The transmembrane and flanking sequences of beta 1,2-N-acetylglucosaminyltransferase I specify medial-Golgi localization.
UDP-GlcNAc:alpha 3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I (GnTI) is an N(in)/C(out) (type II) membrane protein, localized in the medial-Golgi, that initiates the conversion of high mannose N-glycans to complex N-glycans. Anti-rabbit GnTI antibodies were generated using a purified, enzymatically active, bacterial recombinant fusion protein as immunogen. Rabbit GnTI was effectively retained in the Golgi complex of transfected COS-1 cells and murine L cells, as assessed by indirect immunofluorescence using the species-specific anti-GnTI antibodies; no surface expression of rabbit GnTI could be detected in the transfected cells. Rabbit GnTI, stably expressed in murine L cells, was localized by immunoperoxidase electron microscopy to the medial-cisternae of the Golgi stack. The role of the transmembrane domain of GnTI in Golgi localization was examined by generation of a hybrid construct containing the amino-terminal 31 amino acids of GnTI, corresponding to the 25-residue transmembrane (signal/anchor) domain and flanking hydrophilic sequences, fused with ovalbumin; this ovalbumin/GnTI hybrid molecule was retained in the Golgi complex of transfected COS cells and stably transfected murine L cells. No surface expression of ovalbumin/GnTI was detected. In contrast, ovalbumin fused to the equivalent domains of the human transferrin receptor, a type II cell-surface protein, was efficiently expressed on the cell surface of transfected cells. The ovalbumin/GnTI hybrid molecules in the transfected L cells were N-glycosylated, indicating an N(in)/C(out) membrane orientation, and were localized by immunoperoxidase electron microscopy to one or two cisternae of the medial-Golgi (90% of stained Golgi profiles showed medial-cisternae staining). These results show that a signal contained within the transmembrane domain and flanking residues of GnTI specifies medial-Golgi localization.
Burke
J
J
Department of Pathology and Immunology, Monash University Medical School, Alfred Hospital, Melbourne, Australia.
Pettitt
J M
JM
Schachter
H
H
Sarkar
M
M
Gleeson
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000201:enzymology*; D007124:Immunoenzyme Techniques; D007668:Kidney; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010047:Ovalbumin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D010047:Ovalbumin; D004247:DNA; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I