Both Svp26 and Mnn6 are required for the efficient ER exit of Mnn4 in Saccharomyces cerevisiae.

Incorporation of membrane and secretory proteins into COPII vesicles are facilitated either by the direct interaction of cargo proteins with COPII coat proteins, or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in the yeast Saccharomyces cerevisiae. The ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that the efficient incorporation of Mnn4, a type II membrane protein required for mannosyl phosphate transfer to glycoprotein-linked oligosaccharides, into COPII vesicles is also dependent on the function of Svp26. We show that Mnn4 is localized to the Golgi. In addition to Mnn4, Mnn6 is known to be also required for the transfer of mannosyl phosphate to the glycans. We show, by indirect immunofluorescence, that Mnn6 localizes to the ER. As in the case with Svp26, deletion of the MNN6 gene results in the accumulation of Mnn4 in ER. In vitro COPII vesicle budding assays show that Svp26 and Mnn6 facilitate the incorporation of Mnn4 into COPII vesicles. In contrast to Svp26, which is itself efficiently captured into the COPII vesicles, Mnn6 was not incorporated into the COPII vesicles. Mnn4 and Mnn6 have the DXD motif which is often found in the many glycosyltransferases and functions to coordinate a divalent cation essential for the reaction. Alcian blue dye binding assay shows that substitution of the first D in this motif present in Mnn4 by A impairs the Mnn4 function. In contrast, amino acid substitutions in DXD motifs present in Mnn6 did not affect the function of Mnn6. These results suggest that Mnn4 may be directly involved in the mannosyl phosphate transfer reaction.

Svp26 facilitates ER exit of mannosyltransferases Mnt2 and Mnt3 in Saccharomyces cerevisiae.

After being translocated into the ER lumen, membrane and secretory proteins are transported from the ER to the early Golgi by COPII vesicles. Incorporation of these cargo proteins into COPII vesicles are facilitated either by direct interaction of cargo proteins with COPII coat proteins or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in yeast Saccharomyces cerevisiae. ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that efficient incorporation of Mnt2 and Mnt3 into COPII vesicles is also dependent on the function of Svp26. Mnt2 and Mnt3 are Golgi-localized α-1,3-mannosyltransferases with type II membrane topology involved in protein O-glycosylation. Immunoisolation of the yeast Golgi subcompartments quantitatively showed that Mnt2 and Mnt3 are more abundant in the early Golgi fraction than in the late Golgi fraction. Subcellular fractionation and fluorescence microscopy showed that deletion of the SVP26 gene results in the accumulation of Mnt2 and Mnt3 in ER. Using an in vitro COPII vesicle formation assay, we further demonstrate that Svp26 facilitates incorporation of Mnt2 and Mnt3 into COPII vesicles. Finally, we showed that Mnt2 and Mnt3 were co-immunoprecipitated with Svp26 from digitonin-solubilized membranes. These results indicate that Svp26 functions as an ER exit adaptor protein of Mnt2 and Mnt3.

The F-actin-binding RapGEF GflB is required for efficient macropinocytosis in Dictyostelium.

Macropinocytosis involves the uptake of large volumes of fluid, which is regulated by various small GTPases. The Dictyostelium discoideum protein GflB is a guanine nucleotide exchange factor (GEF) of Rap1, and is involved in chemotaxis. Here, we studied the role of GflB in macropinocytosis, phagocytosis and cytokinesis. In plate culture of vegetative cells, compared with the parental strain AX2, gflB-knockout (KO) cells were flatter and more polarized, whereas GflB-overproducing cells were rounder. The gflB-KO cells exhibited impaired crown formation and retraction, particularly retraction, resulting in more crowns (macropinocytic cups) per cell and longer crown lifetimes. Accordingly, gflB-KO cells showed defects in macropinocytosis and also in phagocytosis and cytokinesis. F-actin levels were elevated in gflB-KO cells. GflB localized to the actin cortex most prominently at crowns and phagocytic cups. The villin headpiece domain (VHP)-like N-terminal domain of GflB directly interacted with F-actin in vitro Furthermore, a domain enriched in basic amino acids interacted with specific membrane cortex structures such as the cleavage furrow. In conclusion, GflB acts as a key local regulator of actin-driven membrane protrusion possibly by modulating Rap1 signaling pathways.

Distinct adaptor proteins assist exit of Kre2-family proteins from the yeast ER.

