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. 2008 Apr 15;47(15):4535-43.
doi: 10.1021/bi702445j. Epub 2008 Mar 25.

Conventional kinesin holoenzymes are composed of heavy and light chain homodimers

Scott R DeBoer  1 , YiMei You, Anita Szodorai, Agnieszka Kaminska, Gustavo Pigino, Evelyn Nwabuisi, Bin Wang, Tatiana Estrada-Hernandez, Stefan Kins, Scott T Brady, Gerardo Morfini
Affiliations

Conventional kinesin holoenzymes are composed of heavy and light chain homodimers

Scott R DeBoer et al. Biochemistry. .

Abstract

Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations.

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Figures

Figure 1
Figure 1
Characterization of anti-kinesin-1-specific antibodies. (A) Immunoblot analysis of whole mouse brain lysates using anti-kinesin-1 and anti-KLC antibodies. Anti-kinesin-1A, -1B, and -1C antibodies recognized a single band at the expected molecular mass size (approximately 100−110 kDa). Note the slightly lower mobility of kinesin-1A under these SDS–PAGE conditions. H2 antibody against all kinesin-1s recognizes a band doublet corresponding to kinesin-1A (upper band, arrowhead) and kinesin-1C/B (lower band, arrow). Anti-kinesin-1B PA1−643 antibody recognized a major band with a slower mobility to those recognized by other kinesin-1 antibodies, raising questions against its specificity (see text). (B) Aliquots of mouse brain cytosol (Cyt) were incubated with Taxol and either 2 mM ATP or 2 mM AMP-PNP, a nonhydrolyzable ATP analogue. After 20 min incubation, samples were spun to produce a MT-enriched pellet (P) and a corresponding supernatant (S). In the presence of AMP-PNP, this procedure allows for quantitative recovery of conventional kinesin in association to MT-enriched pellets. Aliquots of each fraction were analyzed by immunoblotting using H2 antibody, and various novel antibodies against kinesin-1s and KLC2 (see Table 1). Note that with the only exception of PA1−643 antibody, all antibodies tested recognized polypeptides which biochemically behave as expected for conventional kinesin. (C) COS-7 cells were transfected with plasmids encoding His-tagged, full-length versions of kinesin-1A, -1B, and -1C and lysed for 24 h after transfection. COS-7 lysates were first normalized to similar kinesin-1 levels using anti-His antibody (Tet-His) and then analyzed by immunoblot using anti-kinesin-1A (PA1−642), anti-kinesin-1B (UIC 81), and anti-kinesin-1C (uKHC) (see Table 1). These antibodies recognized a single band at the expected molecular mass size (approximately 100−110 kDa), and no cross-reactivity with other kinesin-1s was observed. The monoclonal antibody H2 recognized all kinesin-1s, albeit with different affinities (1A > 1C >> 1B). After very long exposure of film, a very faint band was recognized by H2 antibody in untransfected (Neg) cells (data not shown). Arrowheads and arrows indicate the positions of kinesin 1A and kinesin-1B/C, respectively.
Figure 2
Figure 2
Kinesin-1s exist as homodimers in brain tissue. (A) Validated anti-kinesin-1 antibodies were used for immunoprecipitation from mouse brain lysates. Non-immune IgGs were used as controls for immunoprecipitation specificity. A lane loaded with mouse brain lysate (Input) is shown. Immunoblot analysis indicates that each antibody exclusively immunoprecipitated their respective antigen. As expected from its specificity, H2 antibody immunoprecipitated all kinesin-1s. The presence of KLC1 and KLC2 in these immunoprecipitates confirmed the native conditions of the methods herein. Key: NR IgG, normal rabbit IgG; NM IgG, normal mouse IgG. (B) Three rounds of immunoprecipitation were carried out as described in (A), and aliquots of the lysate supernatants obtained after each round were analyzed by immunoblot. Depletion of each kinesin-1A and kinesin-1C from brain lysates does not significantly affect the levels of all other kinesin-1 proteins, indicating that kinesin-1s exist primarily as homodimers.
Figure 3
Figure 3
Kinesin-1 homodimers interact with both KLC1 and KLC2 homodimers. Immunoblots with 63−90 antibody show that anti-KLC1 (L2) and anti-KLC2 (B2A5) antibodies selectively immunoprecipitated their corresponding antigen, whereas KLC-All antibody raised against the TR domain common to all KLCs immunoprecipitated both. These data confirmed previous reports of KLC homodimerization. Significantly, kinesin-1A, -1B, and -1C could all be found in both anti-KLC1 and anti-KLC2 immunoprecipitates, suggesting there is no specificity in the interactions between KLCs and kinesin-1 homodimers.
Figure 4
Figure 4
Biochemically heterogeneous forms of conventional kinesin associate to different MBOs. (A) Microsomal membranes from mouse brain were fractionated by iodixanol density gradient centrifugation, and several fractions along the iodixanol gradient were analyzed by immunoblotting using antibodies against kinesin-1s, KLCs, and various membrane-associated proteins corresponding to different MBO markers. (B, C) Quantitation of blots in (A) shows different distribution profiles of kinesin-1s (B) but not KLCs (C) along the iodixanol gradient, suggesting a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to biochemically heterogeneous membrane-bounded organelle cargoes. The distribution of all kinesin-1s plotted together (1A + 1B + 1C) resembled that of KLCs, confirming the heterotetrameric composition of conventional kinesin in association to these MBOs. IU = intensity units.
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