Beta 1,4 N-acetylgalactosaminyltransferase (GM2/GD2/GA2 synthase) forms homodimers in the endoplasmic reticulum: a strategy to test for dimerization of Golgi membrane proteins.
Many Golgi membrane-bound glycosyltransferases exist as intermolecular disulfide bonded species, some of which have been demonstrated to be homodimers. Evidence for homodimer formation has come primarily from radiation inactivation experiments. We utilized an alternative strategy to test for homodimer formation of the cloned beta 1,4 N-acetylgalactosaminyltransferase (GalNAcT) responsible for synthesis of the glycosphingolipids GM2, GD2, and GA2. We stably transfected CHO cells with myc epitopetagged GalNAcT, which localizes primarily to the Golgi, and a hemagglutinin (HA) epitope-tagged GalNAcT fusion protein in which the cytoplasmic domain of GalNAcT was replaced by an ER retention signal. We then sought evidence for dimer formation between the two forms of GalNAcT. Immunoprecipitation with anti-myc or anti-HA co-immunoprecipitated the HA-tagged form or the myc-tagged form, respectively, providing evidence for the physical association of the two forms of GalNAcT. As a result of this association, GalNAcT/myc increased in the ER as demonstrated by Western blots and immunofluorescence. The rapid formation of dimers provided further evidence for dimer formation occurring in the ER. In summary, these results demonstrate that GalNAcT forms homodimers as a result of intermolecular disulfide bond formation in the ER. Furthermore, this ER motif strategy is potentially useful for demonstrating homodimer formation of other Golgi enzymes.
Zhu
G
G
Department of Biological and Biophysical Sciences, School of Dentistry, University of Louisville, KY 40292, USA.
Jaskiewicz
E
E
Bassi
R
R
Darling
D S
DS
Young
W W
WW
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002457:Cell Extracts; D006224:Cricetinae; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D011233:Precipitin Tests; D014162:Transfection; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D002457:Cell Extracts; D008565:Membrane Proteins; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase