Content area
Full Text
A nuclear isoform of myosin I beta that contains a unique 16-amino acid aminoterminal extension has been identified. An affinity-purified antibody to the 16-amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I beta colocalized with RNA polymerase II in an alpha-amanitin- and actinomycin D-sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I beta appears to be in a complex with RNA polymerase II and may affect transcription.
Myosin I is a single-headed, nonfilamentous member of the myosin superfamily of actin-- based molecular motors (1, 2). There are at least four different subclasses of myosin I proteins, all containing a 110- to 150-kD heavy chain and one to six light chains. Myosin I is diffusely distributed throughout the cytoplasm (3). It concentrates near cortical surfaces and in the perinuclear region (3), and it appears to mediate plasma membrane extension (3, 4), vesicle and organelle transport (5), and mechanochemical regulation of calcium channels in hair cells (6).
Affinity-purified polyclonal antibodies to bovine adrenal myosin I recognized a 120-kD protein that is larger than the antigen (116 kD) (7). Confocal and electron microscopy showed cytoplasmic and nuclear staining with these antibodies. Biochemical assays on nuclei demonstrated that the 120-kD protein binds adenosine triphosphate (ATP) and calmodulin, is associated with K+-EDTA ATPase activity, and binds actin only in the absence of ATP (7). Because these characteristics are defining features of the myosin superfamily of proteins (1), we considered the possibility that the 120-kD protein is a myosin I isoform that is a nuclear molecular motor.
The 120-kD protein was immunoprecipitated from nuclei isolated from mouse fibroblasts (8). SDS-polyacrylamide gel electrophoresis (PAGE) showed a 120-kD band by Coomassie blue staining in the immunoprecipitates. Microsequencing of the 120-kD protein (8) revealed high sequence homology (>98%) with myosin I beta. It also revealed the presence of 12 amino acids preceding the consensus initiator methionine (9-11) of myosin I beta.
Analysis of an embryonic mouse cDNA library with mouse myosin I beta primers (12) yielded two sequences. The shorter of the two sequences encoded a peptide that contained the consensus mouse myosin I beta start site (9, 10). The longer sequence contained another upstream start site...