A specific light chain of kinesin associates with mitochondria in cultured cells

Abstract

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1-3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.

Figure 1

Characterization of the α-KLC antibody by immunoblotting. (A) Rat brain homogenate Coomassie R-250-stained gel; (B) corresponding immunoblot, probed with α-KLC antibody; and (C) immunoblot of purified bovine brain kinesin probed with α-KLC antibody. The positions of the molecular mass markers are shown on the left.

Figure 2

Alignment of the deduced amino acid sequences of rat KLC-A and hamster KLC-D. Asterisks indicate identical amino acids.

Figure 3

Deduced carboxyl-terminal amino acid sequences of different CHO KLC isoforms compared with the unique regions of the rat KLC-B and -C, the human, and the squid KLCs.

Figure 4

Characterization of α-B antibody by immunoblotting with total cell homogenates of CHO-K1, human skin fibroblasts, and CV-1 cells. (A) Coomassie R-250 stained gel; (B) immunoblot probed with α-KLC antibody; (C) immunoblot probed with α-B antibody; and (D) immunoblot, probed with α-B* antibody. The positions of the molecular mass markers are shown on the left.

Figure 5

Immunolocalization of different KLC isoforms in CHO-K1 cells (A–D), human skin fibroblasts (E–H), and CV-1 cells (I–L). (A, E, and I) α-KLC staining; (B, F, and J) α-B staining; (C, G, and K) MitoTracker staining of the corresponding cells presented in B, F, and J. (D, H, and L) α-B* staining. Bars, 10 μm.

Figure 6

Western blot analysis of KLCs in fractions of mitochondria (A), postmitochondrial supernatant (B), microsomes (C), and cytosol (D) of CV-1 cells. Immunoblots probed with α-KLC, α-B, and α-B* antibodies. The positions of the molecular mass markers and the Coomassie R-250 stained gel of the mitochondrial fraction (CBB) are shown on the left.

Figure 7

Immunoblot probed with anti-kinesin heavy chain antibody. (A) Purified bovine brain kinesin. (B) CV-1 whole-cell homogenate. (C) Mitochondrial fraction of CV-1 cells. (D) KI extract of CV-1 mitochondrial fraction. (E) Immunoprecipitate of KI extract with α-B antibody. (F) Immunoprecipitate of KI extract with α-B* antibody.