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Published 10 May 2004. doi:10.1083/jcb.200310031
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 165, Number 3, 395-405
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Article

 A role for myosin-1A in the localization of a brush border disaccharidase

Matthew J. Tyska1 and Mark S. Mooseker1,2,3

1 Department of Molecular, Cellular, and Developmental Biology
2 Department of Cell Biology, Yale University, New Haven, CT 06511
3 Department of Pathology, Yale University, New Haven, CT 06511

Address correspondence to Matthew J. Tyska, Department of Molecular, Cellular, and Developmental Biology, Yale University 342 Kline Biology Tower, 266 Whitney Ave., New Haven, CT 06511. Tel.: (203) 432-3469. Fax: (203) 432-6161. email: matthew.tyska{at}yale.edu

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 (~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.

Key Words: actin; membrane; lipid raft; microvillus; epithelium

Abbreviations used in this paper: AP, alkaline phosphatase; APN, aminopeptidase N; BB, brush border; BBE, CACO-2BBE; ConA, concanavalin A; DRM, detergent-resistant membrane; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carboiimide; IEC, intestinal epithelial cell; IP, immunopurified; MßCD, methyl-ß-cyclodextrin; M1A, myosin-1A; MV, microvillus/microvilli; SI, sucrase isomaltase; PAS, protein-A Sepharose; SINT, SI NH2-terminal cytoplasmic domain.


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