scholarly journals Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (1) ◽  
pp. 121-128 ◽  
Author(s):  
J H Cramer ◽  
K Lea ◽  
M D Schaber ◽  
R A Kramer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Signal Peptide ◽  
Regulatory Region ◽  
Cellular Protein ◽  
Proteolytic Processing ◽  
Coding Region ◽  
Molecular Weights ◽  
Amino Terminal ◽  
Acid Phosphatase Gene

We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PHO5) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.

Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae

1987 ◽  
Vol 7 (1) ◽  
pp. 121-128
Author(s):  
J H Cramer ◽  
K Lea ◽  
M D Schaber ◽  
R A Kramer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Signal Peptide ◽  
Regulatory Region ◽  
Cellular Protein ◽  
Proteolytic Processing ◽  
Coding Region ◽  
Molecular Weights ◽  
Amino Terminal ◽  
Acid Phosphatase Gene

We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PHO5) in multicopy expression plasmids. Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein. Phaseolin polypeptides in S. cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed. We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase. Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S. cerevisiae. Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur. Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation. However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein.

scholarly journals The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence.

1983 ◽  
Vol 3 (3) ◽  
pp. 439-447 ◽  
Author(s):  
M Carlson ◽  
R Taussig ◽  
S Kustu ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Signal Peptide ◽  
Sequence Data ◽  
Signal Sequence ◽  
Coding Region ◽  
Nucleotide Sequence Data ◽  
Coding Sequence ◽  
Amino Terminal ◽  
Suc2 Gene

The SUC2 gene of Saccharomyces cerevisiae encodes two differently regulated mRNAs (1.8 and 1.9 kilobases) that differ at their 5' ends. The larger RNA encodes a secreted, glycosylated form of invertase and the smaller RNA encodes an intracellular, nonglycosylated form. We have determined the nucleotide sequence of the amino-terminal coding region of the SUC2 gene and its upstream flanking region and have mapped the 5' ends of the SUC2 mRNAs relative to the DNA sequence. The 1.9-kilobase RNA contains a signal peptide coding sequence and presumably encodes a precursor to secreted invertase. The 1.8-kilobase RNA does not include the complete coding sequence for the signal peptide. The nucleotide sequence data prove that SUC2 is a structural gene for invertase, and translation of the coding information provides the complete amino acid sequence of an S. cerevisiae signal peptide.

The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence

1983 ◽  
Vol 3 (3) ◽  
pp. 439-447
Author(s):  
M Carlson ◽  
R Taussig ◽  
S Kustu ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Signal Peptide ◽  
Sequence Data ◽  
Signal Sequence ◽  
Coding Region ◽  
Nucleotide Sequence Data ◽  
Coding Sequence ◽  
Amino Terminal ◽  
Suc2 Gene

The SUC2 gene of Saccharomyces cerevisiae encodes two differently regulated mRNAs (1.8 and 1.9 kilobases) that differ at their 5' ends. The larger RNA encodes a secreted, glycosylated form of invertase and the smaller RNA encodes an intracellular, nonglycosylated form. We have determined the nucleotide sequence of the amino-terminal coding region of the SUC2 gene and its upstream flanking region and have mapped the 5' ends of the SUC2 mRNAs relative to the DNA sequence. The 1.9-kilobase RNA contains a signal peptide coding sequence and presumably encodes a precursor to secreted invertase. The 1.8-kilobase RNA does not include the complete coding sequence for the signal peptide. The nucleotide sequence data prove that SUC2 is a structural gene for invertase, and translation of the coding information provides the complete amino acid sequence of an S. cerevisiae signal peptide.

