A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c.

The CYC1-239-O mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu- replacement of the normal -Ala-Gly- sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-1-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA. The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal. However, in contrast to the normal CYC1+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYC1-239-O mRNA is associated with one to four ribosomes. These results suggest that the stable secondary structure within the translated region of the CYC1 mRNA diminishes translation by inhibiting elongation.

Download Full-text

The GTS1 gene, which contains a Gly-Thr repeat, affects the timing of budding and cell size of the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5569 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5569-5578 ◽

Cited By ~ 41

Author(s):

K Mitsui ◽

S Yaguchi ◽

K Tsurugi

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Cell Size ◽

Single Cells ◽

Exponential Growth Phase ◽

Restrictive Temperature ◽

Multicopy Plasmid ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Mean Cell Volume

A gene with an open reading frame encoding a protein of 417 amino acid residues with a Gly-Thr repeat was isolated from the yeast Saccharomyces cerevisiae by using synthetic oligonucleotides encoding three Gly-Thr dimers as probes. The deduced amino acid sequence showed partial homology to the clock-affecting gene, per, of Drosophila melanogaster in the regions including the GT repeat. The function of the gene, named GTS1, was examined by characterizing the phenotypes of transformants with different copy numbers of the GTS1 gene produced either by inactivating the GTS1 gene by gene disruption (TM delta gts1) or by transformation with multicopy plasmid pPER119 (TMpGTS1). They grew at similar rates during the exponential growth phase, but the lag phases were shorter for TM delta gts1 and longer for TMpGTS1 cells than that for the wild type. Analyses of their cell cycle parameters using synchronized cells revealed that the unbudding period changed as a function of gene dosage; that is, the periods of TM delta gts1 and TMpGTS1 were about 20% shorter and longer, respectively, than that of the wild-type. Another significant change in the transformants was detected in the distribution of the cell size. The mean cell volume of the TM delta gts1 cells in the unbudded period (single cells) was 27% smaller than that of single wild-type cells, whereas that of single TMpGTS1 cells was 48% larger. Furthermore, in the temperature-sensitive cdc4 mutant, the GTS1 gene affected the timing of budding at the restrictive temperature. Thus, the GTS1 gene product appears to modulate the timing of budding to obtain an appropriate cell size independent of the DNA replication cycle.

Download Full-text

Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5139-5153.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5139-5153

Author(s):

L Feng ◽

H Yoon ◽

T F Donahue

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Isoelectric Focusing ◽

Mutational Analysis ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Initiation Factor ◽

Protein Species ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae

Previous studies have demonstrated that the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha), encoded by the SUI2 gene in the yeast Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control. Here we describe that yeast eIF-2 alpha is a constitutively phosphorylated protein species that is multiply phosphorylated by a GCN2-independent mechanism. 32Pi labeling and isoelectric focusing analysis of a SUI2+ delta gcn2 strain identifies eIF-2 alpha as radiolabeled and a single isoelectric protein species. Treatment of SUI2+ delta gcn2 strain extracts with phosphatase results in the identification of three additional isoelectric forms of eIF-2 alpha that correspond to the stepwise removal of three phosphates from the protein. Mutational analysis of SUI2 coupled with biochemical analysis of eIF-2 alpha maps the sites to the carboxyl region of SUI2 that correspond to Ser residues at amino acid positions 292, 294, and 301 that compose consensus casein kinase II sequences. 32Pi labeling or isoelectric focusing analysis of eIF-2 alpha from conditional casein kinase II mutants indicated that phosphorylation of eIF-2 alpha is abolished or dephosphorylated forms of eIF-2 alpha are detected when these strains are grown at the restrictive growth conditions. Furthermore, yeast casein kinase II phosphorylates recombinant wild-type eIF-2 alpha protein in vitro but does not phosphorylate recombinant eIF-2 alpha that contains Ser-to-Ala mutations at all three consensus casein kinase II sequences. These data strongly support the conclusion that casein kinase II directly phosphorylates eIF-2 alpha at one or all of these Ser amino acids in vivo. Although substitution of SUI2 genes mutated at these sites for the wild-type gene have no obvious effect on cell growth, one test that we have used appears to demonstrate that the inability to phosphorylate these sites has a physiological consequence on eIF-2 function in S. cerevisiae. Haploid strains constructed to contain Ser-to-Ala mutations at the consensus casein kinase II sequences in SUI2 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects. These genetic data appear to indicate that the modifications that we describe at the carboxyl end of the eIF-2 alpha protein are required for optimal eIF-2 function in S. cerevisiae.