The Svp26 protein of S. cerevisiae is an ER- and Golgi-localized integral membrane protein with 4 potential membrane-spanning domains. It functions as an adaptor protein that facilitates the ER exit of Ktr3, a mannosyltransferase required for biosynthesis of O-linked oligosaccharides, and the ER exit of Mnn2 and Mnn5, mannosyltransferases, which participate in the biosynthesis of N-linked oligosaccharides. Ktr3 belongs to the Kre2 family, which consists of 9 members of type-II membrane proteins sharing sequence similarities. In this report, we examined all Kre2 family members and found that the Golgi localizations of two others, Kre2 and Ktr1, were dependent on Svp26 by immunofluorescence microscopy and cell fractionations in sucrose density gradients. We show that Svp26 functions in facilitating the ER exit of Kre2 and Ktr1 by an in vitro COPII budding assay. Golgi localization of Ktr4 was not dependent on Svp26. Screening null mutants of the genes encoding abundant COPII membrane proteins for those showing mislocalization of Ktr4 in the ER revealed that Erv41 and Erv46 are required for the correct Golgi localization of Ktr4. We provide biochemical evidence that the Erv41-Erv46 complex functions as an adaptor protein for ER exit of Ktr4. This is the first demonstration of the molecular function of this evolutionally conserved protein complex. The domain switching experiments show that the lumenal domain of Ktr4 is responsible for recognition by the Erv41-Erv46 complex. Thus, ER exit of Kre2-family proteins is dependent on distinct adaptor proteins and our results provide new insights into the traffic of Kre2-family mannosyltransferases.

Molecular mechanisms of the localization of membrane proteins in the yeast Golgi compartments.

The Golgi apparatus of the eukaryotic cell is an essential organelle at the center of the network of vesicular transport delivering proteins and lipids to the correct locations in the cell. There are several Golgi compartments that have distinct resident proteins and functions, but the mechanism creating and maintaining the differences has long been an unsolved mystery in cell biology. After the discovery and molecular characterization of the transport vesicles and their coat proteins, we realized that the Golgi is an extremely dynamic organelle existing as repeating cycles of appearance, maturation, and disappearance. In this review, we describe essential findings as to the Golgi apparatus uncovered by work on an excellent model microorganism, the yeast Saccharomyces cerevisiae, with special reference to the results of our studies.

Action of multiple endoplasmic reticulum chaperon-like proteins is required for proper folding and polarized localization of Kre6 protein essential in yeast cell wall ß-1,6-glucan synthesis.

Saccharomyces cerevisiae Kre6 is a type II membrane protein essential for cell wall ß-1,6-glucan synthesis. Recently we reported that the majority of Kre6 is in the endoplasmic reticulum (ER), but a significant portion of Kre6 is found in the plasma membrane of buds, and this polarized appearance of Kre6 is required for ß-1,6-glucan synthesis. An essential membrane protein, Keg1, and ER chaperon Rot1 bind to Kre6. In this study we found that in mutant keg1-1 cells, accumulation of Kre6 at the buds is diminished, binding of Kre6 to Keg1 is decreased, and Kre6 becomes susceptible to ER-associated degradation (ERAD), which suggests Keg1 participates in folding and transport of Kre6. All mutants of the calnexin cycle member homologues (cwh41, rot2, kre5, and cne1) showed defects in ß-1,6-glucan synthesis, although the calnexin chaperon system is considered not functional in yeast. We found synthetic defects between them and keg1-1, and Cne1 co-immunoprecipitated with Keg1 and Kre6. A stronger binding of Cne1 to Kre6 was detected when two glucosidases (Cwh41 and Rot2) that remove glucose on N-glycan were functional. Skn1, a Kre6 homologue, was not detected by immunofluorescence in the wild type yeast, but in kre6Δ cells it became detectable and behaved like Kre6. In conclusion, the action of multiple ER chaperon-like proteins is required for proper folding and localization of Kre6 and probably Skn1 to function in ß-1,6-glucan synthesis.

Whole-genome sequencing of sake yeast Saccharomyces cerevisiae Kyokai no. 7.

The term 'sake yeast' is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.

Kre6 protein essential for yeast cell wall beta-1,6-glucan synthesis accumulates at sites of polarized growth.

