Identification and characterization of Saccharomyces cerevisiae mutants defective in fluid-phase endocytosis

A mutant library generated by the European Functional Analysis Network (EUROFAN) was screened for strains defective in fluid-phase endocytosis. Accumulation of Lucifer yellow in the vacuole was used as a marker for efficient endocytosis. Fourteen mutants, including ede1Delta, rcy1Delta, sys1Delta and tlg2Delta, previously described to be involved in membrane trafficking, were identified in this screen. alpha-Factor uptake, endocytosis of FM4-64, carboxypeptidase Y secretion, vacuolar morphology, and a vma2 synthetic growth defect were used as criteria to characterize the endocytic defect of the mutant strains obtained. Accordingly, eight mutant strains have endocytic phenotypes in addition to their defect in Lucifer yellow accumulation. These fluid-phase endocytosis mutants are defective at different steps of the endocytic pathway. Interestingly, only two mutants were defective for internalization, two for vacuolar protein sorting and four mutants had aberrant vacuolar morphologies. Some of the mutants identified in this screen that sort carboxypeptidase Y correctly may affect endocytosis at an early post-internalization step before the intersection of the endocytic with the vacuolar protein-sorting pathway.     

Schizosaccharomyces pombe Pep12p is required for vacuolar protein transport and vacuolar homotypic fusion

In eukaryotic cells, SNARE proteins are essential for intracellular vesicle trafficking. Several SNARE proteins are required for vacuolar protein transport and vacuolar biogenesis in Saccharomyces cerevisiae. Previously we demonstrated that one of the fission yeast SNARE proteins, Pep12p, is not required for vacuolar fusion process in Schizosaccharomyces pombe. We have re-examined the function of S. pombe Pep12p using the newly created pep12(+) deletion strain. Deletion of the fission yeast pep12(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y-and various ion- and drug-sensitivities. GFP-Pep12 fusion protein is mostly localized at the vacuolar membrane and the prevacuolar compartment. The S. pombe pep12Δ mutation phenocopies that of vps33Δ, suggesting that both Pep12p and Vps33p act at the same membrane fusion step in S. pombe, and both mutations cause vacuolar deficiency.     

Characterization of vps33+, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe

From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33(+) gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4-64 in the cells. Deletion of the Sz. pombe vps33(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells.     

Endocytosis and vacuolar morphology in Saccharomyces cerevisiae are altered in response to ethanol stress or heat shock

The vital lipophilic dye N‐(3‐triethylammoniumpropyl)‐4‐[6‐(4‐(diethylamino)phenyl]hexatrienyl) pyridinium dibromide (FM 4‐64) was used to study the effect of ethanol stress and heat shock on endocytosis in the yeast Saccharomyces cerevisiae. Yeast cells stained with FM 4‐64 were placed in a culture chamber and the internalization of the dye was monitored by fluorescence microscopy during perfusion of the cells with fresh growth medium. In the absence of ethanol in the perfusion medium, the internalization of FM 4‐64 from the plasma membrane to the vacuolar membrane by yeast cells harvested from the exponential phase of growth was completed in 30 min. The presence of 6% (v/v) ethanol in the perfusion medium had no obvious effect on the internalization of FM 4‐64 from the plasma membrane, but did lead to an accumulation of the dye in endocytic intermediates. Consequently, vacuolar membrane staining was delayed. Cells stained with FM 4‐64 and subjected to heat shock displayed a similar effect, with endocytic intermediates becoming more prominent with the severity of the heat shock. For both ethanol stress and heat shock, vacuolar morphology altered from segregated structures to a single, large organelle. The findings of this study reinforce previous observations that ethanol stress and heat shock induce similar responses in yeast. Copyright © 1999 John Wiley & Sons, Ltd.     

A novel vacuolar protein encoded by SSU21 / MCD4 is involved in cell wall integrity in yeast.

Using a screening procedure for obtaining yeast strains with enhanced ability to secrete heterologous protein, we have isolated a mutant with alteration of the cell wall structure. This mutant displayed strong decrease in cell wall mannoprotein content, which was not accompanied by decreased glycosylation of secreted proteins. The mutation defines a gene, designated SSU21(identical to previously characterized MCD4), which encodes a novel vacuolar protein. SSU21 is probably connected to the cell integrity protein kinase C-mediated pathway, since ssu21 and pkc1Delta double mutant is synthetic lethal. To our knowledge, this is the first example of a yeast vacuolar protein whose alteration results in a cell wall defect.     

