Membrane topology and retention of microsomal aldehyde dehydrogenase in the endoplasmic reticulum.
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.
Masaki
R
R
Department of Physiology and Division of Cell Biology, Liver Research Center, Kansai Medical University, Moriguchi, Osaka 570, Japan.
Yamamoto
A
A
Tashiro
Y
Y
D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002451:Cell Compartmentation; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D019084:Fluorescent Antibody Technique, Indirect; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017299:Rod Opsins / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D017299:Rod Opsins; D000444:Aldehyde Dehydrogenase