Medial-Golgi retention of N-acetylglucosaminyltransferase I. Contribution from all domains of the enzyme

• Published in: The Journal of biological chemistry Vol. 269; no. 16; pp. 12049 - 12059
• Main Authors: BURKE, J, PETTITT, J. M, HUMPHRIS, D, GLEESON, P. A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 22.04.1994
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Cytoplasm - enzymology; DNA Primers; Enzymes and enzyme inhibitors; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Golgi Apparatus - enzymology; Immunoenzyme Techniques; Immunohistochemistry; Intracellular Membranes - enzymology; L Cells; Mice; Molecular Sequence Data; N-Acetylglucosaminyltransferases - analysis; N-Acetylglucosaminyltransferases - biosynthesis; N-Acetylglucosaminyltransferases - metabolism; Ovalbumin - analysis; Ovalbumin - biosynthesis; Ovalbumin - metabolism; Polymerase Chain Reaction; Protein Multimerization; Rabbits; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - biosynthesis; Transfection; Transferases; α-1,3-Mannosyl-glycoprotein β-1,2-N-acetylglucosaminyltransferase; Enzyme; Transferases; Glycosyltransferases; Rodentia; Electron microscopy; Structure function relationship; Cell surface; Golgi apparatus; Vertebrata; Mammalia; Mouse; Hexosyltransferases; Immunofluorescence; Localization; Hybrid gene
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)32679-0

We have previously shown that the transmembrane domain and flanking residues of beta-1,2-N-acetylglucosaminyltransferase I (GnTI) can localize a hybrid molecule to medial-Golgi cisternae (Burke, J., Pettitt, J. M., Schachter, H., Sarkar, M., and Gleeson, P.A. (1992) J. Biol. Chem. 267, 24433-24440). Here, we have further examined the contribution of the cytoplasmic tail, transmembrane domain, and the catalytically active, luminal domain of GnTI in medial-Golgi localization, by analyzing the localization of hybrid molecules stably expressed in murine cells. In contrast to wild-type GnTI, which was efficiently localized to the medial-Golgi and not detected at the cell surface, hybrid molecules containing any two of the three domains of GnTI were localized to the medial-Golgi and were also present at low levels at the cell surface. Hybrid molecules containing only the transmembrane domain or the luminal domain of GnTI showed partial Golgi retention together with an increased level of cell surface expression compared with molecules containing two GnTI domains. The cytoplasmic tail independently was unable to retain reporter sequences to the Golgi but increased the ability of constructs containing either the luminal or transmembrane domain of GnTI to localize to the Golgi apparatus. Therefore, all three domains of GnTI contribute significantly to medial-Golgi localization. Furthermore, GnTI hybrid molecules showing increased cell surface expression were more readily extracted in a low salt buffer, suggesting that Golgi localized GnTI differs in physicochemical properties from cell surface GnTI. Based on an aggregation model of localization, we propose that Golgi retention of these hybrid molecules is mediated by the interaction of their GnTI domains with the corresponding domains of endogenous glycosyltransferase aggregates within the Golgi membranes of the transfected cell.