IMPAIRED SECRETION OF A HYDROPHOBIC CUTINASE BY SACCHAROMYCES-CEREVISIAE CORRELATES WITH AN INCREASED ASSOCIATION WITH IMMUNOGLOBULIN HEAVY-CHAIN BINDING-PROTEIN (BIP)

This study focuses on the different efficiencies of secretion of two f ungal cutinases by Saccharomyces cerevisiae, a wild-type cutinase (CY0 00) and a hydrophobic mutant cutinase (CY028). Both cutinases are plac ed under control of the GAL7 promoter, by which the expression levels can be regulated, Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level , Pulse-chase experiments revealed that whereas CY000 cutinase is secr eted, CY028 is irreversibly retained in the cell, Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic r eticulum (ER), The increase of wild-type cutinase expression did not r esult in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase. Immunoprecipitation studies showed that in contrast to the wild-type c utinase, the hydrophobic mutant cutinase interacts with BiP. These res ults indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause th e retention of this mutant cutinase in the ER.