Download Full-text

Membrane proteins associated with amino acid transport by yeast (Saccharomyces cerevisiae)

Biochemical Journal ◽

10.1042/bj1920659 ◽

1980 ◽

Vol 192 (2) ◽

pp. 659-664 ◽

Cited By ~ 10

Author(s):

J R Woodward ◽

H L Kornberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Membrane Proteins ◽

Wild Type Strain ◽

Osmotic Shock ◽

Wild Type ◽

Amino Acid Permease ◽

Yeast Saccharomyces Cerevisiae ◽

General Amino Acid Permease ◽

Wild Type Yeast

Cells of the wild-type yeast (Saccharomyces cerevisiae) strain Y185, grown under conditions that de-repress the formation of a general amino acid permease (‘Gap’) system, bind delta-N-chloroacetyl[1-(14)C]ornithine; L- and D-amino acid substrates of the general amino acid permease system protect against this binding. The protein responsible is released from the cells by homogenization or by preparation of protoplasts; it is not released by osmotic shock. This protein is virtually absent from the wild-type strain when it is grown under conditions that repress the general amino acid permease system, and is also absent from a Gap- mutant Y185-His3, selected by its resistance to D-amino acids. This mutant and repressed wild-type cells also fail to form a number of membrane proteins elaborated by de-repressed wild-type cells. It is possible that all these proteins are components of the general amino acid permease system.

Download Full-text

Casein kinase II mediates multiple phosphorylation of Saccharomyces cerevisiae eIF-2 alpha (encoded by SUI2), which is required for optimal eIF-2 function in S. cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5139 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5139-5153 ◽

Cited By ~ 29

Author(s):

L Feng ◽

H Yoon ◽

T F Donahue

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Isoelectric Focusing ◽

Mutational Analysis ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Initiation Factor ◽

Protein Species ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae

Previous studies have demonstrated that the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha), encoded by the SUI2 gene in the yeast Saccharomyces cerevisiae, is phosphorylated at Ser-51 by the GCN2 kinase in response to general amino acid control. Here we describe that yeast eIF-2 alpha is a constitutively phosphorylated protein species that is multiply phosphorylated by a GCN2-independent mechanism. 32Pi labeling and isoelectric focusing analysis of a SUI2+ delta gcn2 strain identifies eIF-2 alpha as radiolabeled and a single isoelectric protein species. Treatment of SUI2+ delta gcn2 strain extracts with phosphatase results in the identification of three additional isoelectric forms of eIF-2 alpha that correspond to the stepwise removal of three phosphates from the protein. Mutational analysis of SUI2 coupled with biochemical analysis of eIF-2 alpha maps the sites to the carboxyl region of SUI2 that correspond to Ser residues at amino acid positions 292, 294, and 301 that compose consensus casein kinase II sequences. 32Pi labeling or isoelectric focusing analysis of eIF-2 alpha from conditional casein kinase II mutants indicated that phosphorylation of eIF-2 alpha is abolished or dephosphorylated forms of eIF-2 alpha are detected when these strains are grown at the restrictive growth conditions. Furthermore, yeast casein kinase II phosphorylates recombinant wild-type eIF-2 alpha protein in vitro but does not phosphorylate recombinant eIF-2 alpha that contains Ser-to-Ala mutations at all three consensus casein kinase II sequences. These data strongly support the conclusion that casein kinase II directly phosphorylates eIF-2 alpha at one or all of these Ser amino acids in vivo. Although substitution of SUI2 genes mutated at these sites for the wild-type gene have no obvious effect on cell growth, one test that we have used appears to demonstrate that the inability to phosphorylate these sites has a physiological consequence on eIF-2 function in S. cerevisiae. Haploid strains constructed to contain Ser-to-Ala mutations at the consensus casein kinase II sequences in SUI2 in combination with a mutated allele of either the GCN2, GCN3, or GCD7 gene have synthetic growth defects. These genetic data appear to indicate that the modifications that we describe at the carboxyl end of the eIF-2 alpha protein are required for optimal eIF-2 function in S. cerevisiae.

Download Full-text

Changes in membrane proteins associated with inhibition of the general amino acid permease of yeast (Saccharomyces cerevisiae)

Biochemical Journal ◽

10.1042/bj1960531 ◽

1981 ◽

Vol 196 (2) ◽

pp. 531-536 ◽

Cited By ~ 10

Author(s):

J R Woodward ◽

H L Kornberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Membrane Proteins ◽

Membrane Protein ◽

Wild Type ◽

Amino Acid Permease ◽

Yeast Saccharomyces Cerevisiae ◽

General Amino Acid Permease ◽

Wild Type Yeast

The general amino acid permease (‘Gap’) system of the wild-type yeast (Saccharomyces cerevisiae) strain Y185 is inhibited by the uptake and accumulation of its substrate amino acids. Surprisingly, this inhibition persists even after ‘pools’ of amino acids, accumulated initially, have returned to normal sizes. Recovery from this inhibition depends on a supply of energy and involves the synthesis of a membrane protein component of the Gap system.

