Elizabeth W. Jones

Fluorescence microscopy methods for yeast.

Publisher Summary This chapter reviews and provides detailed protocols for the application of immunofluorescence and other fluorescence-microscopic procedures to yeast. These procedures play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light (including fluorescence) microscopy. The potential advantages of light-microscopic approaches derive from the facts (1) that they can be applied to lightly processed or living cells, (2) that much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) that some structures have simply been easier to see by light microscopy than by electron microscopy. The methods are also effective with other yeasts such as Schizosaccharomyces pombe and Candida albicans .

Regulation of Amino Acid and Nucleotide Biosynthesis in Yeast

INTRODUCTION In this paper we attempt to summarize the current status of our knowledge of regulation of anabolic pathways. The discussion includes pathways of biosynthesis of all 20 amino acids, of purine and pyrimidine nucleotides, and of single carbon metabolism. Some discussion is devoted to the pathways themselves. We consider this essential, for, in our opinion, doubt exists as to what the actual pathway of synthesis is for some metabolites (e.g., methionine and cysteine). We have also devoted some attention to assignment of genetic blocks. For many genes, assignment is based on enzyme assay, but for a substantial number, assignments are based on indirect evidence such as accumulations and feeding tests. Regulation of biosynthesis occurs at two levels: the regulation of enzyme formation by control of gene expression and the regulation of enzyme activity that controls flow of metabolites. It seems likely that an essential aspect of the latter control is the regulation of the flow of metabolites between compartments of the cell. Substantial portions of the intracellular amino acids are compartmentalized within the cell, largely in the vacuole (for review, see Wiemken 1980; also see Messenguy et al. 1980). Redistribution of amino acids between compartments in response to metabolic signals has been demonstrated (Messenguy et al. 1980; Wiemken 1980). To what extent compartmentalization of metabolites may play a role in regulation for pathways other than amino acid biosynthesis is unknown. Compartmentalization within the mitochondrion has also been demonstrated for some anabolic enzymes. Only for the arginine and branched-chain amino...

Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases.

The PRB1 gene of Saccharomyces cerevisiae encodes the vacuolar endoprotease protease B. We have determined the DNA sequence of the PRB1 gene and the amino acid sequence of the amino terminus of mature protease B. The deduced amino acid sequence of this serine protease shares extensive homology with those of subtilisin, proteinase K, and related proteases. The open reading frame of PRB1 consists of 635 codons and, therefore, encodes a very large protein (molecular weight, greater than 69,000) relative to the observed size of mature protease B (molecular weight, 33,000). Examination of the gene sequence, the determined amino-terminal sequence, and empirical molecular weight determinations suggests that the preproenzyme must be processed at both amino and carboxy termini and that asparagine-linked glycosylation occurs at an unusual tripeptide acceptor sequence.

A rainbow of fluoromodules: a promiscuous scFv protein binds to and activates a diverse set of fluorogenic cyanine dyes.

Combined magnetic and fluorescence cell sorting were used to select Fluorogen Activating Proteins (FAPs) from a yeast surface-displayed library for binding to the fluorogenic cyanine dye Dimethyl Indole Red (DIR). Several FAPs were selected that bind to the dye with low nanomolar Kd values and enhance fluorescence more than 100-fold. One of these FAPs also exhibits considerable promiscuity, binding with high affinity to several other fluorogenic cyanine dyes with emission wavelengths covering most of the visible and near-IR regions of the spectrum. This significantly expands the number and wavelength range of scFv-based fluoromodules.

4 Biogenesis and Function of the Yeast Vacuole

I. INTRODUCTION A defining characteristic of all eukaryotic cells is the presence of distinct endomembrane organelles. These compartments allow a cell to carry out competing processes in separate environments suited to the particular requirements of each process (e.g., biosynthesis vs. degradation). The presence of endomembrane organelles requires not only the formation and maintenance of the lipid components that delineate the different compartments, but also the synthesis and targeting of proteins that serve either structural or enzymatic functions in the organelles. The vacuole of Saccharomyces cerevisiae is a large organelle, comprising as much as 25% of the cellular volume (Wiemken and Durr 1974). There are some indications that vacuolar morphology is correlated with the cell’s position in the cell cycle (Hartwell 1970; Wiemken et al. 1970; Jones et al. 1993); logarithmically growing cells often have a multilobed vacuole, with interconnections between lobes indicative of a reticulum, whereas stationary-phase cells and cells in G 1 typically have a single large vacuole that is relatively less dense (Hartwell 1970; Wiemken et al. 1970; Preston et al. 1989; Pringle et al. 1989; Jones et al. 1993). The vacuole is a very dynamic organelle. When cells are starved of a carbon source, the vacuolar lobes rapidly fuse to give a single large vacuole that is less dense than prior to fusion; it will be phase bright, whereas the reticular vacuole is not. Refeeding results in a rapid return to the denser reticular form (Pringle et al. 1989). The yeast vacuole shares features with lysosomes. It contains...

Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes). Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.

Genetic Approaches to the Study of Protease Function and Proteolysis in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, proteases have been implicated in septum formation (Ulane and Cabib 1976), sporulation (Chen and Miller 1968; Hopper et al. 1974; Klar and Halvorson 1975; Betz and Weiser 1976; Zubenko and Jones 1981), protein turnover (Lopez and Gancedo 1979; Hansen et al. 1977), catabolite inactivation (Hagele et al. 1978; Neeff et al. 1978; Funayama et al. 1980; Holzer 1976; Jusik et al. 1976; Molano and Gancedo 1974; Magni et al. 1977; Magni et al. 1978), carbon starvation-induced degradation of NADP-dependent glutamate dehydrogenase (Hemmings and Mazon 1979; Mazon and Hemmings 1979), nitrogen starvation-induced degradation of NAD-dependent glutamate dehydrogenase (Hemmings 1980), enzyme secretion (Perlman and Halvorson 1981), localization of mitochondrial enzymes (Neupert and Schatz 1981), processing of enzyme precursors (Hemmings et al. 1981; Hasilik and Tanner 1978a, Jones et al. 1981; Zubenko et al. 1981), production of the pheromone α factor (J. Kurjan and I. Herskowitz 1982; D. Julius, A. Brake, L. Blair, and J. Thorner, personal communication), destruction of α factor (Maness and Edelman 1978; Ciejek and Thorner 1979; Ciejek 1980) and degradation of missense proteins (Donahue and Henry 1981) and nonsense fragments (Bigelis and Burridge 1978; Bigelis and Fink 1981).

Proteolytic inactivation of the NADP-dependent glutamate dehydrogenase in proteinase-deficient mutants of Saccharomyces cerevisiae

Abstract The proteolytic inactivation of NADP-dependent glutamate dehydrogenase ( l -glutamate: NADP + oxidoreductase, EC 1.4.1.4) during carbon starvation was studied using several proteinase-deficient mutants of Saccharomyces cerevisiae . Strains bearing mutations in the structural genes for proteinase B, proteinase C (carboxypeptidase Y), or in both genes catalyzed the inactivation and initial proteolytic cleavage of NADP-glutamate dehydrogenase at a rate indistinguishable from that of the wild-type parent strain. In addition, the pleiotropic mutation, pep4-3 , which results in a deficiency for proteinases A, B, and C, did not affect the inactivation or initial proteolytic cleavage of NADP-glutamate dehydrogenase.

The spectrum of Trp- mutants isolated as 5-fluoroanthranilate-resistant clones in Saccharomyces bayanus, S. mikatae and S. paradoxus.

5‐Fluoroanthranilic acid (FAA)‐resistant mutants were selected in homothallic diploids of three Saccharomyces species, taking care to isolate mutants of independent origin. Mutations were assigned to complementation groups by interspecific complementation with S. cerevisiae tester strains. In all three species, trp3, trp4 and trp5 mutants were recovered. trp1 mutants were also recovered if the selection was imposed on a haploid strain. Thus, FAA selection may be more generally applicable than was previously described. Copyright

The Membrane-Bound 95 KDA Subunit of the Yeast Vacuolar Proton-Pumping ATPase is Required for Enzyme Assembly and Activity

Yeast vacuoles, like mammalian lysosomes, are maintained at an acidic pH by a vacuolar-type proton-pumping ATPase (V-ATPase). A genetic screen using the pH sensitive fluorescence of 6-carboxyfluorescein diacetate was developed to identity yeast defective in vacuolar acidification (Preston et al., 1992). Yeast bearing the vphl-1 (Vacuolar pH1-1) mutation have neutral vacuoles in vivo (Preston et al., 1989) and have no detectable bafilomycin-sensitive ATPase activity or ATP dependent proton-pumping associated with purified vacuoles. The peripherally bound nucleotide-binding subunits (Vmaip and Vma2p) are present in wild type levels in yeast whole cell extract yet are not associated with the vacuolar membrane. The VPH1 gene was cloned by screening a λgt11 expression library with antibodies directed against a 95 kDa vacuolar integral membrane protein and independently cloned by complementation of the vphl-1 mutation. Disruption of the gene revealed that the VPH1 gene product is required for V-ATPase assembly and vacuolar acidification but is not essential for cell viability or for targeting and maturation of vacuolar proteases. Through cell fractionation and immunobloting, the Vphlp antigen was shown to co-purify with alkaline phosphatase activity, a specific marker for vacuolar membranes. Furthermore, the Vphlp antigen was enriched and co-purified with vacuolar ATPase activity. H+-ATPase activity. The inability of alkaline Na2CO3