Identification of functionally important regions of the Saccharomyces cerevisiae mitochondrial translational activator Cbs1p

Translation of cytochrome b mRNA in yeast mitochondria requires activation by the nuclear-encoded Cbs1p. According to the current model, Cbs1p tethers cytochrome b mRNA to the inner mitochondrial membrane via interaction with the 5'-untranslated leader. Cbs1p is predicted to be a hydrophilic protein with two hydrophobic segments near the carboxyl-terminal end, which are both too short to span the membrane. Nevertheless Cbs1p is tightly associated with the mitochondrial membrane, as shown by its behaviour in extraction experiments with taurodeoxycholate. In an attempt to define functionally important regions of Cbs1p, we created a number of mutant alleles by random and directed mutagenesis. We report that a Cbs1p mutant protein lacking the mitochondrial presequence is still able to complement a Deltacbs1 strain, suggesting that the presequence does not contain essential mitochondrial targeting information. Mutations in a cluster of positively charged amino acids at the extremeC-terminus have no effect on Cbs1p function, but removal of this segment severely impairs Cbs1p function. Truncation of 12 or more amino acids from the C-terminus results in a completely defective protein. We further show that both short hydrophobic regions are essential for Cbs1p function, although membrane association is observed even in the absence of these regions.     

Genetic interaction between YPT6 and YPT1 in Saccharomyces cerevisiae

Ypt6p, the yeast homologue of human RAB6, is required for protein trafficking at elevated temperatures. Biochemical data provide evidence that Ypt6p plays a role in an early step(s) of the secretory pathway: from ER to Golgi, or from cis to medial Golgi, or both. Here we show that overexpression of YPT1 suppresses the growth and secretion defects of a ypt6 temperature-sensitive (ts) strain. SLY1-20, encoding a dominant mutant allele that suppresses the lethal effect of YPT1, also suppresses the growth defect of a ypt6 ts strain. Conversely, SSD1, isolated as a suppressor of ypt6 ts, can suppress the growth defect of a ypt1 ts allele. These data suggest that Ypt6p has some redundant function with Ypt1p. However, overexpression of Ypt6p is toxic to a ypt1 ts strain, although it does not affect the growth of wild-type cells, suggesting that Ypt6p may sequester proteins shared with Ypt1p. This genetic evidence confirms the conclusion that Ypt6p is involved in an early step of the secretory pathway. © 1998 John Wiley & Sons, Ltd.     

The end1 gene of Schizosaccharomyces pombe coding for a DNase is identical with the pnu1 gene coding for an RNase

The DNA nuclease activity encoded by the end1 gene, and its inactivation by mutation, was described in connection with the characterization of DNA topoisomerases in the fission yeast Schizosaccharomyces pombe (Uemura and Yanagida, 1984). Subsequently, end1 mutant strains were used for the preparation of cell extracts for the study of enzymes and intermediates involved in DNA metabolism. The molecular identification of the end1 gene and its identity with the pnu1 gene is presented. The end1-458 mutation alters glycine to glutamate in the conserved motif TGPYLP. The pnu1 gene codes for an RNase that is induced by nitrogen starvation (Nakashima et al., 2002b). Thus, the End1/Pnu1 protein, like related mitochondrial proteins in other organisms, is an example of a sugar-non-specific nuclease. The analysis of strains carrying a pnu1 deletion revealed no defects in meiotic recombination and spore viability.     

Yeast GGA proteins interact with GTP-bound Arf and facilitate transport through the Golgi

ARF proteins regulate the formation of transport vesicles at many steps of the secretory and endocytic pathways. A recently identified family of ARF effectors, named GGAs, appears to regulate membrane traffic exiting the trans-Golgi network in mammalian cells (Boman et al., 2000). We have identified two GGA homologues in the yeast S. cerevisiae. These previously uncharacterized open reading frames, YDR358w and YHR108w, have been named GGA1 and GGA2, respectively. Using the two-hybrid assay and GST-affinity chromatography, we show that Gga1p and Gga2p interact with Arf1p and Arf2p in a GTP-dependent manner, suggesting that both are functional homologues of the human GGA proteins. The Arf-binding domain resides in the amino-terminal half of Gga1p (amino acids 170-330), and the carboxy-terminal 100 amino acids resemble the gamma-adaptin 'ear domain'. Gene deletion experiments indicate that GGA1 and GGA2 are not essential genes, as single and double knockouts are viable at both 30 degrees C and 37 degrees C. However, cells lacking GGA1 and GGA2 exhibit defects in invertase processing and CPY sorting, but not endocytosis. We conclude that yeast Gga proteins are effectors of Arf in yeast that facilitate traffic through the late Golgi.     