Download Full-text

A DELETION MAP OF cyc1 MUTANTS AND ITS CORRESPONDENCE TO MUTATIONALLY ALTERED ISO-1-CYTOCHROMES c OF YEAST

Genetics ◽

10.1093/genetics/81.1.51 ◽

1975 ◽

Vol 81 (1) ◽

pp. 51-73 ◽

Cited By ~ 2

Author(s):

Fred Sherman ◽

Mary Jackson ◽

Susan W Liebman ◽

Ann Marie Schweingruber ◽

John W Stewart

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Cytochrome C ◽

Primary Structure ◽

Recombination Rates ◽

Yeast Saccharomyces Cerevisiae ◽

X Ray ◽

Cytochromes C ◽

Adjacent Point

ABSTRACT Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants.

Download Full-text

Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.390 ◽

1989 ◽

Vol 9 (2) ◽

pp. 390-395 ◽

Cited By ~ 98

Author(s):

S Powers ◽

K O'Neill ◽

M Wigler

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Substitution ◽

Yeast Cells ◽

Wild Type ◽

Inhibitory Effects ◽

Yeast Saccharomyces Cerevisiae ◽

Ras Genes ◽

Evolutionarily Conserved

Two mutant alleles of RAS2 were discovered that dominantly interfere with wild-type RAS function in the yeast Saccharomyces cerevisiae. An amino acid substitution which caused the dominant interference was an alanine for glycine at position 22 or a proline for alanine at position 25. Analogous mutations in human H-ras also dominantly inhibited RAS function when expressed in yeast cells. The inhibitory effects of the mutant RAS2 or H-ras genes could be overcome by overexpression of CDC25, but only in the presence of wild-type RAS. These results suggest that these mutant RAS genes interfere with the normal interaction of RAS and CDC25 proteins and suggest that this interaction is direct and has evolutionarily conserved features.

Download Full-text

Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.390-395.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 390-395

Author(s):

S Powers ◽

K O'Neill ◽

M Wigler

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Substitution ◽

Yeast Cells ◽

Wild Type ◽

Inhibitory Effects ◽

Yeast Saccharomyces Cerevisiae ◽

Ras Genes ◽

Evolutionarily Conserved

Two mutant alleles of RAS2 were discovered that dominantly interfere with wild-type RAS function in the yeast Saccharomyces cerevisiae. An amino acid substitution which caused the dominant interference was an alanine for glycine at position 22 or a proline for alanine at position 25. Analogous mutations in human H-ras also dominantly inhibited RAS function when expressed in yeast cells. The inhibitory effects of the mutant RAS2 or H-ras genes could be overcome by overexpression of CDC25, but only in the presence of wild-type RAS. These results suggest that these mutant RAS genes interfere with the normal interaction of RAS and CDC25 proteins and suggest that this interaction is direct and has evolutionarily conserved features.

Download Full-text

The GTS1 gene, which contains a Gly-Thr repeat, affects the timing of budding and cell size of the yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5569-5578.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5569-5578

Author(s):

K Mitsui ◽

S Yaguchi ◽

K Tsurugi

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Cell Size ◽

Single Cells ◽

Exponential Growth Phase ◽

Restrictive Temperature ◽

Multicopy Plasmid ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Mean Cell Volume

A gene with an open reading frame encoding a protein of 417 amino acid residues with a Gly-Thr repeat was isolated from the yeast Saccharomyces cerevisiae by using synthetic oligonucleotides encoding three Gly-Thr dimers as probes. The deduced amino acid sequence showed partial homology to the clock-affecting gene, per, of Drosophila melanogaster in the regions including the GT repeat. The function of the gene, named GTS1, was examined by characterizing the phenotypes of transformants with different copy numbers of the GTS1 gene produced either by inactivating the GTS1 gene by gene disruption (TM delta gts1) or by transformation with multicopy plasmid pPER119 (TMpGTS1). They grew at similar rates during the exponential growth phase, but the lag phases were shorter for TM delta gts1 and longer for TMpGTS1 cells than that for the wild type. Analyses of their cell cycle parameters using synchronized cells revealed that the unbudding period changed as a function of gene dosage; that is, the periods of TM delta gts1 and TMpGTS1 were about 20% shorter and longer, respectively, than that of the wild-type. Another significant change in the transformants was detected in the distribution of the cell size. The mean cell volume of the TM delta gts1 cells in the unbudded period (single cells) was 27% smaller than that of single wild-type cells, whereas that of single TMpGTS1 cells was 48% larger. Furthermore, in the temperature-sensitive cdc4 mutant, the GTS1 gene affected the timing of budding at the restrictive temperature. Thus, the GTS1 gene product appears to modulate the timing of budding to obtain an appropriate cell size independent of the DNA replication cycle.

Download Full-text