Effects of Calnexin Deletion in Saccharomyces cerevisiae on the Secretion of Glycosylated Lysozymes

Abstract

Disruption of the calnexin gene in Saccharomyces cerevisiae did not lead to gross effects on the levels of cell growth and secretion of wild-type hen egg white lysozymes (HEWL). To investigate the function of calnexin in relation to the secretion of glycoproteins, we expressed both stable and unstable mutant glycosylated lysozymes in calnexin-disrupted S. cerevisiae. The secreted amounts of stable mutant glycosylated lysozymes (G49N and S91T/G49N) were almost the same in both wild-type and calnexin-disrupted S. cerevisiae. In contrast, the secretion of unstable mutant glycosylated lysozymes (K13D/G49N, C76A/G49N, and D66H/G49N) greatly increased in calnexin-disrupted S. cerevisiae, although their secretion was very low in the wild-type strain. This indicates that calnexin may act in the quality control of glycoproteins. We further investigated the expression level of the mRNA of the molecular chaperones BiP and PDI, which play a major role in the protein folding process in the ER, when glycosylated lysozymes were expressed in wild-type and calnexin-disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when the glycosylated lysozymes were expressed in calnexin-disrupted S. cerevisiae. This observation indicates that BiP and PDI may be induced by the accumulation of unfolded glycosylated lysozymes due to the deletion of calnexin.

The effect of calnexin deletion on the expression level of binding protein (BiP) under heat stress conditions in Saccharomyces cerevisiae

“…The calnexin-disrupted S. cerevisiae exhibited a great increase in the secretion of unstable glycosylated lysozyme mutants, despite the low level of secretion by the wild type strain. In addition, we also found that highlevel induction of BiP only occurred for those glycosylated proteins overexpressed in the calnexin-disrupted strains [13]. Combined with the results that we obtained in this study, it seems that both the external and internal ER stresses (overexpressing glycoproteins in the ER) could lead to an increase in the induced level of the ER molecular chaperone BiP in calnexin-disrupted S. cerevisiae (Tab.…”

“…In our previous study, we investigated the levels of cell growth of the calnexindisrupted strain under the internal stress conditions of overexpressing nonglycosylated or glycosylated lysozymes in the ER. There was no evident difference between the growth curve of the calnexin-disrupted strain and that of the wild-type strain [13]. These results indicated that calnexin was not always essential in S. cerevisiae.…”

“…The values are the means of three measurements. a Revised data from our previous study [13]. b Growth rates were measured in liquid YPD at 30ºC, except for those for the heat stress conditions (at 37ºC).…”

“…Protein disulfide isomerase (PDI) and binding protein (BiP) are two major molecular chaperones which play significant roles in the protein-folding process in the ER. It appears that the deletion of calnexin results in the induction of the unfolded protein response (UPR), and increases the levels of PDI and BiP to fold the unfolded glycosylated lysozymes (internal stress) [13]. We found that the induction of PDI in the ER could recover part of the function of calnexin in calnexin-disrupted yeast under heat stress conditions [14].…”

The effect of calnexin deletion on the expression level of PDI in Saccharomyces cerevisiae under heat stress conditions

“…There was no evident difference between the growth curve of the calnexin-disrupted strain and that of the wild-type strain [11]. These results indicate that calnexin is not always essential in S. cerevisiae.…”