The cell wall sensor Wsc1p is involved in reorganization of actin cytoskeleton in response to hypo-osmotic shock in Saccharomyces cerevisiae

The cell wall is essential to preserve osmotic integrity of yeast cells. Some phenotypic traits of cell wall mutants suggest that, as a result of a weakening of the cell wall, hypo-osmotic stress-like conditions are created. Consequent expansion of the cell wall and stretching of the plasma membrane trigger a complex response to prevent cell lysis. In this work we examined two conditions that generate a cell wall and membrane stress: one is represented by the cell wall mutant gas1Delta and the other by a hypo-osmotic shock. We examined the actin cytoskeleton and the role of the cell wall sensors Wsc1p and Mid2p in these stress conditions. In the gas1 null mutant cells, which lack a beta(1,3)-glucanosyltransferase activity required for cell wall assembly, a constitutive marked depolarization of actin cytoskeleton was found. In a hypo-osmotic shock wild-type cells showed a transient depolarization of actin cytoskeleton. The percentage of depolarized cells was maximal at 30 min after the shift and then progressively decreased until cells reached a new steady-state condition. The maximal response was proportional to the magnitude of the difference in the external osmolarity before and after the shift within a given range of osmolarities. Loss of Wsc1p specifically delayed the repolarization of the actin cytoskeleton, whereas Wsc1p and Mid2p were essential for the maintenance of cell integrity in gas1Delta cells. The control of actin cytoskeleton is an important element in the context of the compensatory response to cell wall weakening. Wsc1p appears to be an important regulator of the actin network rearrangements in conditions of cell wall expansion and membrane stretching.     

Functional characterization of Gms1p/UDP-galactose transporter in Schizosaccharomyces pombe

Galactosylation of glycoproteins in the fission yeast Schizosaccharomyces pombe requires the transport of UDP-galactose as substrate for the galactosyltransferase into the lumen of the Golgi apparatus, which is achieved by the UDP-galactose transporter. We isolated a mutant (gms1) that is deficient in galactosylation of cell surface glycoproteins in Sz.pombe, and found that the gms1(+) gene encodes a UDP-galactose transporter. In the prediction of secondary structure of the Gms1 protein, an eight-membrane-spanning structure was obtained. Fluorescent microscopy revealed the functional Gms1-GFP fusion protein to be stably localized at the Golgi membrane. Sequencing analysis of the coding region of Gms1p derived from galactosylation-defective mutants identified a single amino acid mutation (A102T or A258E) located within the putative transmembrane region, helix 2 or helix 7, respectively. The mutagenized Gms1(A102T or A258E)p exhibited loss of UDP-galactose transport activity but no change in the localization to the Golgi membrane. The C-terminal truncated Gms1p mutants demonstrated that the C-terminal hydrophilic region was dispensable for targeting and function as UDP-galactose transporter at the Golgi membrane.We suggest that the putative eighth (the most C-terminus-proximal) transmembrane helix of Gms1p is critical to targeting from ER to the Golgi membrane.     

The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts

The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature‐sensitive fission yeast strains, rrs1‐D14/22G and rrs1‐L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1‐84 (D22/30G) and rrs1‐124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two‐hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts. Copyright © 2015 John Wiley & Sons, Ltd.     

The open reading frame YAL048c affects the secretion of proteinase A in S. cerevisiae

Over-expression of the yeast PEP4 gene encoding the vacuolar aspartic protease proteinase A (PrA) leads to saturation of the vacuolar targeting system of the cell and missorting of PrA to the growth medium. In a screen for genes affecting the secretion of over-expressed PrA we found that multiple copies of the open reading frame (ORF) YAL048c enhanced PrA secretion. Since no function has hitherto been ascribed to YAL048c, we undertook further studies of this ORF. Deletion of YAL048c resulted in slightly reduced secretion of over-produced PrA. Furthermore, strains deleted for YAL048c showed a growth inhibition phenotype resulting in wrinkled colony morphology when grown on rich medium containing high concentrations of calcium. YAL048c is predicted to encode a polypeptide of 662 amino acid residues containing two consensus ATP/GTP-binding site motifs and a putative carboxy-terminal transmembrane region. In addition, the amino acid sequence contains two putative calcium-binding domains. The YAL048c protein may be evolutionarily conserved, as homologues exist in humans and Caenorhabditis elegans. We suggest that the YAL048c protein is involved in vesicle transport in the secretory pathway.     

