Non-muscle myosin IIA transports a Golgi glycosyltransferase to the endoplasmic reticulum by binding to its cytoplasmic tail

• Published in: The international journal of biochemistry & cell biology 2012-07, Vol.44 (7), p.1153-1165
• Main Authors: Petrosyan, Armen, Ali, Mohamed F., Verma, Shailendra Kumar, Cheng, Helen, Cheng, Pi-Wan
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acids; Animals; antibodies; Cell Culture Techniques; Cell Line; coat proteins; Coating; Collapse; Cytoplasm - enzymology; Cytoplasm - metabolism; Degradation; Electrophoresis, Polyacrylamide Gel; endoplasmic reticulum; Endoplasmic Reticulum - enzymology; Endoplasmic Reticulum - metabolism; Enzymes; Glycosyltransferase Golgi-to-ER transport; glycosyltransferases; Glycosyltransferases - metabolism; Golgi Apparatus - enzymology; Golgi Apparatus - metabolism; Golgi remodeling; Heat shock proteins; Humans; Inhibition; Mice; Microscopy, Confocal; Molecular Sequence Data; Myosin; Non-muscle myosin IIA; Nonmuscle Myosin Type IIA - metabolism; precipitin tests; Proteasome; proteasome endopeptidase complex; Protein Transport; Proteins; proteomics; Transfection; Transport; ubiquitin-protein ligase
• ISSN: 1357-2725; 1878-5875
• DOI: 10.1016/j.biocel.2012.04.004

[Display omitted] ► Non-muscle myosin IIA is involved in Golgi remodeling. ► Non-muscle myosin IIA transports Golgi proteins to ER for proteasomal degradation. ► Inhibition of non-muscle myosin IIA prevents brefeldin A-induced Golgi collapse. ► Inhibition of heat shock proteins distinguishes Golgi matrix and non-matrix proteins. 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.