MEMBRANE TOPOLOGY AND RETENTION OF MICROSOMAL ALDEHYDE DEHYDROGENASE IN THE ENDOPLASMIC-RETICULUM

Microsomal aldehyde dehydrogenase (msALDH) is anchored to the endoplas mic 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, 1407-142 0), To determine the membrane topology and the intracellular localizat ion 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 expres sed in COS cells, Indirect immunofluorescence microscopy showed the ER localization of all of the chimeric proteins similar to wildtype msAL DH. 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 gl ycosylated, These results indicate that the membrane binding domain of msALDH spans the bilayer of the ER, The carbohydrate chain of the chi meras was sensitive to endoglycosidase H but resistant to endoglycosid ase D. Upon treatment of transfected COS cells with brefeldin A, the c arbohydrate chain was processed to an endoglycosidase II-resistant for m, presumably by cis/medial Golgi-specific enzymes redistributed in th e ER, These biochemical results in addition to immunofluorescence micr oscopic observations suggest that msALDH is retained in the ER by bloc kading of the exit from the ER.