Membrane Topology and Retention of Microsomal Aldehyde Dehydrogenase in the Endoplasmic Reticulum

doi:10.1074/jbc.271.28.16939

Membrane Topology and Retention of Microsomal Aldehyde Dehydrogenase in the Endoplasmic Reticulum*

From the Department of Physiology and Division of Cell Biology, Liver Research Center, Kansai Medical University, Moriguchi, Osaka 570, Japan

^‡ To whom correspondence should be addressed:
Dept. of Physiology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi, Osaka 570, Japan.
Fax: 81-6-993-5319.

Abstract

Microsomal aldehyde dehydrogenase (msALDH) is anchored to the endoplasmic reticulum (ER) membrane by the hydrophobic domain at its carboxyl terminus, and most of the molecule is exposed to the cytoplasm (Masaki, R., Yamamoto, A., and Tashiro, Y. (1994) J. Cell Biol. 126, 1407-1420). To determine the membrane topology and the intracellular localization of msALDH, the amino-terminal region of bovine opsin containing N-glycosylation sites was fused to the carboxyl terminus of msALDH, and three chimeric proteins with extensions of different sizes were expressed in COS cells. Indirect immunofluorescence microscopy showed the ER localization of all of the chimeric proteins similar to wild-type msALDH. Immunoblotting revealed that the two chimeric proteins containing longer extensions, those with the N-glycosylation site at distances of 13 and 21 amino acids from the membrane anchor, respectively, were glycosylated. These results indicate that the membrane binding domain of msALDH spans the bilayer of the ER. The carbohydrate chain of the chimeras was sensitive to endoglycosidase H but resistant to endoglycosidase D. Upon treatment of transfected COS cells with brefeldin A, the carbohydrate chain was processed to an endoglycosidase H-resistant form, presumably by cis/medial Golgi-specific enzymes redistributed in the ER. These biochemical results in addition to immunofluorescence microscopic observations suggest that msALDH is retained in the ER by blockading of the exit from the ER.

Footnotes

↵* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and by a grant from the Naito Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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

P450

cytochrome P450

msALDH

microsomal aldehyde dehydrogenase

endo D

endoglycosidase D

endo H

endoglycosidase H

PAGE

polyacrylamide gel electrophoresis

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate.
↵² R. Masaki, A. Yamamoto, K. Akagawa, and Y. Tashiro, manuscript in preparation.
- Received April 8, 1996.