title: Non-muscle myosin IIA transports a Golgi glycosyltransferase to the endoplasmic reticulum by binding to its cytoplasmic tail.
abstract: The mechanism of the Golgi-to-ER transport of Golgi glycosyltransferases is not clear. We utilize a cell line expressing the core 2 N-acetylglucosaminyltransferase-M (C2GnT-M) tagged with c-Myc to explore this mechanism. By immunoprecipitation using anti-c-Myc antibodies coupled with proteomics analysis, we have identified several proteins including non-muscle myosin IIA (NMIIA), heat shock protein (HSP)-70 and ubiquitin activating enzyme E1 in the immunoprecipitate. Employing yeast-two-hybrid analysis and pulldown experiments, we show that the C-terminal region of the NMIIA heavy chain binds to the 1-6 amino acids in the cytoplasmic tail of C2GnT-M. We have found that NMIIA co-localizes with C2GnT-M at the periphery of the Golgi. In addition, inhibition or knockdown of NMIIA prevents the brefeldin A-induced collapse of the Golgi as shown by the inhibition of the migration of both Giantin, a Golgi matrix protein, and C2GnT-M, a Golgi non-matrix protein, to the ER. In contrast, knockdown of HSP70 retains Giantin in the Golgi but moves C2GnT-M to the ER, a process also blocked by inhibition or knockdown of NMIIA. Also, the intracellular distribution of C2GnT-M is not affected by knockdown of β-coatomer protein with or without inhibition of HSPs, suggesting that the Golgi-to-ER trafficking of C2GnT-M does not depend on coat protein complex-I. Further, inhibition of proteasome results in accumulation of ubiquitinated C2GnT-M, suggesting its degradation by proteasome. Therefore, NMIIA and not coat protein complex-I is responsible for transporting the Golgi glycosyltransferase to the ER for proteasomal degradation. The data suggest that NMIIA is involved in the Golgi remodeling.
authors.affiliation: Omaha Western Iowa Health System, VA Service, Department of Veterans Affairs Medical Center, Omaha, NE, USA.
authors.forename: Armen
authors.initials: A
authors.lastname: Petrosyan
authors.forename: Mohamed F
authors.initials: MF
authors.lastname: Ali
authors.forename: Shailendra Kumar
authors.initials: SK
authors.lastname: Verma
authors.forename: Helen
authors.initials: H
authors.lastname: Cheng
authors.forename: Pi-Wan
authors.initials: PW
authors.lastname: Cheng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D016695:Glycosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism*; D021381:Protein Transport; D014162:Transfection
chemical_list: D016695:Glycosyltransferases; D024507:Nonmuscle Myosin Type IIA