Impaired Cutinase Secretion in Saccharomyces cerevisiae Induces Irregular Endoplasmic Reticulum (ER) Membrane Proliferation, Oxidative Stress, and ER-Associated Degradation

Abstract

Impaired secretion of the hydrophobic CY028 cutinase invokes an unfolded protein response (UPR) inSaccharomyces cerevisiae cells. Here we show that the UPR in CY028-expressing S. cerevisiae cells is manifested as an aberrant morphology of the endoplasmic reticulum (ER) and as extensive membrane proliferation compared to the ER morphology and membrane proliferation of wild-type CY000-producing S. cerevisiae cells. In addition, we observed oxidative stress, which resulted in a 21-fold increase in carbonylated proteins in the CY028-producing S. cerevisiae cells. Moreover, CY028-producing S. cerevisiae cells use proteasomal degradation to reduce the amount of accumulated CY028 cutinase, thereby attenuating the stress invoked by CY028 cutinase expression. This proteasomal degradation occurs within minutes and is characteristic of ER-associated degradation (ERAD). Our results clearly show that impaired secretion of the heterologous, hydrophobic CY028 cutinase in S. cerevisiae cells leads to protein aggregation in the ER, aberrant ER morphology and proliferation, and oxidative stress, as well as a UPR and ERAD.The yeast Saccharomyces cerevisiae is often used as a cell factory. When strong promoters and high gene copy numbers are used, high levels of heterologous proteins are obtained (14). To make such a process feasible for industrial applications, it is desirable that the cells secrete heterologous proteins into the culture medium, allowing easy purification. The secretion process, however, is complicated, and many factors are involved.Secretion of heterologous proteins by S. cerevisiae has been studied by using cutinase as a model protein (21,22). Cutinase is a lipase which originates from the fungus Fusarium solani subsp. pisi (15). Unlike other lipases, the catalytic serine of cutinase is not buried under surface loops but is accessible to solvents. Therefore, cutinase does not need interfacial activation (18), which makes it an important protein for industrial applications.Sagt et al. found that CY000 cutinase was produced and secreted at a concentration of 30 mg/g of cells and had a specific activity of 325 specific lipase units (SLU)/mg of protein (21). To increase the specific activity of the cutinase, hydrophobic patches were introduced around the active site (G82A, A85F, V184I, A185L, L189F), which resulted in CY028 cutinase. The specific activity of CY028 cutinase was 1,093 SLU/mg of protein; however, this hydrophobic cutinase was produced at a concentration of only 10 mg/g of cells and was secreted at a concentration of 3 mg/g of cells. The difference in production between CY000 and CY028 in S. cerevisiae was not due to a difference in copy number or mRNA abundance. It has been shown that hydrophobic cutinase CY028 is retained in the endoplasmic reticulum (ER) in association with the upregulated molecular chaperone BiP (21), which results in impaired secretion. However, secretion of the hydrophobic cutinase could be increased by introduction of an N-glycosylation site (22).Recently, Travers et al. (29) dis...

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