Degradation of Rhizopus niveus aspartic proteinase-I with mutated prosequences occurs in the endoplasmic reticulum of Saccharomyces cerevisiae.
Rhizopus niveus aspartic proteinase-I (RNAP-I) is secreted by Saccharomyces cerevisiae extracellularly (Horiuchi, H., Ashikari, T., Amachi, T., Yoshizumi, H., Takagi, M., and Yano, K. (1990) Agric. Biol. Chem. 54, 1771-1779). The prosequence of RNAP-I has the function to promote correct folding of its mature part. Deletion (Deltapro) and amino acid substitutions (M1) in the prosequence block secretion of RNAP-I (Fukuda, R., Horiuchi, H., Ohta, A., and Takagi, M. (1994) J. Biol. Chem. 269, 9556-9561). In this study, little accumulation of Deltapro was observed in Western blot analysis of the cell extracts of the transformants producing Deltapro using anti-RNAP-I antisera. In contrast, M1 was accumulated in the yeast cells. Pulse-chase analysis revealed that they were synthesized at almost the same rates and that Deltapro was degraded in the cells more rapidly than M1. In subcellular fractionation analysis, Deltapro was found in the fraction that contained most of the activity of an endoplasmic reticulum (ER) marker enzyme, NADPH-cytochrome c reductase. In indirect immunofluorescence microscopy, Deltapro was observed in the ER. Similar result was also observed in a mutant which is deficient of the two vacuolar proteases, proteinase A and proteinase B. So, the vacuolar proteases are not involved in degradation of Deltapro. From these results, we concluded that RNAP-Is with the mutated prosequences, which probably could not be folded correctly, were retained and degraded in the ER.
Fukuda
R
R
Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan.
Umebayashi
K
K
Horiuchi
H
H
Ohta
A
A
Takagi
M
M
D000409:Alanine; D016282:Aspartic Acid Endopeptidases / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism*; D001483:Base Sequence; D015415:Biomarkers; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000378:metabolism*; D004926:Escherichia coli; D019084:Fluorescent Antibody Technique, Indirect; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012233:Rhizopus / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology
D015415:Biomarkers; D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C100354:aspartic proteinase-I; D000409:Alanine