Cloning of novel kinectin splice variants with alternative C-termini: structure, distribution and evolution of mouse kinectin.
The analysis of cDNA clones encoding novel variant forms of mouse kinectin, an endoplasmic reticulum (ER)-bound receptor for the motor protein kinesin, is reported. Kinesin and cytoplasmic dynein are involved in mediating the anterograde and retrograde movements of intracellular vesicles along the microtubule network. The amino acid sequence deduced from kinectin cDNA isolated from mouse spleen cell and testis libraries revealed a long signal peptide or transmembrane sequence, and a 328 amino acid residue globular N-terminal domain adjacent to a much larger 858-999-residue C-terminal coiled-coil rod domain. The C-terminal domain was composed of 18 coiled-coil regions formed from multiple contiguous heptad repeats which undergo alternative splicing as evidenced by the presence of at least five small (23-33 amino acid residue) insertion sequences scattered throughout. The inserts are present in any one of a number of combinations, generating an array of novel kinectin variants. Insert 5 contains a termination codon, producing a C-terminus that is highly homologous to that of human kinectin. Three out of five mouse kinectin clones lack insert 5, generating a novel eleven amino acid C-terminus encoded by sequence that extends past the insertion site. The existence of alternative C-termini may have functional relevance given that the C-termini are exposed for interaction with kinesin, whereas the globular N-terminus is embedded in the ER membrane. Alternative C-termini represent candidate modifications that could determine specificity of binding to kinesin or cytoplasmic dynein, and the switching of directionality of movement. The cDNA hybridized to 4.5 kb transcripts expressed in all mouse cell lines and tissues examined, which provides the first indication that the kinectins are very widely distributed. Mouse kinectin is 42% similar over a 203 amino acid region to the chicken extracellular cardiac morphogen ES/130, whose canine homologue containing an inserted sequence of 10 amino acids repeated 54 times in tandem, is a ribosome receptor expressed on the ER. Mouse kinectin shares 64 and 83% identity, respectively, with its M(r) 160000 chicken and human kinectin homologues. There is a two-fold molar excess of kinectin over kinesin in unextracted vesicles, suggesting that kinectin might be a dimer. The electrostatic properties of the coiled-coil region of mouse kinectin, together with the relative frequencies of residues in particular positions within the heptad repeats support this notion.
Department of Molecular Medicine, School of Medicine, University of Auckland, New Zealand.
E
E
Leung
C G
CG
Print
D A
DA
Parry
D N
DN
Closey
P J
PJ
Lockhart
S J
SJ
Skinner
D C
DC
Batchelor
G W
GW
Krissansen
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D019143:Evolution, Molecular; D016326:Extracellular Matrix Proteins / Q000737:chemistry; D006801:Humans; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D017510:Protein Folding; D012333:RNA, Messenger; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D030161:Avian Proteins; D018076:DNA, Complementary; C081857:ES-130 protein, Gallus gallus; D016326:Extracellular Matrix Proteins; C088547:KTN1 protein, human; C511012:Ktn1 protein, mouse; D008565:Membrane Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface