EVIDENCE FOR THE PRESENCE OF MYOSIN-I IN THE NUCLEUS

We produced and affinity-purified polyclonal antibodies to adrenal myo sin I, These antibodies recognize adrenal myosin I by Western blot ana lysis (116 kDa) and inhibit the actin-activated ATPase activity of pur ified adrenal myosin I. They also recognize a 120-kDa protein in extra cts prepared from many different cell lines, Fluorescence microscopy d emonstrated the presence of immunoreactive material in the perinuclear region, the leading edges, and the nuclei of 3T3 cells. Fluorescence microscopy also demonstrated nuclear staining in mouse oocytes at the germinal vesicle stage and in the pronuclei during fertilization, Conf ocal and immuno-electron microscopy confirmed the intranuclear localiz ation, Electron microscopy also demonstrated staining of structures in nucleoli that are thought to be associated with rDNA transcription, W estern blot analyses revealed the presence of the 120-kDa protein in e xtracts prepared from nuclei that are apparently free of cytosolic con tamination, The same nuclear protein binds I-125-calmodulin and is pho toaffinity labeled with [alpha-P-32]ATP. The 120-kDa protein was parti ally purified from twice washed nuclei using ammonium sulfate fraction ation and gel filtration chromatography. Column fractions containing 1 20-kDa protein as revealed by Western blot analysis also contain K+-ED TA ATPase activity. The 120-kDa protein was also shown to bind actin i n the absence, but not the presence, of ATP, Since K+-EDTA ATPase acti vity, actin, and ATP binding are defining features of the members of t he myosin superfamily of proteins, we propose that the 120-kDa protein is a previously undescribed myosin I isoform that is an intranuclear actin-based molecular motor.