Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

Abstract

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.

Kinectin Anchors the Translation Elongation Factor-1δ to the Endoplasmic Reticulum

“…The expression of baits A, B, and D fusion proteins in yeast were determined by immunoblotting the yeast extract with GAL4 DNA-BD monoclonal antibody (Clontech). 3 The kinectin bait D was used to screen a human fetal brain Matchmaker cDNA library fused to the GAL4 DNA-activation domain (AD) of the pACT2 vector (Clontech). The bait and the amplified library were sequentially transformed into yeast strain Y190 (Clontech) using the lithium acetate method (29).…”

“…Baits A and B proteins were produced with the predicted molecular mass in yeast. 3 GST-Kinectin Bait D Specifically Interacts with EF-1␦ in Vitro-To verify that kinectin bait D binds to the full-length EF-1␦ in vitro, we first performed a GST pull-down analysis. GST fusion constructs with the kinectin baits A, B, and D were made and expressed in E. coli.…”

“…It is an integral membrane protein largely confined to the endoplasmic reticulum (ER) 1 (1,3). Kinectin consists of a 120-kDa polypeptide and a 160-kDa polypeptide interacting through an ␣-helical coiled-coil domain to form a heterodimer (4).…”

“…The 120-kDa polypeptide is the truncated version of the 160-kDa polypeptide, lacking the first 232 amino acids, in the N terminus (4). The N terminus of the 160-kDa polypeptide consists of a transmembrane domain that anchors kinectin to organelle membranes, potentially with the help of the 7 myristylation sites throughout the molecule (3,4). The C terminus of kinectin consists of two functional domains.…”

Expression of Neuronal Kinesin Heavy Chain Is Developmentally Regulated in the Central Nervous System of the Rat

“…Neuronal kinesin heavy chain (nKHC) is encoded by a gene distinct from the one specifying the ubiquitous kinesin heavy chain (uKHC), although the two proteins share a 65% amino acid sequence identity and a very similar domain organization (Niclas et al, 1994). Both their molecular structures show an N-terminal motor domain (Yang et al, 1989;Nakata and Hirokawa, 1995), an a-helical coiled coil rod (De Cuevas et al, 1992), and a small Cterminal globular domain that, despite its still unclear function, is thought to be involved in the interaction with intracellular cargo (Navone et al, 1992;Toyoshima et al, 1992;Andrews et al, 1993;Skoufias et al, 1994;Yu et al, 1995). The sequence of nKHC has the unique feature of a 70-amino-acid C-terminal stretch that is absent in the uKHC sequence (Niclas et al, 1994).…”