Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast.
The fate of a mutant form of each of the two yeast vacuolar enzymes proteinase yscA (PrA) and carboxypeptidase yscY (CPY) has been investigated. Both mutant proteins are rapidly degraded after entering the secretory pathway. Mutant PrA is deleted in 37 amino acids spanning the processing site region of the PrA pro-peptide. The mutant enzyme shows no activity towards maturation of itself or other vacuolar hydrolases, a function of wild-type PrA. Mutant CPY carries an Arg instead of a Gly residue in a highly conserved region, two positions distant from the active-site Ser. In contrast to wild-type CPY, the mutant form was quickly degraded by trypsin in vitro, indicating an altered structure. Using antisera specific for alpha-1-->6 and alpha-1-->3 outer-chain mannose linkages, no Golgi-specific carbohydrate modification could be detected on either mutant protein. Subcellular fractionation studies located both mutant enzymes in the endoplasmic reticulum. Degradation kinetics of both proteins show the same characteristics, indicating similar degradation pathways. The degradation process was shown to be independent of a functional sec18 gene product and takes place before Golgi-specific carbohydrate modifications occur. The proteasome, the major proteolytic activity of the cytoplasm, is not involved in this degradation event. All degradation characteristics of the two mutant proteins are consistent with a degradation process within the endoplasmic reticulum ('ER degradation').
Finger
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Institut für Biochemie, Universität Stuttgart, Germany.
Knop
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Wolf
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D000251:Adenosine Triphosphatases; D000596:Amino Acids / Q000235:genetics; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D043402:Cathepsin A; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism; D033921:Vesicular Transport Proteins
D000596:Amino Acids; D004792:Enzyme Precursors; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase; D014357:Trypsin; D003546:Cysteine Endopeptidases; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; D008358:Mannose