Analysis of a processing system for proteases using yeast cell surface engineering: conversion of precursor of proteinase A to active proteinase A.
The display of a protease, carboxypeptidase Y (CPY) or procarboxypeptidase Y (proCPY), which is the vacuolar protease, on the yeast-cell surface was successfully performed using yeast-cell-surface engineering for the first time. Through that we could confirm the processing of vacuolar proteases containing proteinase A (PrA) and proteinase B (PrB) which are related to the maturation of proCPY, using a novel cell-surface engineering technique. Various protease-knockout strains of Saccharomyces cerevisiae with the CPY-displaying system were constructed to evaluate the operation of the activation process of CPY. The display of CPY (CPY-agg, which is a fusion protein of CPY with C-terminal half of alpha-agglutinin) on the cell surface was confirmed by immunofluorescence staining. The activity of the CPY-agg was determined after the conversion of proCPY to active CPY by treatment of whole cells with proteinase K. In the proCPY-displaying CPY-knockout strain and PrB-knockout strain, CPY was displayed as an active (mature) form, but in the proCPY-displaying PrA-knockout strain, CPY was present as an inactive form (proCPY). These facts indicate that PrA had been already activated before its transport to the vacuole and that active mature PrA might convert proCPY to CPY before the transport of proCPY to the vacuole. From these results, it was suggested that by using the yeast-cell-surface engineering at the location of the initial step, the autocatalytic activation from proPrA to PrA might occur before the vacuolar branch separates from the main secretory pathway.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. mk@kais.kyoto-u.ac.jp
Michiko
M
Kato
Yuko
Y
Kuzuhara
Haruko
H
Maeda
Seizaburo
S
Shiraga
Mitsuyoshi
M
Ueda
D000954:Antigens, Surface / Q000032:analysis; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D043402:Cathepsin A / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D008856:Microscopy, Fluorescence; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D000954:Antigens, Surface; D016282:Aspartic Acid Endopeptidases; D043402:Cathepsin A; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C023302:yeast proteinase B; D029701:Saccharomyces cerevisiae Proteins; C022442:aspergillopepsin II