The activity of a model heterologous protein in pep4-3 mutants of Saccharomyces cerevisiae.
The bacterial lacZ gene was introduced into two sibling strains of Saccharomyces cerevisiae, one a wild-type strain with normal proteinase activity and the other a pep4-3 mutant strain. The pep4-3 mutation resulted in 90% reduced activity of the four major vacuolar proteinases. By comparing the activity of the lacZ gene product (beta-galactosidase) in both strains the degradative effect of the major vacuolar proteinases on a heterologous protein was estimated. The mutant strain with reduced proteinase activity had higher beta-galactosidase activity under all the test conditions. In the most productive case the pep4-3 mutant had 55% higher beta-galactosidase activity than the wild-type. Batch cultures of the two strains were evaluated for growth characteristics. The strain with reduced proteinase activity grew to higher optical densities than the wild-type. Upon further examination it was found that not only were the optical densities of pep4-3 cultures greater but the cell numbers were much greater than expected due to the smaller size of pep4-3 cells. It is concluded that the strain lacking vacuolar proteinases maintained increased levels of beta-galactosidase and is physiologically as healthy as the wild-type.
Wingfield
J M
JM
School of Pure and Applied Biology, University of Wales College of Cardiff, UK.
Dickinson
J R
JR
D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D007763:Lac Operon; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D001616:beta-Galactosidase; D010450:Endopeptidases