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 fungal cutinases by Saccharomyces cerevisiae, a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028). Both cutinases are placed 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 secreted, 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 reticulum (ER). The increase of wild-type cutinase expression did not result 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 cutinase, the hydrophobic mutant cutinase interacts with BiP. These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.
Sagt
C M
CM
Department of Molecular Cell Biology, Utrecht University, The Netherlands. cees@emsaserv.biol.ruu.nl
Müller
W H
WH
Boonstra
J
J
Verkleij
A J
AJ
Verrips
C T
CT
D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D000091342:Endoplasmic Reticulum Chaperone BiP; D015966:Gene Expression Regulation, Fungal; D006360:Heat-Shock Proteins; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism*; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D002265:Carboxylic Ester Hydrolases; C019952:cutinase