Functional Analysis of an Anchoring Protein CG-NAP in the Golgi Apparatus.

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CG-NAP is a giant coiled-coil protein localized at centrosome and the Golgi apparatus. To investigate the role of CG-NAP in the Golgi apparatus, the Golgi-targeting mechanism was analyzed and the effects of dissociation of CG-NAP from the Golgi were examined on the Golgi morphology and on the membrane traffic.1.By immunofluorescence microscopy of CG-NAP in intact and semi-intact cells, it was revealed that CG-NAP begins to dissociate from the Golgi at late G2 phase and completely dissociates at prophase.2.The Golgi-targeting region was identified to the amino-terminal region of CG-NAP by examining the localization of various deletion mutants. This region was effectively phosphorylated by mitotically activated protein kinase plk, which might cause dissociation of CG-NAP from the Golgi.3.Overexpression of the Golgi-targeting region resulted in dissociation of endogenous CG-NAP from the Golgi in COS cells. In these cells, Golgi marker proteins represented fragmented localization, suggesting that the Golgi apparatus is fragmented. Further, GFP-tagged Golgi marker proteins showed unstable movements in live-cell imaging. On the other hand, the transport of VSV-Gts protein from endoplasmic reticulum to plasma membrane was not inhibited.4.When CG-NAP expression was suppressed by siRNA, similar effects were observed on the Golgi morphology and on the VSV-Gts traffic.These results suggest that CG-NAP is involved in the formation and/or maintenance of the Golgi apparatus at pericentrosomal area.