EVIDENCE FOR RECYCLING OF THE RESIDENT MEDIAL TRANS GOLGI ENZYME, N-ACETYLGLUCOSAMINYLTRANSFERASE-I, IN LDLD-CELLS/

ldlD cells, which lack the UDP-Gal/UDP-GalNAc 4-epimerase, were stably transfected with a Myc-tagged version of N-acetylglucosaminyltransfer ase I (Myc-Glc-NAc-T I). In the absence of GalNAc and Gal, newly synth esized GlcNAc-T I did not acquire O-linked oligosaccharides but was ca talytically active and was transported to the Gels region as defined u sing both immunofluorescence and immunoelectron microscopy. After addi tion of cycloheximide to prevent further synthesis, GalNAc and Gal wer e added, and the unglycosylated GlcNAc-T I was found to acquire mature , O-linked oligosaccharides with a half-time of about 150 min. The add ition of these sugars was sensitive to N-ethylmaleimide and okadaic ac id, both inhibitors of vesicle-mediated traffic. Together, these resul ts suggest that Myc-GlcNAc-T I undergoes retrograde transport to the e arly part of the Golgi apparatus where the first O-linked sugar, GalNA c, is added followed by anterograde transport back to the Golgi stack, where addition of Gal and sialic acid occurs.