Medial Golgi but Not Late Golgi Glycosyltransferases Exist as High Molecular Weight Complexes

To investigate the organization of Golgi glycosyltransferases and their mechanism of localization, we have compared the properties of a number of medial and late acting Golgi enzymes. The medial Golgi enzymes,N-acetylglucosaminyltransferase I and II (GnTI and GnTII) required high salt for solubilization and migrated as high molecular weight complexes on sucrose density gradients. In contrast, the late acting Golgi enzymes, β1,4-galactosyltransferase and α1,2-fucosyltransferase, were readily solubilized in low salt and migrated as monomers/dimers by sucrose density gradient centrifugation. Analysis of membrane-bound GnTI chimeras indicates that the formation of high molecular weight complexes does not require the transmembrane domain and cytoplasmic tail sequences of GnTI. Furthermore, a soluble form of GnTI, containing the stem region and catalytic domain, accumulated in the Golgi prior to secretion, in contrast to β1,4-galactosyltransferase. Soluble GnTI, which also associated with high molecular weight complexes, was comparable with membrane-bound GnTI in its ability to glycosylate newly synthesized glycoproteinsin vivo. Mutation of charged residues within the stem region of GnTI, known to be important for “kin recognition”, had no effect on the efficiency of Golgi localization, the inclusion into high molecular weight complexes, nor functional activity in vivo. The differences in behavior between the medialand late acting Golgi enzymes may contribute to their differential localization and their ability to glycosylate efficiently in the correct Golgi subcompartment. To investigate the organization of Golgi glycosyltransferases and their mechanism of localization, we have compared the properties of a number of medial and late acting Golgi enzymes. The medial Golgi enzymes,N-acetylglucosaminyltransferase I and II (GnTI and GnTII) required high salt for solubilization and migrated as high molecular weight complexes on sucrose density gradients. In contrast, the late acting Golgi enzymes, β1,4-galactosyltransferase and α1,2-fucosyltransferase, were readily solubilized in low salt and migrated as monomers/dimers by sucrose density gradient centrifugation. Analysis of membrane-bound GnTI chimeras indicates that the formation of high molecular weight complexes does not require the transmembrane domain and cytoplasmic tail sequences of GnTI. Furthermore, a soluble form of GnTI, containing the stem region and catalytic domain, accumulated in the Golgi prior to secretion, in contrast to β1,4-galactosyltransferase. Soluble GnTI, which also associated with high molecular weight complexes, was comparable with membrane-bound GnTI in its ability to glycosylate newly synthesized glycoproteinsin vivo. Mutation of charged residues within the stem region of GnTI, known to be important for “kin recognition”, had no effect on the efficiency of Golgi localization, the inclusion into high molecular weight complexes, nor functional activity in vivo. The differences in behavior between the medialand late acting Golgi enzymes may contribute to their differential localization and their ability to glycosylate efficiently in the correct Golgi subcompartment. green fluorescent protein β1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101) soluble GnTI β1,2-N-acetylglucosaminyltransferase II (EC 2.4.1.143) β1,4-galactosyltransferase (EC 2.4.1.38) blood group H α1,2-fucosyltransferase (EC 2.4.1.69) Chinese hamster ovary fetal calf serum leuco-phytohemagglutinin transferrin receptor phosphate-buffered saline polymerase chain reaction endoplasmic reticulum polyacrylamide gel electrophoresis fluorescein isothiocyanate base pair 4-morpholineethanesulfonic acid The Golgi apparatus is a highly complex and dynamic organelle consisting of a series of flattened cisternae associated with numerous vesicles and membrane tubules. The Golgi is central to the secretory pathway, since it plays important roles in the maturation and sorting of newly synthesized secretory and membrane proteins and also in the recycling of proteins and lipids to the endoplasmic reticulum (1.Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1993) Google Scholar, 2.Farquhar M. Hauri H.-P. Berger E.G. Roth J. The Golgi Apparatus. Birhäuser Verlag, Basel1997: 63-129Crossref Google Scholar). In addition, the Golgi has a fundamental role in the biosynthesis of the glycan chains of glycoproteins, proteoglycans, and glycolipids in eukaryotic cells. Glycosylation occurs in a highly regulated manner as the newly synthesized molecules move from the cis to thetrans side of the Golgi stack (3.Varki A. Trends Cell Biol. 1998; 8: 34-40Abstract Full Text PDF PubMed Scopus (126) Google Scholar). The synthesis of carbohydrate chains of glycoconjugates in mammalian cells is likely to require more than 200 different glycosyltransferase enzymes distributed throughout the Golgi stack (4.van den Eijnden D. Joziasse D.H. Curr. Opin. Struct. Biol. 1993; 3: 711-721Crossref Scopus (73) Google Scholar, 5.Schachter H. Fukuda M. Hindsgaul O. Molecular Glycobiology. Oxford University Press, Oxford1994: 88-162Google Scholar, 6.Field M.C. Wainwright L.J. Glycobiology. 1995; 5: 463-472Crossref PubMed Scopus (98) Google Scholar). To understand the control of glycan biosynthesis in vivo requires an appreciation of the organization of glycosyltransferases within the membranes of the individual compartments. All Golgi glycosyltransferases cloned to date are Nin/Cout (type II) membrane proteins containing a short N-terminal cytoplasmic domain, a single hydrophobic membrane-spanning domain, and a large carboxyl-terminal catalytic domain situated in the lumen of the Golgi apparatus (4.van den Eijnden D. Joziasse D.H. Curr. Opin. Struct. Biol. 1993; 3: 711-721Crossref Scopus (73) Google Scholar, 5.Schachter H. Fukuda M. Hindsgaul O. Molecular Glycobiology. Oxford University Press, Oxford1994: 88-162Google Scholar, 6.Field M.C. Wainwright L.J. Glycobiology. 