HIGH-LEVEL EXPRESSION OF ENDOGENOUS ACID-PHOSPHATASE INHIBITS GROWTH AND VECTORIAL SECRETION IN SACCHAROMYCES-CEREVISIAE

The secretion pathway of Saccharomyces cerevisiae was challenged by co nstitutively overexpressing plasmid-encoded acid phosphatase, a secret ed endogenous glycoprotein. A 2-mu m-based multicopy plasmid carrying the coding sequence of acid phosphatase under the control of a truncat ed variant of the strong constitutive glyceraldehyde-3-phosphate dehyd rogenase promoter was used for expression. Selection for the promoterl ess dLEU2 marker leads to a growth arrest. This is not per se due to l eucine starvation, but due to intracellular accumulation of highly gly cosylated enzymatically active acid phosphatase. Immunofluorescence an d cytological analysis indicate that intracellular accumulation of aci d phosphatase occurs in a subpopulation of cells. By Ludox-AM density centrifugation, these cells can be enriched on the basis of their high er density. The dense accumulating cells have a higher average plasmid copy number and produce more acid phosphatase than non-accumulating c ells of low density. These cells are defective in directed secretion a nd bud formation, therefore can no longer grow and show dramatic chang es in cell morphology. We suggest that the secretion pathway in these cells is overloaded with the high level of acid phosphatase leading to a shutdown in vectorial secretion, subsequently to a standstill in gr owth and to the intracellular accumulation of further expressed acid p hosphatase. We have indications that accumulation of acid phosphatase occurs in the late Golgi, suggesting a limitation of the overall secre tion at this stage. (C) 1995 Wiley-Liss, Inc.