracellular localization of a highly conserved Saccharomyces cerevisiae Sem1 protein. DSS1, the mammalian homologue of Sem1p, is functionally conserved between yeast and mammalian cells, and in mammalian cells physically interacts with the strong tumour supressor BRCA2. Yeast and the generated intrabodies are thus expected to offer a useful system for studies on Sem1p/DSS1 function. Sem1p-specific antibody isolated from a phage display library was expressed intracellularily and targeted to either the cytosol or the nucleus of yeast cells. Analysis of the applicability of different antibody fragments as intrabodies showed that the Fab intrabody was expressed most efficiently. Expression of nuclear-targeted anti-Sem1p Fab intrabodies inhibited the growth of the sigma1278b yeast strain in a manner similar to deletion of the SEM1 gene. This indicates that the Fab intrabodies interact in vivo with Sem1p and result in inactivation of Sem1p. Localization of the Fab intrabody with or without the nuclear localization signal to the nucleus in Sem1p-dependent manner suggests that Sem1p mediates the nuclear transport of the intrabody without any targeting signal. Our results suggest that Sem1p function in yeast cells is in part manifested in the nucleus.

The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae.

The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery. We show that overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants. In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppressed the growth defects of several exocyst mutants. Seb1p was coimmunoprecipitated from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis. The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression. Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 resulted in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.

Characterisation of two 14-3-3 genes from Trichoderma reesei: interactions with yeast secretory pathway components.

The 14-3-3 proteins are highly conserved, ubiquitously expressed proteins taking part in numerous cellular processes. Two genes encoding 14-3-3 proteins, ftt1 and ftt2, were isolated and characterised from the filamentous fungus Trichoderma reesei. FTTI showed the highest sequence identity (98% at the amino acid level) to the Trichoderma harzianum protein Th1433. FTTII is relatively distinct from FTTI, showing approximately 75% identity to other fungal 14-3-3 proteins. Despite their sequence divergence, both of the T. reesei ftt genes were equally able to complement the yeast bmh1 bmh2 double disruption. The T. reesei ftt genes were also found to be quite closely linked in the genomic DNA. A C-terminally truncated version of ftt1 (ftt1DeltaC) was first isolated as a multicopy suppressor of the growth defect of the temperature-sensitive yeast secretory mutant sec15-1. Overexpression of ftt1DeltaC also suppressed the growth defect of sec2-41, sec3-101, and sec7-1 strains. Overexpression of ftt1DeltaC in sec2-41 and sec15-1 strains could also rescue the secretion of invertase at the restrictive temperatures, and overexpression of full-length ftt1 enhanced invertase secretion by wild-type yeast cells. These findings strongly suggest that the T. reesei ftt1 has a role in protein secretion.

Characterization of temperature-sensitive mutations in the yeast syntaxin 1 homologues Sso1p and Sso2p, and evidence of a distinct function for Sso1p in sporulation.

The duplicated genes SSO1 and SSO2 encode yeast homologues of syntaxin 1 and perform an essential function during fusion of secretory vesicles at the plasma membrane. We have used in vitro mutagenesis to obtain a temperature-sensitive SSO2 allele, sso2-1, in which a conserved arginine has been changed to a lysine. A yeast strain that lacks SSO1 and carries the sso2-1 allele ceases growth and accumulates secretory vesicles at the restrictive temperature. Interestingly, the strain also has a pronounced phenotype at the permissive temperature, causing a defect in bud neck closure that prevents separation of mother and daughter cells. The same mutation was introduced into SSO1, producing the sso1-1 allele, which also has a temperature-sensitive phenotype, although less pronounced than sso2-1. A screen for high copy number suppressors of sso2-1 yielded three genes that are involved in the terminal step of secretion: SNC1, SNC2 and SEC9. The sso1-1 mutation interacts synthetically with a disruption of the MSO1 gene, which encodes a Sec1p interacting protein. Interestingly, we further found that both MSO1 and SSO1, but not SSO2, are required for sporulation. This difference is not due to differential expression, since SSO2 expressed from the SSO1 promoter failed to restore sporulation. We conclude that a functional difference exists between the Sso1 and Sso2 proteins, with the former being specifically required during sporulation.