Saccharomyces cerevisiae Kre6 is a type II membrane protein with amino acid sequence homology with glycoside hydrolase and is essential for ß-1,6-glucan synthesis as revealed by the mutant phenotype, but its biochemical function is still unknown. The localization of Kre6, determined by epitope tagging, is a matter of debate. We raised anti-Kre6 rabbit antiserum and examined the localization of Kre6 and its tagged protein by immunofluorescence microscopy, subcellular fractionation in sucrose density gradients, and immunoelectron microscopy. Integration of the results indicates that the majority of Kre6 is in the endoplasmic reticulum; however, a small but significant portion is also present in the secretory vesicle-like compartments and plasma membrane. Kre6 in the latter compartments is observed as strong signals that accumulate at the sites of polarized growth by immunofluorescence. The truncated Kre6 without the N-terminal 230-amino acid cytoplasmic region did not show this polarized accumulation and had a severe defect in ß-1,6-glucan synthesis. This is the first evidence of a ß-1,6-glucan-related protein showing the polarized membrane localization that correlates with its biological function.

Inhibitory activity of blasticidin A, a strong aflatoxin production inhibitor, on protein synthesis of yeast: selective inhibition of aflatoxin production by protein synthesis inhibitors.

Blasticidin A (BcA), an antibiotic produced by Streptomyces, inhibits aflatoxin production without strong growth inhibition toward aflatoxin-producing fungi. During the course of our study on the mode of action of BcA by two-dimensional differential gel electrophoresis (2D-DIGE), we found a decrease in the abundances of ribosomal proteins in Saccharomyces cerevisiae after exposure to BcA. This phenomenon was also observed by treatment with blasticidin S (BcS) or cycloheximide. BcA inhibited protein synthesis in a galactose-induced expression system in S. cerevisiae similar to BcS and cycloheximide. BcS, but not cycloheximide, inhibited aflatoxin production in Aspergillus parasiticus without inhibition of fungal growth, similar to BcA. A decrease in the abundances of aflatoxin biosynthetic enzymes was observed in 2D-DIGE experiments with Aspergillus flavus after exposure to BcA or BcS. These results suggested that protein synthesis inhibitors are useful to control aflatoxin production.

Svp26 facilitates endoplasmic reticulum to golgi transport of a set of mannosyltransferases in Saccharomyces cerevisiae.

Svp26 is a polytopic integral membrane protein found in the ER and early Golgi compartment. In the Deltasvp26 cell, the Golgi mannosyltransferase Ktr3 remains in the ER. Here, we report that two other Golgi mannosyltransferases, Mnn2 and Mnn5 are also mislocalized and found in the ER in the absence of Svp26 and that localization of other mannosyltransferases including Mnn1 are not affected. Mnn2 and Mnn5 bind to Svp26 in vivo as Ktr3 does. Using an in vitro budding assay, the incorporation of Ktr3 and Mnn2 in the COPII vesicles is greatly stimulated by the presence of Svp26. As Svp26 itself is an efficient cargo, Svp26 is likely to support selective incorporation of a set of mannosyltransferases into COPII vesicles by working as their adaptor protein. The domain switching between Svp26-dependent Mnn2 or Ktr3 and Svp26-independent Mnn1 suggests that the lumenal domain of mannosyltransferases, but not the cytoplasmic or transmembrane domain, is responsible for recognition by Svp26.

Kei1: a novel subunit of inositolphosphorylceramide synthase, essential for its enzyme activity and Golgi localization.

Fungal sphingolipids have inositol-phosphate head groups, which are essential for the viability of cells. These head groups are added by inositol phosphorylceramide (IPC) synthase, and AUR1 has been thought to encode this enzyme. Here, we show that an essential protein encoded by KEI1 is a novel subunit of IPC synthase of Saccharomyces cerevisiae. We find that Kei1 is localized in the medial-Golgi and that Kei1 is cleaved by Kex2, a late Golgi processing endopeptidase; therefore, it recycles between the medial- and late Golgi compartments. The growth defect of kei1-1, a temperature-sensitive mutant, is effectively suppressed by the overexpression of AUR1, and Aur1 and Kei1 proteins form a complex in vivo. The kei1-1 mutant is hypersensitive to aureobasidin A, a specific inhibitor of IPC synthesis, and the IPC synthase activity in the mutant membranes is thermolabile. A part of Aur1 is missorted to the vacuole in kei1-1 cells. We show that the amino acid substitution in kei1-1 causes release of Kei1 during immunoprecipitation of Aur1 and that Aur1 without Kei1 has hardly detectable IPC synthase activity. From these results, we conclude that Kei1 is essential for both the activity and the Golgi localization of IPC synthase.