1995; 5: 463-472Crossref PubMed Scopus (98) Google Scholar). The catalytic domain is linked to the transmembrane domain by a loosely defined “stem” region that may play a role in positioning the catalytic domain away from the lipid bilayer, facilitating access to the substrates. A number of the enzymes involved in the synthesis of complex N-glycans have been precisely localized and that are with their in the Berger E.G. J. Cell 1995; PubMed Google Scholar). The that the of Golgi glycosyltransferases are not Analysis of glycosyltransferase chimeras from eukaryotic cells has that the transmembrane domain of glycosyltransferases plays a role in Golgi in addition, in a number of from the domain and cytoplasmic tail have also been that may be involved in the localization of Golgi enzymes Glycobiology. PubMed Scopus Google and Cell Biol. 1998; PubMed Scopus Google Scholar). have been in it the glycosyltransferases chimeras are that in vivo. A number of for the Golgi of glycosyltransferases have been and Trends Cell Biol. 1998; 8: Full Text PDF PubMed Scopus Google Scholar, J. 1998; PubMed Scopus Google Scholar). are not to be in the of the for maturation O. A. M. A. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, 1998; PubMed Scopus Google and the ability of green fluorescent protein Golgi glycosyltransferases to and in Golgi membranes M. M. J. 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The of GnTI and from is known to be H. J. Biol. Full Text PDF PubMed Google Scholar, J. Cell Biol. 1994; PubMed Scopus Google Scholar). In we have that enzymes in a manner in that is an of In low of a large of GnTI and from cells was salt the enzymes were a of GnTI and was to in high molecular weight complexes that be complexes are large and are of membrane contrast with and that were solubilized in low salt and in the of a high J. Cell Biol. 1994; PubMed Scopus Google have that the medial Golgi enzymes, GnTI and II from membranes with a in the of the of GnTI with the was with J. Cell Biol. 1994; PubMed Scopus Google Scholar). that GnTI is from Golgi membranes in a manner also that GnTI is solubilized as high molecular weight complexes, which in a a behavior by The of Golgi glycosyltransferases as high molecular weight complexes is with on the of that a high form of H. J. Biol. Full Text PDF PubMed Google Scholar). that the domain of GnTI is important for inclusion into the molecular weight properties to GnTI and was in high molecular weight soluble GnTI containing the stem region and catalytic domain was in a high molecular weight the soluble GnTI into the as a Furthermore, soluble GnTI accumulated in the Golgi region prior to as by the localization of soluble GnTI in cells and by which within the Golgi be by with that the molecules are not is that the of soluble GnTI within the Golgi is to the inclusion into high molecular weight membrane Soluble GnTI was to glycosylate efficiently in vivo as by the ability of soluble to the of cells. The ability of soluble GnTI to form high molecular complexes may be important in its ability to the synthesis of complex N-glycans in cells. The of membrane-bound and soluble of Golgi glycosyltransferases to glycosylate newly synthesized proteins have been and differences between enzymes have been J. Biol. Full Text Full Text PDF Scopus Google Scholar, Glycobiology. 1998; 8: PubMed Scopus Google Scholar). A for differences be that the enzymes that glycosylate vivo have sequences in their that to the formation of protein complexes and of the soluble the inclusion of the medial Golgi enzymes into high molecular weight complexes may be J. J. 1998; PubMed Scopus Google has the of complexes with glycosyltransferase activity in the cis Golgi of is that the GnTI and complexes may also the of complexes and Berger E.G. J. 1994; PubMed Scopus Google have that Golgi enzymes may to form large was on the that the of an to GnTI not GnTI to to the also medial Golgi within the The between GnTI and within membranes has been to be by their J. 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The formation of high molecular weight complexes Golgi enzymes may between the protein molecules of the and the charged residues of the GnTI stem not with with of the of the of Golgi enzymes be important in the J. 1995; PubMed Scopus Google not a between the enzymes and is also with that the enzymes, and as within the Golgi and indicates that late Golgi enzymes in a different from medial Golgi enzymes. is the between the high molecular weight complex formation of the medial Golgi enzymes and Golgi is likely that inclusion into the high molecular weight complexes for the Golgi localization of and also enzymes efficiently membrane-bound molecules within the Golgi J. D. J. Biol. 1994; Full Text PDF PubMed Google Scholar, Biol. 1995; PubMed Scopus Google the of late Golgi enzymes to be J. Biol. Full Text PDF PubMed Google Scholar, J. PubMed Scopus Google Scholar). the is that the transmembrane of medial Golgi and Golgi enzymes Golgi Glycobiology. PubMed Scopus Google Scholar, Cell Biol. 1998; PubMed Scopus Google Scholar). The transmembrane domain of medial Golgi enzymes is likely to be acting as a of the domain and may Golgi localization of proteins by into of Golgi lipid Trends Cell Biol. 1998; 8: Full Text PDF PubMed Scopus Google Scholar). The may also into vesicles J. J. D. J. J. Cell Biol. 1998; PubMed Scopus Google Scholar). The ability of medial Golgi enzymes to form complexes does the that sorting may be in of the complex and the of enzymes within the Golgi The of the localization of Golgi enzymes, be the of which are it is that be a to the localization of Golgi enzymes, since that and late Golgi enzymes in different that different may contribute to the localization of glycosyltransferases that in different of the Golgi and for the of for the and for on the