Abstract
In this report, we present the identification of the main polypeptides that are extracted from purified cell walls of a Saccharomyces cerevisiae mnn1 mnn9 strain by reducing agents. Treatment of the purified cell walls of this strain with beta-mercaptoethanol releases several mannoproteins, of which three, with apparent sizes of 120, 45, and 40 kDa, are the most abundant. Analysis of the amino-terminal sequences revealed that the 120-kDa mannoprotein is Bar1p, the protease involved in the so-called barrier activity in yeast cells, and that the 45- and 40-kDa mannoproteins are the Kex2-unprocessed and Kex2-processed forms of the gene product of open reading frame (ORF) YJL158c, an ORF that belongs to the PIR (protein with internal repeats) family of genes, composed thus far of PIR1, PIR2/HSP150, and PIR3. Accordingly we have named this gene PIR4, and Pir4 denotes the 40-kDa Kex2-processed form of the mannoprotein. We have characterized Pir4 and have shown the feasibility of using it as a fusion partner for the targeting of recombinant proteins to the cell wall.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Aspartic Acid Endopeptidases*
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Biological Transport / drug effects
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Blotting, Western
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Cell Wall / chemistry*
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Cell Wall / genetics
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Cell Wall / metabolism
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Cytoskeletal Proteins / analysis
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Cytoskeletal Proteins / genetics
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Cytoskeletal Proteins / metabolism
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Electrophoresis, Polyacrylamide Gel
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Endopeptidases / chemistry
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Endopeptidases / genetics
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Endopeptidases / metabolism
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Fluorescent Antibody Technique, Indirect
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Fungal Proteins / chemistry
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Fungal Proteins / genetics*
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Fungal Proteins / metabolism
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Gene Deletion
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Gene Expression Regulation, Enzymologic
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Gene Expression Regulation, Fungal
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Glycosylation
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Membrane Glycoproteins / analysis*
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Membrane Glycoproteins / metabolism
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Mercaptoethanol
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Molecular Sequence Data
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Molecular Weight
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Mutagenesis
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Open Reading Frames / genetics
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Phenotype
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Proprotein Convertases*
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Protein Tyrosine Phosphatases / chemistry
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Protein Tyrosine Phosphatases / genetics
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Protein Tyrosine Phosphatases / metabolism
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Recombinant Proteins / genetics
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Reducing Agents / pharmacology*
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Saccharomyces cerevisiae / chemistry*
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Saccharomyces cerevisiae / enzymology
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae Proteins*
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Sodium Dodecyl Sulfate
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Subtilisins / metabolism
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Surface-Active Agents
Substances
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Cytoskeletal Proteins
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Fungal Proteins
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Membrane Glycoproteins
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Recombinant Proteins
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Reducing Agents
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Saccharomyces cerevisiae Proteins
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Surface-Active Agents
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mannoproteins
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Sodium Dodecyl Sulfate
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Mercaptoethanol
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Protein Tyrosine Phosphatases
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Endopeptidases
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Proprotein Convertases
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Subtilisins
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KEX2 protein, S cerevisiae
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Aspartic Acid Endopeptidases
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BAR1 protein, S cerevisiae