Abstract
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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U15617
N-terminal Sequencing of the 160- and 120-kDa Kinectin—For Nterminal sequencing of the 160- and 120-kDa proteins, approximately 20 pmol of protein was purified by immunoprecipitation, as described earlier, from the Triton X-100 soluble fraction of vesicles (CEB P2) by using mAb KR160.9, which cross-reacts with both forms of kinectin
The 120-kDa kinectin was found in the low density Nycodenz peak fractions, whereas high density fractions contained both the 120- and 160-kDa kinectin
Summary
Distribution of 160- and 120-kDa Kinectin on CEF Vesicles—Chick embryo fibroblast (CEF) vesicles were prepared as described previously [13]. N-terminal Sequencing of the 160- and 120-kDa Kinectin—For Nterminal sequencing of the 160- and 120-kDa proteins, approximately 20 pmol of protein was purified by immunoprecipitation, as described earlier, from the Triton X-100 soluble fraction of vesicles (CEB P2) by using mAb KR160.9, which cross-reacts with both forms of kinectin. P2) was immunoprecipitated as described above except that the solubilized vesicles were diluted with an equal volume of homogenization buffer and centrifuged at 60,000 rpm in a TLA 100.3 rotor (Beckman Instruments) for 30 min at 4 °C. The supernatant was collected and immunoprecipitated with KR160.9 mAb using protein A-agarose as described above except that washed protein A beads were solubilized in SDS sample buffer and separated on 7.5% SDS-PAGE gel in duplicate as described in the legend to Fig. 8.
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