A role for myosin-1A in the localization of a brush border disaccharidase.
To gain insight regarding myosin-1A (M1A) function, we expressed a dominant negative fragment of this motor in the intestinal epithelial cell line, CACO-2BBE. Sucrase isomaltase (SI), a transmembrane disaccharidase found in microvillar lipid rafts, was missing from the brush border (BB) in cells expressing this fragment. Density gradient centrifugation, affinity purification, and immunopurification of detergent-resistant membranes isolated from CACO-2BBE cells and rat microvilli (MV) all indicate that M1A and SI reside on the same population of low density (approximately 1.12 g/ml) membranes. Chemical cross-linking of detergent-resistant membranes from rat MV indicates that SI and M1A may interact in a lipid raft complex. The functional significance of such a complex is highlighted by expression of the cytoplasmic domain of SI, which results in lower levels of M1A and a loss of SI from the BB. Together, these studies are the first to assign a specific role to M1A and suggest that this motor is involved in the retention of SI within the BB.
Tyska
Matthew J
MJ
Department of Molecular, Cellular, and Developmental Biology, Yale University 342 Kline Biology Tower, 266 Whitney Ave., New Haven, CT 06511,USA. matthew.tyska@yale.edu
Mooseker
Mark S
MS
D000818:Animals; D018938:Caco-2 Cells; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D004063:Digestion / Q000502:physiology; D004187:Disaccharides / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000648:ultrastructure; D021962:Membrane Microdomains / Q000378:metabolism / Q000648:ultrastructure; D008871:Microvilli / Q000201:enzymology / Q000648:ultrastructure; D018995:Myosin Heavy Chains; D024461:Myosin Type I; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D013347:Subcellular Fractions; D013394:Sucrase-Isomaltase Complex / Q000378:metabolism*
D002148:Calmodulin-Binding Proteins; D004187:Disaccharides; C062291:MYO1A protein, human; D013394:Sucrase-Isomaltase Complex; D024461:Myosin Type I; D018995:Myosin Heavy Chains