Ypt11 functions in bud-directed transport of the Golgi by linking Myo2 to the coatomer subunit Ret2.

A yeast class V myosin Myo2 transports the Golgi into the bud during its inheritance. However, the mechanism that links the Golgi to Myo2 is unknown. Here, we report that Ypt11, a Rab GTPase that reportedly interacts with Myo2, binds to Ret2, a subunit of the coatomer complex. When Ypt11 is overproduced, Ret2 and the Golgi markers, Och1 and Sft2, are accumulated in the growing bud and are lost in the mother cell. In a ret2 mutant that produces the Ret2 protein with reduced affinity to Ypt11, no such accumulation is observed upon overproduction of Ypt11. At a certain stage of budding, it is known that the late Golgi cisternae labeled with Sec7-GFP show polarized distribution in the bud. We find that this polarization of late Golgi cisternae is not observed in the ypt11Delta mutant. Indeed, analyses of Sec7-GFP dynamics with spatio-temporal image correlation spectroscopy (STICS) and fluorescence loss in photobleaching (FLIP) reveals that Ypt11 is required for the vectorial actin-dependent movement of the late Golgi from the mother cell toward the emerging bud. These results indicate that the Ypt11 and Ret2 are components of a Myo2 receptor complex that functions during the Golgi inheritance into the growing bud.

KEG1/YFR042w encodes a novel Kre6-binding endoplasmic reticulum membrane protein responsible for beta-1,6-glucan synthesis in Saccharomyces cerevisiae.

KEG1/YFR042w of Saccharomyces cerevisiae is an essential gene that encodes a 200-amino acid polypeptide with four predicted transmembrane domains. The green fluorescent protein- or Myc(6)-tagged Keg1 protein showed the typical characteristics of an integral membrane protein and was found in the endoplasmic reticulum by fluorescence imaging. Immunoprecipitation from the Triton X-100-solubilized cell lysate revealed that Keg1 binds to Kre6, which has been known to participate in beta-1,6-glucan synthesis. To analyze the essential function of Keg1 in more detail, we constructed temperature-sensitive mutant alleles by error-prone polymerase chain reaction. The keg1-1 mutant cells showed a common phenotype with Deltakre6 mutant including hypersensitivity to Calcofluor white, reduced sensitivity to the K1 killer toxin, and reduced content of beta-1,6-glucan in the cell wall. These results suggest that Keg1 and Kre6 have a cooperative role in beta-1,6-glucan synthesis in S. cerevisiae.

Pga1 is an essential component of Glycosylphosphatidylinositol-mannosyltransferase II of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae essential gene YNL158w/PGA1 encodes an endoplasmic reticulum (ER)-localized membrane protein. We constructed temperature-sensitive alleles of PGA1 by error-prone polymerase chain reaction mutagenesis to explore its biological role. Pulse-chase experiments revealed that the pga1(ts) mutants accumulated the ER-form precursor of Gas1 protein at the restrictive temperature. Transport of invertase and carboxypeptidase Y were not affected. Triton X-114 phase separation and [(3)H]inositol labeling indicated that the glycosylphosphatidylinositol (GPI)-anchoring was defective in the pga1(ts) mutants, suggesting that Pga1 is involved in GPI synthesis or its transfer to target proteins. We found GPI18, which was recently reported to encode GPI-mannosyltransferase II (GPI-MT II), as a high-copy suppressor of the temperature sensitivity of pga1(ts). Both Gpi18 and Pga1 were detected in the ER by immunofluorescence, and they were coprecipitated from the Triton X-100-solubilized membrane. The gpi18(ts) and pga1(ts) mutants accumulated the same GPI synthetic intermediate at the restrictive temperature. From these results, we concluded that Pga1 is an additional essential component of the yeast GPI-MT II.

Peculiar protein-protein interactions of the novel endoplasmic reticulum membrane protein Rcr1 and ubiquitin ligase Rsp5.

Overproduction of the ER membrane protein Rcr1 makes Saccharomyces cerevisiae resistant to Congo red by reducing the chitin content through a unknown mechanism. By both co-immunoprecipitation and yeast two-hybrid experiments, specific interaction between Rcr1 and the ubiquitin ligase Rsp5 was found. This binding was largely mediated by a singular VPEY sequence in Rcr1 in addition to PPSY, the consensus ligand motif of the WW domains. Mutant analysis indicated that Rsp5 and other Rcr1-interacting proteins discovered in the current screen were not engaged in Congo red resistance.

