scholarly journals Giant peroxisomes in oleic acid-induced Saccharomyces cerevisiae lacking the peroxisomal membrane protein Pmp27p.

1995 ◽  
Vol 128 (4) ◽  
pp. 509-523 ◽  
Author(s):  
R Erdmann ◽  
G Blobel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Growth Defect ◽  
Wild Type ◽  
Peroxisomal Membrane ◽  
Daughter Cells ◽  
Sds Page ◽  
Multiple Buds ◽  
Media Form ◽  
Tubular Membranes

We have purified peroxisomal membranes from Saccharomyces cerevisiae after induction of peroxisomes in oleic acid-containing media. About 30 distinct proteins could be discerned among the HPLC- and SDS-PAGE-separated proteins of the high salt-extracted peroxisomal membranes. The most abundant of these, Pmp27p, was purified and the corresponding gene PMP27 was cloned and sequenced. Its primary structure is 32% identical to PMP31 and PMP32 of the yeast Candida biodinii (Moreno, M., R. Lark, K. L. Campbell, and M. J. Goodman. 1994. Yeast. 10:1447-1457). Immunoelectron microscopic localization of Pmp27p showed labeling of the peroxisomal membrane, but also of matrix-less and matrix containing tubular membranes nearby. Electronmicroscopical data suggest that some of these tubular extensions might interconnect peroxisomes to form a peroxisomal reticulum. Cells with a disrupted PMP27 gene (delta pmp27) still grew well on glucose or ethanol, but they failed to grow on oleate although peroxisomes were still induced by transfer to oleate-containing media. The induced peroxisomes of delta pmp27 cells were fewer but considerably larger than those of wild-type cells, suggesting that Pmp27p may be involved in parceling of peroxisomes into regular quanta. delta pmp27 cells cultured in oleate-containing media form multiple buds, of which virtually all are peroxisome deficient. The growth defect of delta pmp27 cells on oleic acid appears to result from the inability to segregate the giant peroxisomes to daughter cells.

scholarly journals The PAL1 gene product is a peroxisomal ATP-binding cassette transporter in the yeast Saccharomyces cerevisiae.

1996 ◽  
Vol 132 (4) ◽  
pp. 549-563 ◽  
Author(s):  
E E Swartzman ◽  
M N Viswanathan ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Amino Acid ◽  
Carbon Source ◽  
Amino Acid Sequence ◽  
Gene Product ◽  
Atp Binding ◽  
Wild Type ◽  
Peroxisomal Membrane ◽  
Atp Binding Cassette

The PAL1 gene was isolated using PCR and degenerate oligonucleotide primers corresponding to highly conserved amino acid sequence motifs diagnostic of the ATP-binding cassette domain of the superfamily of membrane-bound transport proteins typified by mammalian multidrug resistance transporter 1 and Saccharomyces cerevisiae Ste6. The deduced PAL1 gene product is similar in length to, has the same predicted topology as, and shares the highest degree of amino acid sequence identity with two human proteins, adrenoleukodystrophy protein and peroxisomal membrane protein (70 kD), which are both presumptive ATP-binding cassette transporters thought to be constituents of the peroxisomal membrane. As judged by hybridization of a PAL1 probe to isolated RNA and by expression of a PAL1-lacZ fusion, a PAL1 transcript was only detectable when cells were grown on oleic acid, a carbon source which requires the biogenesis of functional peroxisomes for its metabolism. A pal1delta mutant grew normally on either glucose- or glycerol-containing media; however, unlike PAL1+ cells (or the pal1delta mutant carrying the PAL1 gene on a plasmid), pal1delta cells were unable to grow on either a solid medium or a liquid medium containing oleic acid as the sole carbon source. Antibodies raised against a chimeric protein in which the COOH-terminal domain of Pal1 was fused to glutathione S-transferase specifically recognized a protein in extracts from wild-type cells only when grown on oleic acid; this species represents the PAL1 gene product because it was missing in pal1delta cells and more abundant in pal1delta cells expressing PAL1 from a multicopy plasmid. The Pal1 polypeptide was highly enriched in the organellar pellet fraction prepared from wild-type cells by differential centrifugation and comigrated upon velocity sedimentation in a Nycodenz gradient with a known component of the peroxisomal matrix, e-oxoacyl-CoA thiolase. As judged by both subcellular fractionation and indirect immunofluorescence, localization of 3-oxoacyl-CoA thiolase to peroxisomes was unchanged whether Pal1 was present, absent, or overexpressed. These findings demonstrate that Pal1 is a peroxisome-specific protein, that it is required for peroxisome function, but that it is not necessary for the biogenesis of peroxisomes or for the import of 3-oxoacyl-CoA thiolase (and at least two other peroxisomal matrix proteins).

Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 45-56
Author(s):  
Luther Davis ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heterologous Expression ◽  
Protein Translation ◽  
Developmental Pathways ◽  
G1 Phase ◽  
Growth Defect ◽  
Wild Type ◽  
Drosophila Homolog ◽  
Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

scholarly journals Enlarged Peroxisomes Are Present in Oleic Acid–grown Yarrowia lipolytica Overexpressing the PEX16 Gene Encoding an Intraperoxisomal Peripheral Membrane Peroxin

1997 ◽  
Vol 137 (6) ◽  
pp. 1265-1278 ◽  
Author(s):  
Gary A. Eitzen ◽  
Rachel K. Szilard ◽  
Richard A. Rachubinski
Keyword(s):  
Oleic Acid ◽  
Yarrowia Lipolytica ◽  
Consensus Sequence ◽  
Wild Type ◽  
Gene Encoding ◽  
Peroxisomal Membrane ◽  
Peripheral Protein ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signal

Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid–containing medium. Overexpression of the PEX16 gene in oleic acid– grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.

scholarly journals Redox-sensitive homodimerization of Pex11p: a proposed mechanism to regulate peroxisomal division.

1996 ◽  
Vol 135 (1) ◽  
pp. 123-137 ◽  
Author(s):  
P A Marshall ◽  
J M Dyer ◽  
M E Quick ◽  
J M Goodman
Keyword(s):  
Oleic Acid ◽  
Active Species ◽  
Monomeric Form ◽  
Wild Type ◽  
Dimer Formation ◽  
Peroxisomal Membrane ◽  
Cell Biol ◽  
Organelle Division ◽  
Inner Surface

Pex11p (formerly Pmp27) has been implicated in peroxisomal proliferation (Erdmann, R., and G. Blobel. 1995. J. Cell Biol. 128; 509-523; Marshall, P.A., Y.I. Krimkevich, R.H. Lark, J.M. Dyer, M. Veenhuis, and J.M. Goodman, 1995. J. Cell Biol. 129; 345-355). In its absence, peroxisomes in Saccharomyces cerevisiae fail to proliferate in response to oleic acid; instead, one or two large peroxisomes are formed. Conversely, overproduction of Pex11p causes an increase in peroxisomal number. In this report, we confirm the function of Pex11p in organelle proliferation by demonstrating that this protein can cause fragmentation in vivo of large peroxisomes into smaller organelles. Pex11p is on the inner surface of the peroxisomal membrane. It can form homodimers, and this species is more abundant in mature peroxisomes than in proliferating organelles. Removing one of the three cysteines in the protein inhibits homodimerization. This cysteine 3-->alanine mutation leads to an increase in number and a decrease in peroxisomal density, compared with the wild-type protein, in response to oleic acid. We propose that the active species is the "monomeric" form, and that the increasing oxidative metabolism within maturing peroxisomes causes dimer formation and inhibition of further organelle division.

scholarly journals SYM1 Is the Stress-Induced Saccharomyces cerevisiae Ortholog of the Mammalian Kidney Disease Gene Mpv17 and Is Required for Ethanol Metabolism and Tolerance during Heat Shock

2004 ◽  
Vol 3 (3) ◽  
pp. 620-631 ◽  
Author(s):  
Amy Trott ◽  
Kevin A. Morano
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Kidney Disease ◽  
Membrane Protein ◽  
Cellular Response ◽  
Ethanol Metabolism ◽  
Growth Defect ◽  
Inner Mitochondrial Membrane ◽  
Peroxisomal Membrane ◽  
Metabolic Role

ABSTRACT Organisms rapidly adapt to severe environmental stress by inducing the expression of a wide array of heat shock proteins as part of a larger cellular response program. We have used a genomics approach to identify novel heat shock-induced genes in Saccharomyces cerevisiae. The uncharacterized open reading frame (ORF) YLR251W was found to be required for both metabolism and tolerance of ethanol during heat shock. YLR251W has significant homology to the mammalian peroxisomal membrane protein Mpv17, and Mpv17−/− mice exhibit age-onset glomerulosclerosis, deafness, hypertension, and, ultimately, death by renal failure. Expression of Mpv17 in ylr251wΔ cells complements the 37°C ethanol growth defect, suggesting that these proteins are functional orthologs. We have therefore renamed ORF YLR251W as SYM1 (for “stress-inducible yeast Mpv17”). In contrast to the peroxisomal localization of Mpv17, we find that Sym1 is an integral membrane protein of the inner mitochondrial membrane. In addition, transcriptional profiling of sym1Δ cells uncovered changes in gene expression, including dysregulation of a number of ethanol-repressed genes, exclusively at 37°C relative to wild-type results. Together, these data suggest an important metabolic role for Sym1 in mitochondrial function during heat shock. Furthermore, this study establishes Sym1 as a potential model for understanding the role of Mpv17 in kidney disease and cardiovascular biology.

