Molecular cloning and characterization of human kinectin.
We have identified a human cDNA that is homologous to the chicken kinectin, a putative receptor for the organelle motor kinesin. The human cDNA clone hybridized to a single 4.6-kb mRNA species that codes for a protein of 156 kDa molecular mass. The predicted primary translation product contains an N-terminal transmembrane helix followed by a bipartite nuclear localization sequence and two further C-terminal leucine zipper motifs. In addition, the aminoacid sequence revealed a large region (327-1362) of predicted alpha-helical coiled coils. A monoclonal antibody CT-1 raised against a GST-kinectin fusion protein produced a perinuclear, endoplasmic reticulum-like staining pattern in diverse cell types from different species, indicating evolutionary conservation. Monoclonal antibody CT-1 and anti-chicken kinectin antibodies cross-reacted both in Western blotting and immunoprecipitation with a 160-kDa protein, confirming the antigenic identity of this 160-kDa protein with chicken kinectin. Epitope tagging studies revealed that the nuclear localization sequence motif of kinectin is not functional. Furthermore, a truncated kinesin cDNA lacking the N-terminal hydrophobic domain revealed a nonspecific cytoplasmic staining pattern. Together the data suggest that kinectin is an integral membrane protein anchored in the endoplasmic reticulum via a transmembrane domain.
Fütterer
A
A
Institut für Medizinische Mikrobiologie und Hygiene, Technische Universität München, Germany.
Kruppa
G
G
Krämer
B
B
Lemke
H
H
Krönke
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D005075:Biological Evolution; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000302:isolation & purification; D006801:Humans; D016547:Kinesins / Q000378:metabolism*; D016350:Leucine Zippers; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011956:Receptors, Cell Surface / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; C088547:KTN1 protein, human; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D016547:Kinesins