Protease B of Saccharomyces cerevisiae: Isolation and Regulation of the PRB1 Structural Gene

Genetics ◽  
1987 ◽  
Vol 115 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Charles M Moehle ◽  
Martha W Aynardi ◽  
Michael R Kolodny ◽  
Frances J Park ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Structural Gene ◽  
Genomic Library ◽  
Time Lag ◽  
Proteolytic Processing ◽  
Coding Region ◽  
Protease B ◽  
Endonuclease Mapping ◽  
Protein Molecular Weight

ABSTRACT We have isolated the structural gene, PRB1, for the vacuolar protease B of Saccharomyces cerevisiae from a genomic library by complementation of the prb1-1122 mutation. Deletion analysis localized the complementing activity to a 3.2-kilobase pair XhoI-HindIII restriction enzyme fragment. The fragment was used to identify a 2.3-kilobase mRNA. S1 endonuclease mapping indicated that the mRNA and the gene were colinear. No introns were detected. The mRNA is of sufficient size to encode a protein of about 69,000 molecular weight, a number much larger than either the mature enzyme (≃30,000 protein molecular weight) or the sole reported precursor (≃39,000 protein molecular weight). These results suggest that proteolytic processing steps beyond that thought to be catalyzed by protease A may be required to convert the initial glycosylated translation product into mature protease B. The PRB1 mRNA is made in substantial amounts only when the cells have exhausted the glucose supply and enter the diauxic plateau. There is an extended time lag between PRB1 transcription and expression of protease B activity. A deletion that removes about 83% of the coding region was constructed as a diploid heterozygote. Spores bearing the deletion germinate, grow at normal rates into colonies, and have no obvious phenotype beyond protease B deficiency.

scholarly journals Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (1) ◽  
pp. 38-46 ◽  
Author(s):  
L W Bergman ◽  
M C Stranathan ◽  
L H Preis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Copy Number ◽  
Regulatory Control ◽  
Gene Copy ◽  
Growth Media ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Acid Phosphatase Gene ◽  
Negative Supercoils

We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.

Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae

1986 ◽  
Vol 6 (1) ◽  
pp. 38-46
Author(s):  
L W Bergman ◽  
M C Stranathan ◽  
L H Preis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Copy Number ◽  
Regulatory Control ◽  
Gene Copy ◽  
Growth Media ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Acid Phosphatase Gene ◽  
Negative Supercoils

We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.

Regulation of repressible acid phosphatase gene transcription in Saccharomyces cerevisiae

1985 ◽  
Vol 5 (8) ◽  
pp. 2131-2141
Author(s):  
J M Lemire ◽  
T Willcocks ◽  
H O Halvorson ◽  
K A Bostian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Gene Transcription ◽  
Transcriptional Activation ◽  
Regulatory Gene ◽  
Genetic System ◽  
Yeast Genome ◽  
Mrna Synthesis ◽  
Protein Factor ◽  
Acid Phosphatase Gene

We examined the genetic system responsible for transcriptional regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum, EC 3.1.3.2]) in Saccharomyces cerevisiae at the molecular level by analysis of previously isolated and genetically well-defined regulatory gene mutants known to affect APase expression. These mutants identify numerous positive- (PHO4, PHO2, PHO81) and negative-acting (PHO80, PHO85) regulatory loci dispersed throughout the yeast genome. We showed that the interplay of these positive and negative regulatory genes occurs before or during APase gene transcription and that their functions are all indispensible for normal regulation of mRNA synthesis. Biochemical evidence suggests that the regulatory gene products they encode are expressed constitutively. More detailed investigation of APase synthesis is a conditional PHO80(Ts) mutant indicated that neither PHO4 nor any other protein factor necessary for APase mRNA synthesis is transcriptionally regulated by PHO80. Moreover, in the absence of PHO80, the corepressor, presumed to be a metabolite of Pi, did not inhibit their function in the transcriptional activation of APase.

Comparative analysis of the 5'-end regions of two repressible acid phosphatase genes in Saccharomyces cerevisiae

1983 ◽  
Vol 3 (4) ◽  
pp. 570-579
Author(s):  
G P Thill ◽  
R A Kramer ◽  
K J Turner ◽  
K A Bostian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Amino Acid ◽  
Dna Sequences ◽  
Amino Acid Sequences ◽  
Molecular Weights ◽  
Coding Regions ◽  
A Cell ◽  
Tata Sequence ◽  
Flanking Regions

The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion.

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