The membrane anchor for the molecular motor kinesin is a critical site involved in intracellular membrane trafficking. Monoclonal antibodies specific for the cytoplasmic surface of chick brain microsomes were used to define proteins involved in microtubule-dependent transport. One of four antibodies tested inhibited plus-end-directed vesicle motility by approximately 90 percent even as a monovalent Fab fragment and reduced kinesin binding to vesicles. This antibody bound to the cytoplasmic domain of kinectin, an integral membrane protein of the endoplasmic reticulum that binds to kinesin. Thus, kinectin acted as a membrane anchor protein for kinesin-driven vesicle motility.

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 embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.