Erythrocyte protein 4.1 binds and regulates myosin.
Myosin was recently identified in erythrocytes and was shown to partition both with membrane and cytosolic fractions, suggesting that it may be loosely bound to membranes [Fowler, V. M., Davis, J. Q. & Bennett, V. (1985) J. Cell Biol. 100, 47-55, and Wong, A. J., Kiehart, D. P. & Pollard, T. D. (1985) J. Biol. Chem. 260, 46-49]; however, the molecular basis for this binding was unclear. The present studies employed immobilized monomeric myosin to examine the interaction of myosin with erythrocyte protein 4.1. In human erythrocytes, protein 4.1 binds to integral membrane proteins and mediates spectrin-actin assembly. Protein 4.1 binds to rabbit skeletal muscle myosin with a Kd = 140 nM and a stoichiometry consistent with 1:1 binding. Heavy meromyosin competes for protein 4.1 binding with Ki = 36-54 nM; however, the S1 fragment (the myosin head) competes less efficiently. Affinity chromatography of partial chymotryptic digests of protein 4.1 on immobilized myosin identified a 10-kDa domain of protein 4.1 as the myosin-binding site. In functional studies, protein 4.1 partially inhibited the actin-activated Mg2+-ATPase activity of rabbit skeletal muscle myosin with Ki = 51 nM. Liver cytosolic and erythrocyte myosins preactivated with myosin light-chain kinase were similarly inhibited by protein 4.1. These studies show that protein 4.1 binds, modulates, and thus may regulate myosin. This interaction might serve to generate the contractile forces involved in Mg2+-ATP-dependent shape changes in erythrocytes and may additionally serve as a model for myosin organization and regulation in non-muscle cells.
Pasternack
G R
GR
Department of Pathology, Johns Hopkins University School of Medicine, MD 21205.
Racusen
R H
RH
D000818:Animals; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D003598:Cytoskeletal Proteins; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D009479:Neuropeptides; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011485:Protein Binding; D011817:Rabbits
D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D002918:Chymotrypsin; D009218:Myosins