CHARACTERIZATION OF KINECTIN, A KINESIN-BINDING PROTEIN - PRIMARY SEQUENCE AND N-TERMINAL TOPOGENIC SIGNAL ANALYSIS

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embry onic chick brain cDNA Library by immunoscreening with a panel of monoc lonal antibodies. The cDNA contained an open reading frame of 1364 ami no acids encoding a protein of 156 kDa. A bacterially expressed produc t of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depende d upon the N-terminal domain. Analysis of the predicted amino acid seq uence indicated a highly hydrophobic near N-terminal stretch of 28 ami no acids and a large portion (326-1248) of predicted alpha helical coi led coils. The 30-kDa fragment containing the N-terminal hydrophobic r egion was produced by cell-free in vitro translation and found to asse mble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane dom ain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral m embrane protein that binds kinesin and is likely to be a coiled-coil d imer.