Specific membrane recruitment of Uso1 protein, the essential endoplasmic reticulum-to-Golgi tethering factor in yeast vesicular transport.

Uso1 is a yeast essential protein that functions to tether vesicles in the ER-to-Golgi transport. Its recruitment to the ER-derived vesicles has been demonstrated in in vitro membrane transport systems using semi-intact cells. Here we report that the binding of Uso1 to specific membranes can be detected through simple sucrose density block centrifugation. The purified Uso1 protein binds to slowly sedimenting membranes generated from rapidly sedimenting P10 membranes. These membranes were produced dependent on ATP hydrolysis, contained COPII vesicle components, but had neither of the coat subunits or ER proteins, which indicates that they were representative of the uncoated ER-derived COPII vesicles. The slowly sedimenting membranes of different origins were physically linked when they were mixed in the presence of Uso1. The C-terminal acidic region was not required in membrane binding. The presence of membranes to which Uso1 could bind in the yeast cell lysate was detected using the current method.

Tvp38, Tvp23, Tvp18 and Tvp15: novel membrane proteins in the Tlg2-containing Golgi/endosome compartments of Saccharomyces cerevisiae.

Four previously uncharacterized proteins (Tvp38, Tvp23, Tvp18 and Tvp15) were found in Tlg2-containing membrane by proteomic analysis of immunoisolated Golgi subcompartments of Saccharomyces cerevisiae (Inadome et al., Mol. Cell. Biol., 25 (2005) 7696-7710). Immunofluorescence double staining of HA-tagged Tvp proteins and myc-tagged tSNAREs supported that these proteins mainly localize in the Tlg2-containing compartments. Conserved sequences of Tvp38, Tvp23 and Tvp18 are found in higher eukaryotes, but these homologues have not been characterized yet. All Tvp proteins were nonessential for growth under laboratory conditions. Immunoprecipitation of Tvp proteins indicated that Tvp23, Tvp18 and Tvp15 are in an interactive network with Yip1-family proteins, Yip4 and Yip5. They may collectively assist in the effective maintenance/function of the late Golgi/endosomal compartments. Disruptions of tvp15 and tvp23 showed synthetic aggravation with ypt6 or ric1 null mutation. Processing of carboxypeptidase Y and alkaline phosphatase in tvp disruptants occurred as in the wild type.

Molecular cloning and characterization of a Pichia pastoris ortholog of the yeast Golgi GDP-mannose transporter gene.

There are two structural profiles in the yeast Golgi. The Golgi of Saccharomyces cerevisiae is composed of a number of vesicular compartments dispersed in the cytoplasm as recognized by a large number of Golgi marker proteins. In contrast, the Golgi of Pichia pastoris was reported to be organized in a small number of stacked cisternae located near the transitional endoplasmic reticulum (tER) sites by electron microscopy and immunofluorescent staining of a few marker proteins. The guanosine diphosphate (GDP)-mannose transporter (GMT) is an essential component in the yeast Golgi apparatus. We isolated an ortholog of the GMT gene of P. pastoris and visualized the gene product by epitope tagging to verify the structural characteristics of the Golgi. The tagged product in P. pastoris cell was observed in rod-like compartments in which Och1 mannosyltransferase was also found and the tER marker Sec12 and Sec13 proteins localized very close to them. The present results add further evidence of the restricted localization of the Golgi in P. pastoris cell.

Isolation and characterization of promoters suitable for a multidrug-resistant marker CuYAP1 in the yeast Candida utilis.

The overexpression of CuYAP1 by the CuGAP1 promoter (Pgap) was recently shown to function as a drug-resistant selection marker for the industrially important yeast Candida utilis. In order to increase the efficiency of selection, we screened for promoters better than Pgap to express CuYAP1. Two restriction fragments, P2-1-2 (0.5 kbp) and P2-33-2 (1.4 kbp), gave higher cycloheximide resistance, and five- to 10-fold of the transformants were selectable by using these fragments. These promoters were found to be at the 5' of the ribosomal protein genes, RPL31 and RPL29, respectively. Interestingly, their transcription activity was less than one-tenth that of Pgap in the absence of cycloheximide. The transcription also increased by the addition of blasticidin S or hygromycin B and heat shock. These novel characteristics will be suitable for an economical marker of the recombinant cell. The DDBJ/EMBL/GenBank Accession Nos. for P2-1, P2-33-2, RPL31 and RPL29 are AB206952, AB206953, AB208646 and AB208647, respectively.