Random substitution of large parts of the propeptide of yeast proteinase A.
The yeast aspartic protease, proteinase A, has a 54 amino-acid propeptide, which is removed during activation of the zymogen in the vacuole. Apart from being involved inhibition/activation, the propeptide has been shown to be essential for formation of a stable active enzyme (van den Hazel, H. B., Kielland-Brandt, M. C., and Winther, J. R. (1993) J. Biol. Chem. 268, 18002-18007). We have investigated the sequence requirements for function of the propeptide. The N-terminal half and the C-terminal half of the propeptide were replaced by random sequences at the genetic level, and collections of the mutants were subjected to a colony screen for ones exhibiting activity. A high frequency (around 1%) of active constructs was found, which indicates a very high tolerance for mutations in the propeptide. Thirty-nine functional mutant forms containing random sequence at either the N- or C-terminal half of the propeptide were characterized. Comparison of the propeptides of the active constructs suggests that a particular lysine residue is important for efficient biosynthesis of proteinase A.
van den Hazel
H B
HB
Department of Yeast Genetics, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Kielland-Brandt
M C
MC
Winther
J R
JR
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004792:Enzyme Precursors / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011498:Protein Precursors; D029701:Saccharomyces cerevisiae Proteins; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases