THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. Vol. 258, No. 5, Issue of March 10, pp. 3159-3165, 1983 Evidence for Extensive Subcellular Organization of Asparagine-linked Oligosaccharide Processing and Lysosomal Enzyme Phosphorylation* (Received for publication, August 9, 1982) Daniel E. Goldberg and Stuart Kornfeld From the Washinpton University School of Medicine, Departments of Internal Medicine and Biological Chemistry, Division of Hematology-Oncology, St. Louis, Missouri 63110 Membranes prepared from mouse lymphoma BW5147.3 cells and P388D1 macrophages were fraction- ated on a continuous sucrose gradient and assayed for enzymes involved in the processing of asparagine- linked oligosaccharides. The order in which these en- zymes distributed from dense to light membranes cor- related with the established sequence of events in gly- coprotein biosynthesis. A number of enzymes which have been previously localized to the Golgi separated into four regions on the gradient. UDP-N-acetylgluco- samine:lysosomal enzyme N-acetylglucosamine-l- phosphotransferase, the enzyme which catalyzes the selective phosphorylation of the high mannose oligo- saccharides of lysosomal enzymes, was present in the densest membranes. N-Acetylglucosamine l-phospho- diester a-N-acetylglucosaminidase was in the next re- gion. Several enzymes involved in the late stages of asparagine-linked oligosaccharide processing were lo- calized to the third region. UDP-ga1actose:N-acetylglu- cosamine galactosyltransferase was present in the lightest membranes (region IV). Pulse-chase experiments utilizing [2-3H]mannose demonstrated that the distribution of in vivo labeled asparagine-linked oligosaccharide intermediates cor- relates with the distribution of these processing en- zymes. Analysis of the phosphorylated oligosaccha- rides of lysosomal enzymes which were bound to the phosphomannosyl receptor indicated that these en- zymes had already passed through the region of the Golgi which contains galactosyltransferase and sialyl- transferase. These findings are consistent with there being a high degree of organization within the Golgi complex. The physical separation of processing enzymes could serve as one mechanism for the control of asparagine-linked oligosaccharide biosynthesis. The participation of the Golgi complex in glycoprotein biosynthesis was first indicated by the [’Hlhexose autoradi- ographic studies of Neutra and Leblond (1-3). Since then numerous morphological and biochemical studies have added to our understanding of the role of the Golgi in oligosaccharide processing (reviewed in Ref. 4). Galactosyltransferase was the first enzyme to be localized to a Golgi subcellular fraction (3, * This investigation was supported by Grants R01 CA 08759 and 5T05GM02016 from the United States Public Health Service and in part by National Institutes of Health Service Award GM 07200, Medical Scientist, from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and since then sialyltransferase (6), fucosyltransferase (7), N- acetylglucosaminyltransferase I (6), a-mannosidase I (8), N- acetylglucosamine 1-phosphodiester a-N-acetylglucosamini- dase’ (9, lo), and N-acetylglucosamine 1-phosphotransferase (10) have all been shown to fractionate with Golgi membranes. Evidence for functional specialization of different regions of the Golgi has come from 5 separate approaches: 1) morpho- logical studies which showed differences between the cis and trans faces of the Golgi in appearance and in proximity to transitional endoplasmic reticulum and condensing vacuoles (4); 2) cytochemical studies of thin tissue slices and Golgi subfractions which revealed differential staining of the cis or trans face of Golgi stacks for thiamine pyrophosphatase (ll), acid phosphatase (ll), and NADP phosphatase (12); 3) im- munological studies of It0 and Palade (13) showing that Golgi membranes containing NADPH-cytochrome P-450 reductase could be immunoprecipitated without co-precipitation of ga- lactosyltransferase activity; 4) immunocytochemical localiza- tion of galactosyltransferase to a few cisternae on the trans side of the Golgi by Roth and Berger (14); 5) biochemical characterization of Golgi subfractions by Dunphy et al. (15) which demonstrated that galactosyltransferase and a-man- nosidase I are located on different membranes. Thus, a variety of studies have led to a picture of the directional movement of glycoproteins through the cell start- ing with translation and glycosylation in the rough endoplas- mic reticulum, followed by movement through an ill-defined transitional region to the Golgi. Next, transit through the Golgi takes place, during which time oligosaccharide process- ing and other post-translational modifications occur. The processing of asparagine-linked oligosaccharides requires a great number of enzymes. Fig. 1 shows the steps involved in the conversion of high mannose-type units to complex-type units and to the phosphorylated species present on lysosomal enzymes. Finally, at or near the trans face of the Golgi, sorting occurs and proteins are packaged for secretion, transport to lysosomes, or targeting to various membranes. Lysosomal enzymes are a special class of glycoproteins that possess phosphorylated high mannose units in addition to the usual neutral high mannose and complex-type units. Their oligosaccharides are phosphorylated by the two step reaction shown in Fig. 1 as reactions 3 and 4. First N-acetylglucosamine 1-phosphate is transferred to an acceptor mannose by UDP- N-acetylglucosamine:lysosomalenzymeN-acetylglucosamine- 1-phosphotransferase (N-acetylglucosaminylphosphotrans- ferase), resulting in a phosphate group in diester linkage between the outer “blocking” N-acetylglucosamine and the ’ Formerly called a-N-acetylgiucosaminylphosphodiesterase. New name: N-acetylglucosamine I-phosphodiester a-N-acetylglucosamin- idase, or phosphodiester glycosidase for short. (A. Varki, W. Sherman, and S.