Conventional Kinesin Holoenzymes Are Composed of Heavy and Light Chain Homodimers

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.Molecular motors of the kinesin and dynein superfamilies are responsible for microtubule-(MT-) based motility in cells. Approximately 40−45 kinesin-related polypeptides have been identified in mouse and human (1), with 25 or more being expressed in the developing nervous system (2). From these, conventional kinesin is the most abundant kinesin family member in the adult nervous system (3). Biochemical (4) and electron microscopic studies (5) indicated that the native conventional kinesin holoenzyme exists as a tetramer consisting of two kinesin light chain (KLCs) 1 and two kinesin heavy chain (kinesin-1, KHC, KIF5s) subunits (6). † This work was supported by grants from the Huntington's Disease Society of America (HDSA) and ALSA (to G.M.), grants from NINDS (NS23868, NS23320, NS41170 and NS43408), MDA, and ALSA (to S.T.B.), and the Fitz-Thyssen Foundation and DFG (to S.K.).* To whom correspondence should be addressed. Phone: (312) 996−6791. Fax: (312) 413−0354. E-mail: gmorfini@uic.edu.. ‡ These authors contributed equally to this paper. § University of Illinois at Chicago. ∥ University of Heidelberg.1 Abbreviations: KHC, kinesin heavy chain; KLC, kinesin light chain; TR, tandem repeats; PIPES, 1,4-piperazinediethanesulfonic acid; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; GST, glutathione Stransferase; SDS-PAGE, sodium d...

Phosphorylation of kinesin light chain 1 at serine 460 modulates binding and trafficking of calsyntenin-1

“…The functional kinesin-1 tetramer is now known to comprise two homodimers, each containing a single KLC isoform (KLC1 or KLC2); heterotetramers containing both KLC1 and KLC2 do not exist in vivo (DeBoer et al, 2008). Transfection of KLC1wt, KLC1ser460ala or KLC1ser460asp greatly increased KLC1 expression (Fig.…”

“…During the course of our study, others also identified KLC1ser460 as a phosphorylation site through mass spectrometry sequencing of the phosphoproteome (Daub et al, 2008;Dephoure et al, 2008), including in mouse brain (see http://www.phosida.com/). KLC1 and calsyntenin-1 are both enriched in neurons (DeBoer et al, 2008;Hintsch et al, 2002), so we additionally analysed phosphorylation of KLC1ser460 in endogenous KLC1 isolated by immunoprecipitation from cultured rat cortical neurons. Again, we identified KLC1ser460 as a phosphorylated residue (Fig.…”

Mechanisms and functions of lysosome positioning

“…By far the best-characterized kinesin involved in lysosome transport is kinesin-1 (Figs 3 and 4). This kinesin is a heterotetramer composed of two heavy chains (KIF5A, KIF5B or KIF5C) and two light chains (KLC1, KLC2, KLC3 or KLC4) (DeBoer et al, 2008). Kinesin-1 is recruited to lysosomes by a chain of interacting proteins, including the multisubunit BLOC-1-related complex (BORC), the Arf-like small GTPase Arl8 (which has two isoforms in mammals, Arl8a and Arl8b, hereafter generically referred to as Arl8), and the Arl8 effector SifA and kinesin-interacting protein (SKIP, also known as PLEKHM2) (Boucrot et al, 2005;Bagshaw et al, 2006;Hofmann and Munro, 2006;Rosa-Ferreira and Munro, 2011;Pu et al, 2015) (Fig.…”

Tight Functional Coupling of Kinesin-1A and Dynein Motors in the Bidirectional Transport of Neurofilaments

“…In contrast, kinesin-1C knockout mice are viable, although they exhibit loss of some motor neurons and a slight decrease in overall brain size (Kanai et al, 2000). Recent evidence indicates that kinesin-1A, B, and C heavy chains form homodimers (DeBoer et al, 2008).…”

“…In contrast, kinesin-1C knockout mice are viable, although they exhibit loss of some motor neurons and a slight decrease in overall brain size (Kanai et al, 2000). Recent evidence indicates that kinesin-1A, B, and C heavy chains form homodimers (DeBoer et al, 2008).Additional evidence that kinesin-1 is a motor for neurofilaments has come from studies on kinesin-1A knockout mice by Goldstein and colleagues (Xia et al, 2003). The conventional knockouts (type I deletion) died shortly after birth, so the authors created conditional knockouts (type II deletion) by using a Cre-lox strategy.…”