Dependence of Saccharomyces cerevisiae Golgi functions on V-ATPase activity.

The V-ATPase of Saccharomyces cerevisiae is an ATP-dependent proton pump responsible for acidification of the vacuole and other internal compartments including the whole secretory pathway. We have studied the behavior of several glycoprotein processing reactions occurring in different Golgi compartments of representative vmaΔ mutants. We found that outer chain initiation is not altered in the mutants while mannosylphosphate transfer, α(1,3)-linked mannoses addition, and α factor maturation seem to be affected. The results suggest a gradation in the dependence of Golgi functions on V-ATPase activity, from early Golgi (unaffected) to late Golgi (significantly reduced). These findings are in agreement with the internal pH of Golgi cisternae measured in mammalian cells, which is more acidic in the late region. The mutant defects can be partially restored by buffering the external medium to pH 6.0, which supports the existence of a mechanism that, in the absence of a functional V-ATPase, could contribute to pH regulation at least in the late Golgi.

Standard YPD, even supplemented with extra nutrients, does not always compensate growth defects of Saccharomyces cerevisiae auxotrophic strains.

Conventional complex media are routinely used to grow auxotrophic strains under the assumption that they can compensate the latter's nutritional deficiencies. We here demonstrate that this is not always true. This study compares the growth parameters of Saccharomyces cerevisiae (S288C) and its derived auxotrophic strains FY1679-14C and BY4741 in synthetic minimal medium (SD), standard YPD medium from two of the most commonly used suppliers, or modified YPD medium. Maximum specific growth rates of auxotrophic strains were slightly lower than the prototrophic case in all growth conditions tested. Also, the biomass production of auxotrophic strains in synthetic medium was slightly less than the prototrophic case. However in both of the two standard YPD media used, the biomass production of both auxotrophic strains was markedly lower than that of the prototrophic one. The extent of the differences depended on the medium used. Indeed in one of the two YPD media, the lower biomass production of auxotrophic strains was evident even at the diauxic shift. Uracil seems to be the main limiting growth factor for both auxotrophic strains growing in the two standard YPD medium tested. No YPD media or specific supplement was able to compensate for the effect of the auxotrophic mutations in the multiple auxotrophic marker strain BY4741. The fact that auxotrophic strains grew poorly on YPD when compared to their prototrophic counterpart indicates that standard YPD medium is not sufficient to overcome the effect of auxotrophic mutations.

Identification of the MNN3 gene of Saccharomyces cerevisiae.

The MNN3 gene of Saccharomyces cerevisiae has been identified as a synonym of VPS74. We have compared phenotype characteristics of the original mnn3 mutant, including low dye binding phenotype, size of external invertase, clump formation, and sodium orthovanadate resistance and found these to be identical to those shown by vps74Δ. Mating of both haploid strains resulted in non-complementation of mutant phenotypes. Finally, a vector containing wild-type VPS74 complemented the defects of both vps74Δ and mnn3. This work completes the identification of the entire collection of genes that are defective in mnn mutants. In addition, we have identified the mnn3 mutation by sequencing the VPS74 gene from the original mnn3 strain. We found a single amino acid change of Arg97 to Cys. This unique alteration seems to be sufficient to account for the phenotype of mnn3.

Genome-wide expression profile of the mnn2 Delta mutant of Saccharomyces cerevisiae.

The MNN2 gene of S. cerevisiae encodes an alpha (1,2) mannosyl transferase required for branching the outer chain of N-linked oligosaccharides (Rayner J.C. and Munro S. 1998. J. Biol. Chem. 273: 26836-26843) and it also seems to have some effect on the transfer of mannosyl phosphate groups to the inner core (Olivero I. et al. 2000. FEBS Lett. 475: 111-116). In order to reveal possible interactions of MNN2 expression with other cellular pathways, we analyzed the transcriptome of the deletion mutant S. cerevisiae mnn2 Delta using cDNA microarrays. We found 151 genes that showed an altered expression level of > or =2-fold, 58 of them up-regulated and 93 down-regulated. Quite a high proportion of these genes (29%) encode unclassified proteins. In contrast to other defects affecting the integrity of the cell wall, deletion of MNN2 does not stimulate the expression of any of the genes included in the previously defined 'cell wall compensatory cluster' (Lagorce et al. 2003. J. Biol. Chem. 278: 20345-20357). We also found that 15% of the selected genes are related to central metabolic pathways. In addition, the mnn2 Delta strain seems to have a certain level of stimulation of DNA processing reactions while some genes involved in intracellular transport pathways are under-regulated.

A genome-wide screen for Saccharomyces cerevisiae nonessential genes involved in mannosyl phosphate transfer to mannoprotein-linked oligosaccharides.

A collection of haploid Saccharomyces cerevisiae deletion strains--both MAT a and alpha--was screened for mutants that exhibit low dye binding (ldb) phenotype. This phenotype has previously been associated with reduced incorporation of mannosyl phosphate groups into the mannoprotein-linked oligosaccharides. We identified 199 nonessential genes whose deletion resulted in a detectable ldb phenotype. They fell into diverse functional categories, including those involved in protein glycosylation, vacuolar function, intracellular transport, cytoskeleton organization, transcription, signal transduction, among others. The study extends the number of known genes that affect mannosyl phosphorylation of mannoprotein-linked oligosaccharides, and establishes a link with other relevant pathways in the cell, especially vacuolar function. We have assigned an LDB name to four uncharacterized ORFs identified in this study: YCL005W, LDB16; YDL146W, LDB17; YLL049W, LDB18; and YOR322C, LDB19.

Identification of low-dye-binding (ldb) mutants of Saccharomyces cerevisiae.

We have completed the identification of Saccharomyces cerevisiae genes that are defective in previously isolated ldb (low-dye-binding) mutants. This was done by complementation of the mutant's phenotype with DNA fragments from a genomic library and by running standard tests of allelism with single-gene deletion mutants of similar phenotype. The results were as follows: LDB2 is allelic to ERD1; LDB4 to SPC72; LDB5 to RLR1; LDB6 to GON7/YJL184W; LDB7 to YBL006C; LDB9 to ELM1; LDB10 to CWH36; LDB11 to COG1; LDB12 to OCH1; LDB13 to VAN1; LDB14 to BUD32; and LDB15 to PHO85. Since the precise function of some of the genes is not known, these data may contribute to the functional characterization of the S. cerevisiae genome.

The ldb1 mutant of Saccharomyces cerevisiae is defective in Pmr1p, the yeast secretory pathway/Golgi Ca(2+)/Mn(2+)-ATPase.

The LDB1 gene of Saccharomyces cerevisiae was identified by complementation of the ldb1 mutant phenotype with a genomic library. We found that the ldb1 defect is complemented by PMR1 which codes for the yeast secretory pathway/Golgi Ca(2+)/Mn(2+)-ATPase. Besides that, the analysis of a null mutation of the PMR1 gene revealed a phenotype identical to that of ldb1 mutant. Thus, LDB1 must be considered a synonym of PMR1.