Abstract
We previously reported that the reductive activities of yeast protein-disulfide isomerase (PDI) family proteins did not completely explain their contribution to the viability of Saccharomyces cerevisiae (Kimura, T., Hosoda, Y., Kitamura, Y., Nakamura, H., Horibe, T., and Kikuchi, M. (2004) Biochem. Biophys. Res. Commun. 320, 359-365). In this study, we examined oxidative refolding activities and found that Mpd1p, Mpd2, and Eug1p exhibit activities of 13.8, 16.0, and 2.16%, respectively, compared with Pdi1p and that activity for Eps1p is undetectable. In analyses of interactions between yeast PDI proteins and endoplasmic reticulum molecular chaperones, we found that Mpd1p alone does not have chaperone activity but that it interacts with and inhibits the chaperone activity of Cne1p, a homologue of mammalian calnexin, and that Cne1p increases the reductive activity of Mpd1p. These results suggest that the interface between Mpd1p and Cne1p is near the peptide-binding site of Cne1p. In addition, Eps1p interacts with Pdi1p, Eug1p, Mpd1p, and Kar2p with dissociation constants (KD) in the range of 10(-7) to 10(-6). Interestingly, co-chaperone activities were completely suppressed in Eps1p-Pdi1p and Eps1p-Mpd1p complexes, although only Eps1p and Pdi1p have chaperone activity. The in vivo consequences of these results are discussed.
Highlights
An important event in the folding of secretory proteins is the formation of disulfide bonds
In analyses of interactions between yeast protein-disulfide isomerase (PDI) proteins and endoplasmic reticulum molecular chaperones, we found that Mpd1p alone does not have chaperone activity but that it interacts with and inhibits the chaperone activity of Cne1p, a homologue of mammalian calnexin, and that Cne1p increases the reductive activity of Mpd1p
A similar concentration dependence was observed for other yPDI proteins
Summary
PDI, protein-disulfide isomerase; yPDI, yeast PDI; ER, endoplasmic reticulum; CNX, calnexin. Many homologues similar in structure to PDI have been identified These PDI homologues have two or more CXXC motifs (where X is a variable amino acid), each of which is predicted to function as a site for the formation, reduction, or isomerization of a disulfide bond. The interactions of ERp57 with calnexin (CNX) and calreticulin [16, 17], of PDI with peptidyl-prolyl cis-trans isomerase [18] and ERp57 [19] and of JPDI (J domain-containing protein-disulfide isomerase-like protein) with BiP (immunoglobulin heavy chain-binding protein) [20] have been reported in mammalian cells. We found that Mpd1p interacts with Mpd2p but that the two proteins do not influence the reductive or chaperone activities of each other [21]. Eps interactions with Pdi1p, Eug1p, Mpd1p, and Kar2p have different effects on their activities
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