Evidence for Recycling of the Resident medial/trans Golgi Enzyme, N-Acetylglucosaminyltransferase I, in ldlD Cells

doi:10.1074/jbc.270.42.25057

Evidence for Recycling of the Resident medial/trans Golgi Enzyme, N-Acetylglucosaminyltransferase I, in ldlD Cells (*)

From the Cell Biology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A, 3PX, United Kingdom

^§§ To whom correspondence should be addressed. Tel.: 71-269-3561; Fax: 71-269-3417.

↵^§ Present address: Program in Cellular Biochemistry and Biophysics, Rockefeller Research Laboratory, Sloan-Kettering Institute, 1275 York Ave., New York, NY 10021.
↵^¶ Present address: Dept. of Biochemistry, Dartmouth Medical School, 7200 Vail Bldg., Hanover, NH 03755-3844.
↵** Present address: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724.

Abstract

ldlD cells, which lack the UDP-Gal/UDP-GalNAc 4-epimerase, were stably transfected with a Myc-tagged version of N-acetylglucosaminyltransferase I (Myc-GlcNAc-T I). In the absence of GalNAc and Gal, newly synthesized GlcNAc-T I did not acquire O-linked oligosaccharides but was catalytically active and was transported to the Golgi region as defined using both immunofluorescence and immunoelectron microscopy. After addition of cycloheximide to prevent further synthesis, GalNAc and Gal were added, and the unglycosylated GlcNAc-T I was found to acquire mature, O-linked oligosaccharides with a half-time of about 150 min. The addition of these sugars was sensitive to N-ethylmaleimide and okadaic acid, both inhibitors of vesicle-mediated traffic. Together, these results suggest that Myc-GlcNAc-T I undergoes retrograde transport to the early part of the Golgi apparatus where the first O-linked sugar, GalNAc, is added followed by anterograde transport back to the Golgi stack, where addition of Gal and sialic acid occurs.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ER

endoplasmic reticulum

NEM

N-ethylmaleimide

TGN

trans-Golgi network

CGN

cis-Golgi network

GalNAc-T

galactosaminyl transferase

Myc-GlcNAc-T I

Myc-tagged glucosaminyl transferase I

GlcNAc-T I

glucosaminyl transferase I

dFCS

dialyzed fetal calf serum

PAGE

polyacrylamide gel electrophoresis.
↵²Harry Schachter, personal communication.
- Received February 10, 1995.
- Revision received July 27, 1995.