Recombinant expression of the brush border myosin I heavy chain.
Although the specific functions of myosin I motors are not known, their localization to membrane structures suggests a function in membrane motility. Different myosin I isoforms in the same cell or in different cells can possess different localizations. To determine if the localization and biochemical activity of the best-characterized mammalian myosin I, chicken intestinal epithelium brush border myosin I, was dependent on determinants of the membrane or actin cytoskeleton specific to epithelial cells, we transfected the cDNA for the heavy chain of this myosin into COS cells. Transient transfection of COS cells with the chicken brush border myosin heavy chain resulted in the production of recombinant myosin I. Recombinant brush border myosin I localized to protrusions of the plasma membrane, particularly at spreading edges, and also to unknown cytoplasmic structures. Some cells expressing particularly high levels of brush border myosin I possessed a highly irregular surface. Recombinant brush border myosin I purified from COS cells bound to actin filaments in an ATP-dependent manner and decorated actin filaments to form a characteristic appearance. The recombinant myosin also catalyzed calcium-sensitive, actin-activated MgATPase activity similar to that of the native enzyme. Thus, any cellular factor required for the general membrane localization or biochemical activity of brush border myosin I is present in COS cells as well as intestinal epithelium.
Collins
K
K
Whitehead Institute, Massachusetts Institute of Technology, Cambridge, USA.
Matsudaira
P T
PT
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002417:Cattle; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D007422:Intestines / Q000201:enzymology; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D008871:Microvilli / Q000201:enzymology*; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000199:Actins; D018076:DNA, Complementary; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D017301:Ca(2+) Mg(2+)-ATPase; D018995:Myosin Heavy Chains