Vacuolar functions are involved in stress-protective effect of intracellular proline in Saccharomyces cerevisiae

Proline protects yeast cells from damage caused by various stresses. A yeast Saccharomyces cerevisiae mutant with high levels of intracellular proline grown in a minimal medium accumulated proline in its vacuole, but when grown in a nutrient medium, accumulated proline mainly in the cytosol. To understand the role of the proline pool in the vacuole, we examined the stress-protective effect of proline in proline-accumulating yeast cells deficient in vacuolar functions. The disruption of PEP3 encoding a vacuolar membrane protein required for vacuolar biogenesis caused hypersensitivity to heat shock and ethanol stresses, probably due to disappearance of normal vacuoles. The vph1-disrupted cells lacking vacuolar-ATPase activity showed resistance to heat shock without any change in proline localization, but showed severe growth defects in an ethanol-containing medium. These results indicate that vacuolar functions are involved in the stress-protective effect of proline in S. cerevisiae. Also, it appears that excess proline is transported to the vacuole in an ATP-independent manner.     

Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae

In this work, we report results on the functional analysis of Saccharomyces cerevisiae ORF YGR224w, predicted to code for an integral membrane protein, with 14 potential transmembrane segments, belonging to the major facilitator superfamily (MFS) of transporters which are required for multiple-drug resistance (MDR). This MFS-MDR homologue is required for yeast adaptation to high stress imposed by low-chain organic acids, in particular by acetic acid, and for resistance to azoles, especially to ketoconazole and fluconazole; the encoding gene was thus named the AZR1 gene. These conclusions were based on the higher susceptibility to these compounds of an azr1Delta deletion mutant strain compared with the wild-type and on the increased resistance of both azr1Delta and wild-type strains upon increased expression of the AZR1 gene from a centromeric plasmid clone. AZR1 gene expression reduces the duration of acetic acid-induced latency, although the growth kinetics of adapted cells under acetic acid stress is apparently independent of AZR1 expression level. Fluorescence microscopy observation of the distribution of the Azr1-GFP fusion protein in yeast living cells indicated that Azr1 is a plasma membrane protein. Studies carried out to gain some understanding of how this plasma membrane putative transporter facilitates yeast adaptation to acetic acid did not implicate Azr1p in the alteration of acetic acid accumulation into the cell through the active efflux of acetate.     

Characterization and behaviour of alpha-glucan synthase in Schizosaccharomyces pombe as revealed by electron microscopy

Alpha-1,3-Glucan is a cell wall component in Schizosaccharomyces pombe and is exclusive to budding yeast. We analysed the ultrastructure of the cell wall in the alpha-glucan synthase mutant mok1 and determined the role of alpha-1,3-glucan in cell wall formation of Sz. pombe. The mok1 mutant cell has an abnormal shape, with swelling at the tip or at the site of the septum. The cell wall is thicker and looser than that of wild-type cells, and the layered structure of the cell wall is broken. The glucan fibrils forming the protoplast retain a fine fibril structure, although their development into bundles is abnormal. We also report the localization of Mok1p by immunoelectron microscopy using high-pressure freeze substitution and SDS-digested freeze-fracture replica labelling methods. The Mok1p is localized on the cell membrane and moves from the cell tip to the medial region during the cell cycle. These results confirm that Mok1p plays an important role in the normal construction of the cell wall and in the primary step of glucan bundle formation, and that it is required for new cell wall synthesis during vegetative growth. These findings suggest that alpha-1,3-glucan is an essential component for cell wall formation in fission yeast.     

