A Possible Role of ER-60 Protease in the Degradation of Misfolded Proteins in the Endoplasmic Reticulum

doi:10.1074/jbc.270.25.14958

A Possible Role of ER-60 Protease in the Degradation of Misfolded Proteins in the Endoplasmic Reticulum (*)

From the (1) Protein Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565 and the
(2)Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan

^§ To whom correspondence should be addressed:
Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565, Japan.
Tel.: 81-6-872-8208; Fax: 81-6-872-8219.

↵^¶ Present address: Institute for Fundamental Research, Suntory Ltd., Osaka 618, Japan.
↵** Present address: Dept. of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, Shiga 525-77, Japan.

Abstract

Wild-type human lysozyme (hLZM) is secreted when expressed in mouse L cells, whereas misfolded mutant hLZMs are retained and eventually degraded in a pre-Golgi compartment (Omura, F., Otsu, M., Yoshimori, T., Tashiro, Y., and Kikuchi, M.(1992) Eur. J. Biochem. 210, 591-599). These misfolded mutant hLZMs are associated with protein disulfide isomerase (Otsu, M., Omura, F., Yoshimori, T., and Kikuchi, M.(1994) J. Biol. Chem. 269, 6874-6877). From the observation that this degradation is sensitive to cysteine protease inhibitors, such as N-acetyl-leucyl-leucyl-norleucinal and N-acetyl-leucyl-leucyl-methioninal, but not to the serine protease inhibitors, 1-chloro-3-tosylamido-7-amino-2-heptanone and (p-amidinophenyl)methanesulfonyl fluoride, it was suggested that some cysteine proteases are likely responsible for the degradation of abnormal proteins in the endoplasmic reticulum (ER). ER-60 protease (ER-60), an ER resident protein with cysteine protease activity (Urade, R., Nasu, M., Moriyama, T., Wada, K., and Kito, M.(1992) J. Biol. Chem. 267, 15152-15159), was found to associate with misfolded hLZMs, but not with the wild-type protein, in mouse L cells. Furthermore, denatured hLZM is degraded by ER-60 in vitro, whereas native hLZM is not. These results suggest that ER-60 could be a component of the proteolytic machinery for the degradation of misfolded mutant hLZMs in the ER.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ER

endoplasmic reticulum

PDI

protein disulfide isomerase

hLZM

human lysozyme

ALLN

N-acetyl-leucyl-leucyl-norleucinal

ALLM

N-acetyl-leucyl-leucyl-methioninal

TLCK

1-chloro-3-tosylamido-7-amino-2-heptanone

APMSF

(p-amidinophenyl)methanesulfonyl fluoride

DSP

dithiobis(succinimidyl propionate)

PVDF

polyvinylidene difluoride

PBS

phosphate-buffered saline

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis

bis-Tris

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
↵²R. Urade, T. Oda, H. Ito, S. Utsumi, T. Moriyama, and M. Kito, manuscript in preparation.
↵³M. Otsu, R. Urade, M. Kito, T. Hayano, and M. Kikuchi, unpublished data.