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Endoproteolytic processing of recombinant proalbumin variants by the yeast Kex2 protease.

ABSTRACT: The yeast Kex2 protease is regarded as the prototype of the eukaryotic family of subtilisin-like serine proteases involved in processing after dibasic amino acid sequences. Here we investigate the specificity of Kex2 using recombinant human proalbumin variants. Proalbumins with the processing site sequences Arg-Arg and Lys-Arg were cleaved after the dibasic sequence at approximately the same rate by Kex2 in vitro, and yeast expressing either of these sequences secreted mature albumin into the culture medium. As expected, the Arg-Gly-Val-Phe-His-Arg-albumin (proalbumin Lille) was not a substrate for Kex2 and neither was the Arg-Gly-Arg-Phe-His-Arg-albumin. In contrast to the mammalian endoproteases furin and the hepatic proalbumin convertase, the Kex2 protease was adversely affected by a P4 arginine. There was an 85% decrease in the cleavage of Arg-Gly-Arg-Phe-Arg-Arg-albumin compared with normal; also chicken proalbumin with an Arg-Phe-Ala-Arg processing site sequence was not a substrate for Kex2. A P1' arginine had a marked negative effect on processing and N-terminal sequence analysis confirmed that cleavage was occurring at the P1-P1' bond. The sequence context surrounding the classical dibasic site is critical in determining susceptibility to cleavage by the Kex2 protease.

SUBMITTER: Ledgerwood EC

PROVIDER: S-EPMC1136879 | biostudies-other | 1995 May

REPOSITORIES: biostudies-other

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Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24.

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Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris.

Project description:Kexin-like proteinases are a subfamily of the subtilisin-like serine proteinases with multiple regulatory functions in eukaryotes. In the yeast Saccharomyces cerevisiae the Kex2 protein is biochemically well investigated, however, with the exception of a few well known proteins such as the alpha-pheromone precursors, killer toxin precursors and aspartic proteinase propeptides, very few substrates are known. Fungal kex2 deletion mutants display pleiotropic phenotypes that are thought to result from the failure to proteolytically activate such substrates.In this study we have aimed at providing an improved assembly of Kex2 target proteins to explain the phenotypes observed in fungal kex2 deletion mutants by in vitro digestion of recombinant substrates from Candida albicans and C. glabrata. We identified CaEce1, CA0365, one member of the Pry protein family and CaOps4-homolog proteins as novel Kex2 substrates.Statistical analysis of the cleavage sites revealed extended subsite recognition of negatively charged residues in the P1', P2' and P4' positions, which is also reflected in construction of the respective binding pockets in the ScKex2 enzyme. Additionally, we provide evidence for the existence of structural constrains in potential substrates prohibiting proteolysis. Furthermore, by using purified Kex2 proteinases from S. cerevisiae, P. pastoris, C. albicans and C. glabrata, we show that while the substrate specificity is generally conserved between organisms, the proteinases are still distinct from each other and are likely to have additional unique substrate recognition.

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Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant.

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