A fission yeast kinesin affects Golgi membrane recycling

We report here an in vivo study of kinesin heavy chain (KHC) functions in yeast. We have identified in Schizosaccharomyces pombe a kinesin motor gene, klp3(+), which has the highest homology to the Neurospora crassa KHC. Using indirect immunofluorescence, HA epitope-tagged Klp3 protein is cytoplasmic and appears as one to a few distinct patches that are coincident with microtubules. The klp3 null allele is viable. In klp3 deleted cells, ER, Golgi and mitochondrial distribution appear normal. Mitochondrial distribution in S. pombe is known to be microtubule-associated. We show that latrunculin A does not cause mitochondria to aggregate, suggesting that mitochondrial distribution in fission yeast, unlike budding yeast, is not dependent upon actin-based processes. Neither latrunculin A nor thiabendazole affects ER or Golgi distribution. We also used the vital dye FM4-64 to visualize the internalization of the dye and its transport to vacuoles in fission yeast in the presence and absence of Klp3. We observed no significant difference between the wild-type and Klp3 null cells in either the dynamics of endocytosis or the distribution and fusion of vacuoles. The drug brefeldin A causes Golgi-to-ER recycling in wild-type fission yeast cells. Although recycling of Golgi to ER after brefeldin A treatment occurs in klp3 null cells, recycling is defective and the distribution pattern we see is different from that observed in the wild-type strain. We conclude that Klp3 plays a role in BFA-induced membrane transport. The nucleotide sequence of S. pombe klp3(+) was submitted to GenBank under Accession No. AF154055.     

Saccharomyces cerevisiae Big1p,a putative endoplasmic reticulum membrane protein required for normal levels of cell wall beta-1,6-glucan

Deletion of Saccharomyces cerevisiae BIG1 causes an approximately 95% reduction in cell wall beta-1,6-glucan, an essential polymer involved in the cell wall attachment of many surface mannoproteins. The big1 deletion mutant grows very slowly, but growth can be enhanced if cells are given osmotic support. We have begun a cell biological and genetic analysis of its product. We demonstrate, using a Big1p-GFP fusion construct, that Big1p is an N-glycosylated integral membrane protein with a Type I topology that is located in the endoplasmic reticulum (ER). Some phenotypes of a big1Delta mutant resemble those of strains disrupted for KRE5, which encodes another ER protein affecting beta-l,6-glucan levels to a similar extent. In a big1Deltakre5Delta double mutant, both the growth and alkali-soluble beta-l,6-glucan levels were reduced as compared to either single mutant. Thus, while Big1p and Kre5p may have similar effects on beta-l,6-glucan synthesis, these effects are at least partially distinct. Residual beta-l,6-glucan levels in the big1Deltakre5Delta double mutant indicate that these gene products are unlikely to be beta-l,6-glucan synthase subunits, but rather may play some ancillary roles in beta-l,6-glucan synthase assembly or function, or in modifying proteins for attachment of beta-l,6-glucan.     

Involvement of yeast YOL151W/GRE2 in ergosterol metabolism

The Saccharomyces cerevisiae gene YOL151W/GRE2 is widely used as a model gene in studies on yeast regulatory responses to osmotic and oxidative stress. Nevertheless, information concerning the physiological role of this enzyme, a distant homologue of mammalian 3-beta-hydroxysteroid dehydrogenases, is scarce. Combining quantitative phenotypic profiling and protein expression analysis studies, we here report the involvement of yeast Gre2p in ergosterol metabolism. Growth was significantly and exclusively reduced in gre2Delta strains subjected to environmental stress straining the cell membrane. Furthermore, whereas no compensatory mechanisms were activated due to loss of Gre2p during growth in favourable conditions (synthetic defined media, no stress), a striking and highly specific induction of the ergosterol biosynthesis pathway, represented by the enzymes Erg10p, Erg19p and Erg6p, was observed in gre2Delta during growth in a stress condition in which lack of Gre2p significantly affects growth. Involvement of Gre2p in ergosterol metabolism was confirmed by application of an array of selective inhibitors of lipid biosynthesis, as gre2Delta displayed vastly impaired tolerance exclusively to agents targeting the ergosterol biosynthesis. The approach outlined here, combining broad-spectrum phenotypic profiling, expression analysis during conditions reducing the growth of the mutant and functional confirmation by application of highly selective inhibitors, may prove a valuable tool in gene functional analysis.     

The centromere-binding factor Cbf1p from Candida albicans complements the methionine auxotrophic phenotype of Saccharomyces cerevisiae

A gene encoding the centromere binding factor 1 (Cbf1p) of the human pathogenic yeast Candida albicans was cloned and characterized. An open reading-frame was detected which encoded a 223 amino acid protein with a calculated molecular weight of 25.8 kDa and a relative isoelectric point of 5.55. It shares 39% overall amino acid sequence identity with Saccharomyces cerevisiae Cbf1p. We localized the CaCBF1 gene on chromosome 4. Southern analysis indicated that CaCBF1 is probably present as a single copy gene per haploid genome. The CaCBF1 gene under the control of its own promoter was able to complement the methionine auxotrophic growth, the increased mitotic instability of CEN plasmids and the slow growth of a Saccharomyces cerevisiae cbf1Delta mutant strain.