An intrinsic membrane glycoprotein of the golgi apparatus with O-linked N-acetylglucosamine facing the cytosol.

J M Capasso,C Abeijon,C B Hirschberg

doi:10.1016/s0021-9258(19)77702-3

Abstract

We have recently described the occurrence of integral membrane glycoproteins in rat liver smooth and rough endoplasmic reticulum with O-N-acetylglucosamine facing the cytosolic and luminal sides of the membrane (Abeijon, C., and Hirschberg, C. B. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1010-1014). We now report that integral membrane glycoproteins with cytosolic facing O-N-acetylglucosamine also occur in membranes of rat liver Golgi apparatus. This was determined following incubation of vesicles from the Golgi apparatus, which were sealed and of the same membrane topographical orientation as in vivo, with UDP-[14C]galactose and saturating amounts of bovine milk galactosyltransferase. This enzyme does not enter the lumen of the vesicles and specifically catalyzes the addition of galactose, in a beta 1-4 linkage, to terminal N-acetylglucosamine. Under these conditions, galactose was transferred to a glycoprotein of molecular mass of 92 kDa. This protein was insoluble in sodium carbonate, pH 11.5, conditions under which integral membrane proteins remain membrane bound and was insensitive to treatment with peptide:N-glycosidase F. beta Elimination and chromatography showed that radiolabeled galactose was part of a disaccharide which was characterized as Gal beta 1-4GlcNAcitol. This glycoprotein is specific of the Golgi apparatus membrane. Intrinsic membrane glycoproteins with this unusual carbohydrate membrane orientation thus occur in the endoplasmic reticulum and Golgi apparatus of rat liver.

Highlights

We have recently described the occurrenceof inte- questions about the biosynthesis and mechanism of the final gral membrane glycoproteins in rat liver smooth and topographical arrangement of these glycoproteins in memrough endoplasmic reticulumwith O-N-acetylglucosa- branes
Intrinsic membrane glycoproteinswith this unusual carbohydrate membrane orientation occur in ethnedoplasmic reticulum and Golgi apparatus of rlaivt er
Band 4.1,a cytosolicperipheral glycoprotein, has been shown to contain carbohydrate [10].More recently, we have described in ratliver endoplasmic reticulum, integral membrane glycoproteins with their sugars facing the cytosolic and luminal sides of the membrane [11]

Summary

Introduction

We have recently described the occurrenceof inte- questions about the biosynthesis and mechanism of the final gral membrane glycoproteins in rat liver smooth and topographical arrangement of these glycoproteins in memrough endoplasmic reticulumwith O-N-acetylglucosa- branes. Among these questions are ( a )the subcellular organmine facing the cytosolic and lumsiindaelsof the mem- elles where these O-GlcNAc-containingglycoproteins are synbrane We thesized and ( b ) how the topography of these glycoproteins is established so that their carbohydrate is on both sides of a GmUmrtmtoeeDorseeiplommmlkoPglrii-ibbcnt[fagrr’aep4aatacdhCplnniafanaeeo]ctsggrlttOlaaoiooontl-spfwauNtyoecsrli-ggt,tnaaorwrrtgcsaalaiheiennplvtiscyhecmafuhlrinegcebrdGwlamuaaleoctssborieloaorrge.stniaiuasenTemrnaeaaphtilatopniegistfndaeliayogrvaelcnasnenoaotasozpudmsiycrscoc.moiolfnueutTtesernhivhinntefiidsrwvssswoooaoimeat,mfeshswtnbhecditnoetyeeohvr--tineogdtmmecleiyeeWctnemmcuscoerbbtwpeirrhrndaaiaotnnhvtoeeeierntighsonreuaaerxwnictiearheinmtslhlatuleaisignstvsiaeueotordhcrtsnhehwfGeeaaohrnscorediltiingthonehgipaneutlntvrhaaiuesteitsoimnhoic.tceinyrTlciteenhoorsnsiesfdsiotwcioilgctihpmsuclilylcsaceuchisamdomhmrepabb.ivroorcoefahtrnaeyeeidtmnitrcghaedultemyoleucesbmsosrapwan.mrnoaoAeestthe lumen of thevesicles and specificallycatalyzes the appear to occur in the cytosol or in membranes of the endoaddition of galactose, in81a-4 linkage, to terminal N- plasmic reticulum or other organelles

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Conclusion