Binding of brush border myosin I to phospholipid vesicles.
The actin filament core within each microvillus of the intestinal epithelial cell is attached laterally to the plasma membrane by brush border (BB) myosin I, a protein-calmodulin complex belonging to the myosin I class of actin-based mechanoenzymes. In this report, the binding of BB myosin I to pure phospholipid vesicles was examined and characterized. BB myosin I demonstrated saturable binding to liposomes composed of anionic phospholipids, but did not associate with liposomes composed of only neutral phospholipids. The binding of BB myosin I to phosphatidylserine and phosphatidylglycerol vesicles reached saturation at 4-5 x 10(-3) nmol protein/nmol phospholipid, while the apparent dissociation constant was determined to be 1-3 x 10(-7) M. Similar to the free protein, membrane-associated BB myosin I bound F-actin in an ATP-sensitive manner and demonstrated actin-activated Mg-ATPase activity. Immunoblot analysis of peptides generated from controlled proteolysis of vesicle-bound BB myosin I provided structural information concerning the site responsible for the membrane interaction. Immunoblot staining with domain-specific mAbs revealed a series of COOH-terminal, liposome-associated peptides that were protected from digestion, suggesting that the membrane-binding domain is within the carboxy-terminal "tail" of the BB myosin I heavy chain.
Hayden
S M
SM
Department of Biology, Yale University, New Haven, Connecticut 06511.
Wolenski
J S
JS
Mooseker
M S
MS
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000235:genetics; D002645:Chickens; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008871:Microvilli / Q000378:metabolism*; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D009994:Osmolar Concentration; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D012965:Sodium Chloride / Q000494:pharmacology
D000199:Actins; D008081:Liposomes; D010743:Phospholipids; D012965:Sodium Chloride; D009218:Myosins; D000252:Calcium-Transporting ATPases