Dimerization of the Highly Conserved Light Chain Shared by Dynein and Myosin V

Abstract

The M r 8,000 light chain originally identified in Chlamydomonas flagellar dynein is also a component of both cytoplasmic dynein and myosin V. Furthermore, this small protein has been implicated as an inhibitor of neuronal nitric oxide synthase, suggesting that it may play multiple regulatory roles within the cell. Covalent cross-linking of both dynein and myosin V using 1,5-difluoro-2,4-dinitrobenzene revealed that this light chain exists as a dimer in situ. This observation was confirmed using two additional amine-selective crosslinking reagents (dimethyl pimelimidate and disuccinimidyl suberate). When expressed as a C-terminal fusion with maltose-binding protein, the presence of the light chain caused the recombinant molecule to dimerize. Analysis of fusions containing truncated light chains identified the predicted amphiphilic helix (residues 14 -32) as sufficient to cause dimerization; cross-linking required a second helical segment (residues 33-46). Together the data presented suggest that two light chains interact to form a parallel dimeric structure. This arrangement has significant implications for the potential functions of this highly conserved molecule and suggests a mechanism by which it might dissociate nitric oxide synthase.Dyneins are highly complex molecular motors that translocate the cargo to which they are attached along microtubules (see Refs. 1 and 2 for review). Molecular analysis of components from both cytoplasmic and flagellar outer arm dyneins has revealed that these microtubule motors share a M r 8,000 polypeptide (actual mass ϭ 10.3 kDa) that has been very highly conserved throughout evolution (ϳ90% sequence identity between the Chlamydomonas and human proteins) (3, 4). In Drosophila, partial loss-of-function mutations in this protein lead to morphogenetic defects in bristle and wing development, female sterility, and disruption of sensory axon trajectories; total loss induces apoptosis and causes embryonic lethality (5, 6). Mutation of the homologous protein in Aspergillus results in a nuclear migration phenotype similar to that observed with dynein heavy chain null mutants (7).Intriguingly, fractionation of whole mammalian brain extracts revealed at least three biochemically distinct pools of this protein: (i) non-microtubule-associated; (ii) microtubuleassociated, eluted with ATP or salt; and (iii) microtubule-associated, not salt-extractable (4). Only the second pool was cytoplasmic dynein-associated, and indeed, the predominant form of this molecule (pool (i)) did not cosediment with microtubules. Likewise, this polypeptide is associated with at least two distinct components of the Chlamydomonas flagellar axoneme. There are 8 -10 copies of this protein within the outer dynein arm (8); this fraction is absent in axonemes derived from mutants that lack this structure (9), and it may be extracted from wild-type axonemes with 0.6 M NaCl. The second flagellar fraction, which accounts for ϳ50% of the total axonemal M r 8,000 LC, 1 is tightly integrated into the axonemal superstructure...

DYNLL/LC8 Protein Controls Signal Transduction through the Nek9/Nek6 Signaling Module by Regulating Nek6 Binding to Nek9

“…2B). LC8 alone showed a molecular mass of ϳ20 kDa, corresponding to a dimer and consistent with published data (39). Nek9 , with a predicted molecular mass of ϳ10 kDa, oligomerized with an apparent molecular mass of ϳ50 kDa.…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

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Dynein Light Chain 1 Regulates Dynamin-mediated F-Actin Assembly during Sperm Individualization inDrosophila

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