Ultrastructural and biochemical properties of the 120-kDa form of chick kinectin.
Kinectin, an integral membrane protein (160 kDa), was identified as a kinesin-binding protein. Analysis of the predicted amino acid sequence of kinectin cDNA indicated an alpha-helical coiled-coil structure from amino acid 320 to 1310. A 120-kDa kinectin has been observed consistently, and N-terminal sequencing showed that 232 amino acids were missing from the N terminus of full-length kinectin. 120-kDa kinectin was distributed in the supernatant and a low density fraction of vesicles, whereas both forms were in the high density fraction of vesicles. In the electron microscope, the 120-kDa form appeared as a linear molecule of 133 nm in length. In hydrodynamic studies, the cytosolic 120-kDa kinectin was a dimer. Monoclonal antibody molecules (anti-kinectin KR160.9) bound asymmetrically to kinectin often with two antibodies/kinectin, indicative of a parallel coiled-coil. Metabolic labeling with [3H]myristic acid showed that both the 120- and 160-kDa kinectin are myristoylated in chick embryo fibroblasts. The myristoylation of 120-kDa kinectin may provide a mechanism for linking it to a low density fraction of vesicles. Immunoprecipitation with a 160-kDa kinectin-specific antibody brought down the 120-kDa kinectin. Thus, we suggest that kinectin is an extended parallel coiled-coil dimer, often a heterodimer.
Kumar
J
J
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Erickson
H P
HP
Sheetz
M P
MP
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D018076:DNA, Complementary; D008565:Membrane Proteins; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019814:Myristic Acid / Q000378:metabolism; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; C088547:KTN1 protein, human; D008565:Membrane Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D019814:Myristic Acid