MYOSIN-II IS ASSOCIATED WITH GOLGI MEMBRANES - IDENTIFICATION OF P200AS NONMUSCLE MYOSIN-II ON GOLGI-DERIVED VESICLES

A variety of peripheral membrane proteins associate dynamically with G olgi membranes during the budding and trafficking of transport vesicle s in eukaryotic cells, A monoclonal antibody (AD7) raised against Golg i membranes recognizes a peripheral membrane protein, p200, which asso ciates with vesicles budding off the trans-Golgi network (TGN). Based on preliminary findings, a potential association between p200 and myos in on Golgi membranes was investigated, Immunofluorescence staining of cultured cells under a variety of fixation conditions was carried out using an antibody raised against chick brush border nonmuscle myosin II, We show that, in addition to being found in the cytoplasm or assoc iated with stress fibres, nonmuscle myosin II is also specifically loc alized on Golgi membranes, Myosin II was also detected on Golgi membra nes by immunoblotting and by immunogold labeling at the electron micro scopy level where it was found to be concentrated on Golgi-derived ves icles, The association of myosin II with Golgi membranes is dynamic an d was found to be enhanced following activation of G proteins, Myosin II staining of Golgi membranes was also disrupted by brefeldin A (BFA) , Colocalization of the AD7 and myosin II antibodies at the light and electron microscopy levels led us to investigate the nature of the 200 kDa protein recognized by both antibodies. The 200 kDa protein immuno precipiated by the AD7 antibody was isolated from MDCK cells and used for microsequencing. Amino acid sequence data enabled us to identify p 200 as the heavy chain of nonmuscle myosin IIA. In addition, an extra protein (240 kDa) recognized by the AD7 antibody specifically in extra cts of HeLa cells, was sequenced and identified as another actin-bindi ng protein, filamin, These results show that nonmuscle myosin II is as sociated with Golgi membranes and that the vesicle-associated protein p200, is itself a heavy chain of myosin II.