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 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.
Yu
H
H
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Nicchitta
C V
CV
Kumar
J
J
Becker
M
M
Toyoshima
I
I
Sheetz
M P
MP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D004721:Endoplasmic Reticulum; D015723:Gene Library; D006801:Humans; D007668:Kidney; D016547:Kinesins / Q000378:metabolism*; D008565:Membrane Proteins; D008961:Models, Structural; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014158:Transcription, Genetic; D014162:Transfection
C088547:KTN1 protein, human; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D016547:Kinesins