scholarly journals The links between hypertrophy, reproductive potential and longevity in the Saccharomyces cerevisiae yeast.

2016 ◽  
Vol 63 (2) ◽  
Author(s):  
Mateusz Molon ◽  
Renata Zadrag-Tecza
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Volume ◽  
Genetic Background ◽  
Wild Type Strain ◽  
Reproductive Potential ◽  
Model Organism ◽  
Reproductive Capacity ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Daughter Cells

The yeast Saccharomyces cerevisiae has long been used as a model organism for studying the basic mechanisms of aging. However, the main problem with the use of this unicellular fungus is the unit of "longevity". For all organisms, lifespan is expressed in units of time, while in the case of yeast it is defined by the number of daughter cells produced. Additionally, in yeast the phenotypic effects of mutations often show a clear dependence on the genetic background, suggesting the need for an analysis of strains representing different genetic backgrounds. Our results confirm the data presented in earlier papers that the reproductive potential is strongly associated with an increase in cell volume per generation. An excessive cell volume results in the loss of reproductive capacity. These data clearly support the hypertrophy hypothesis. The time of life of all analysed mutants, with the exception of sch9D, is the same as in the case of the wild-type strain. Interestingly, the 121% increase of the fob1D mutant's reproductive potential compared to the sfp1D mutant does not result in prolongation of the mutant's time of life (total lifespan).

scholarly journals An efficient positive selection procedure for the isolation of peroxisomal import and peroxisome assembly mutants of Saccharomyces cerevisiae.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 731-740 ◽  
Author(s):  
Y Elgersma ◽  
M van den Berg ◽  
H F Tabak ◽  
B Distel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Protein Import ◽  
Chimeric Protein ◽  
Selection Procedure ◽  
Peroxisome Biogenesis ◽  
Wild Type ◽  
Peroxisomal Protein ◽  
Complementation Groups ◽  
Resistance Protein

Abstract To study peroxisome biogenesis, we developed a procedure to select for Saccharomyces cerevisiae mutants defective in peroxisomal protein import or peroxisome assembly. For this purpose, a chimeric gene was constructed encoding the bleomycin resistance protein linked to the peroxisomal protein luciferase. In wild-type cells this chimeric protein is imported into the peroxisome, which prevents the neutralizing interaction of the chimeric protein with its toxic phleomycin ligand. Peroxisomal import and peroxisome assembly mutants are unable to import this chimeric protein into their peroxisomes. This enables the bleomycin moiety of the chimeric protein to bind phleomycin, thereby preventing its toxicity. The selection is very efficient: upon mutagenesis, 84 (10%) of 800 phleomycin resistant colonies tested were unable to grow on oleic acid. This rate could be increased to 25% using more stringent selection conditions. The selection procedure is very specific; all oleic acid non utilizing (onu) mutants tested were disturbed in peroxisomal import and/or peroxisome assembly. The pas (peroxisome assembly) mutants that have been used for complementation analysis represent 12 complementation groups including three novel ones, designated pas20, pas21 and pas22.

Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity

1991 ◽  
Vol 11 (7) ◽  
pp. 3537-3544 ◽  
Author(s):  
M Ziman ◽  
J M O'Brien ◽  
L A Ouellette ◽  
W R Church ◽  
D I Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Mutational Analysis ◽  
Mutant Gene ◽  
Biochemical Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dominant Lethal ◽  
Gtp Binding ◽  
Multiple Buds

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

scholarly journals Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity.

1991 ◽  
Vol 11 (7) ◽  
pp. 3537-3544 ◽  
Author(s):  
M Ziman ◽  
J M O'Brien ◽  
L A Ouellette ◽  
W R Church ◽  
D I Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Polarity ◽  
Mutational Analysis ◽  
Mutant Gene ◽  
Biochemical Properties ◽  
Wild Type ◽  
Mutant Proteins ◽  
Dominant Lethal ◽  
Gtp Binding ◽  
Multiple Buds

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

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