BRUSH-BORDER MYOSIN-I TRUNCATED IN THE MOTOR DOMAIN IMPAIRS THE DISTRIBUTION AND THE FUNCTION OF ENDOCYTIC COMPARTMENTS IN AN HEPATOMA-CELLLINE

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin -based motor properties, are associated with plasma and/or vesicular m embranes and have been suggested as players for trafficking events bet ween cell surface and intracellular membranous structures, To investig ate the function of myosins I, rye have transfected a mouse hepatoma c ell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain, One variant is deleted of the first 446 amino acids and thereby lacks the ATP bindin g site, whereas the other is deleted of the entire motor domain and la cks the ATP and actin binding sites. We have observed (i) that signifi cant amounts of the truncated variants are recovered with membrane fra ctions after cell fractionation, (ii) that they codistribute with a co mpartment containing alpha 2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBM I-truncated variants impairs the distribution of the acidic compartmen t and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha 2-macroglobulin degradation whereas the production of t he variant lacking the ATP binding site and the actin binding site inc reases the rate of alpha 2-macroglobulin degradation. These observatio ns indicate that the two truncated variants have a dominant negative e ffect on the distribution and the function of the endocytic compartmen ts. We propose that an unidentified myosin-I might contribute to the d istribution of endocytic compartments in a juxtanuclear position and/o r to the regulation of the delivery of ligands to the degradative comp artment in BWTG3 cells.