TY - JOUR
T1 - Non-muscle myosin IIA transports a Golgi glycosyltransferase to the endoplasmic reticulum by binding to its cytoplasmic tail
AU - Petrosyan, Armen
AU - Ali, Mohamed F.
AU - Verma, Shailendra Kumar
AU - Cheng, Helen
AU - Cheng, Pi Wan
N1 - Funding Information:
The work is supported in part by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs ( VA 1I1BX000985 ), the NIH ( 1R21HL097238 and 2RO1HL48282 ) and the State of Nebraska (NRI-Cancer Glycobiology Program and LB506). We thank Dr. Sandra Wells for providing the human bronchial epithelium paraffin sections, Dr. David Smith for helpful discussion. We also thank the Mass Spectrometry and Proteomics Core Facility, and Janice A. Taylor and James R. Talaska of the Confocal Laser Scanning Microscope Core Facility at the University of Nebraska Medical Center for providing assistance in proteomics analysis and confocal microscopy, respectively, and the Nebraska Research Initiative and the Eppley Cancer Center for their support of the Core Facilities.
PY - 2012/7
Y1 - 2012/7
N2 - 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.
AB - 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.
KW - Glycosyltransferase Golgi-to-ER transport
KW - Golgi remodeling
KW - Heat shock proteins
KW - Non-muscle myosin IIA
KW - Proteasome
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U2 - 10.1016/j.biocel.2012.04.004
DO - 10.1016/j.biocel.2012.04.004
M3 - Article
C2 - 22525330
AN - SCOPUS:84861227003
SN - 1357-2725
VL - 44
SP - 1153
EP - 1165
JO - International Journal of Biochemistry and Cell Biology
JF - International Journal of Biochemistry and Cell Biology
IS - 7
ER -