Light chains of mammalian cytoplasmic dynein: identification and characterization of a family of LC8 light chains.

Cytoplasmic dynein is a large multisubunit motor protein that moves various cargoes toward the minus ends of microtubules. In addition to the previously identified heavy, intermediate, and light intermediate chains, it has recently been recognized that cytoplasmic dynein also has several light chain subunits with apparent molecular weights between 8-20 kDa. To systematically identify the light chains of purified rat brain cytoplasmic dynein, peptide sequences were obtained from each light chain band resolved by gel electrophoresis. Both members of the tctex1 light chain family, tctex1 and rp3, were identified in a single band. Only one member of the roadblock family, roadblock-2, was found. Two members of the LC8 family were resolved as separate bands, the previously identified LC8 subunit, and a second novel cytoplasmic dynein family member, LC8b. The tissue distribution of these two dynein LC8 subunits differed, although LC8b was the major family member in brain. Database searches found that both LC8a and LC8b were also present in several mammalian species, and a third mammalian LC8 sequence, LC8c was found in the human database. The amino acid sequences of both LC8a and LC8b were completely conserved in mammals. LC8a and LC8b differ in only six of the 89 amino acids. The amino acid differences between LC8a and LC8b were located near the N-terminus of the molecules, and most were in the outward facing alpha-helices of the LC8 dimer. When the mammalian LC8a sequence was compared to the LC8 sequences found in six other animal species including Xenopus and Drosophila, there was, on average, 94% sequence identity. More variation was found in LC8 sequences obtained from plants, fungi, and parasites. LC8c differed from the other two human LC8 sequences in that it has amino acid substitutions in the intermediate chain binding domain at the C-terminal of the molecule. The position of amino acid substitutions of the three mammalian LC8 family members is consistent with the hypothesis that they bind to different proteins.     

Dimerization and folding of LC8, a highly conserved light chain of cytoplasmic dynein

Cytoplasmic dynein is a multisubunit ATPase that transforms chemical energy into motion along microtubules. LC8, a 10 kDa light chain subunit of the dynein complex, is highly conserved with 94% sequence identity between Drosophila and human. The precise function of this protein is unknown, but its ubiquitous expression and conservation suggest a critical role in the function of the dynein motor complex. We have overexpressed LC8 from Drosophila melanogaster and characterized its dimerization and folding using analytical ultracentrifugation, size-exclusion chromatography, circular dichroism, and fluorescence spectroscopy. Sedimentation equilibrium measurements of LC8 at pH 7 reveal a reversible monomer-dimer equilibrium with a dissociation constant of 12 microM at 4 degrees C. At lower pH, LC8 dissociates to a monomer, with a transition midpoint at pH 4.8. Far-UV CD and fluorescence spectra demonstrate that pH-dissociated LC8 retains native secondary and tertiary structures, while the diminished near-UV CD signal shows loss of quaternary structure. The observation that dimeric LC8 dissociates at low pH can be explained by titration of a histidine pair in the dimer interface. Equilibrium denaturation experiments with a protein concentration range spanning almost 2 orders of magnitude indicate that unfolding of LC8 dimer is a two-stage process, in which global unfolding is preceded by dissociation to a folded monomer. The nativelike tertiary structure of the monomer suggests a role for the monomer-dimer equilibrium of LC8 in dynein function.     

Interactions of cytoplasmic dynein light chains Tctex-1 and LC8 with the intermediate chain IC74

The interactions of three subunits of cytoplasmic dynein from Drosophila melanogaster, LC8, Tctex-1, and the N-terminal domain of IC74 (N-IC74, residues 1-289), were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. These subunits were chosen for study because they are presumed to promote the assembly of the complex and to be engaged in the controlled binding and release of cargo. Limited proteolysis and mass spectrometry of N-IC74 in the presence of LC8 and Tctex-1 localized binding of Tctex-1 to the vicinity of K104 and K105, and localized binding of LC8 to the region downstream of K130. Circular dichroism, fluorescence, sedimentation velocity, and proteolysis studies indicate that N-IC74 has limited secondary and tertiary structure at near physiological solution conditions. Upon addition of LC8, N-IC74 undergoes a significant conformational change from largely unfolded to a more ordered structure. This conformational change is reflected in increased global protection of N-IC74 from proteolytic digestion following the interaction, and in a significant change in the CD signal. A smaller but reproducible change in the CD spectra was observed upon Tctex-1 binding as well. The increased structure introduced into N-IC74 upon light chain binding suggests a mechanism by which LC8 and Tctex-1 may regulate the assembly of the dynein complex.     

Dephosphorylation of Tctex2-related dynein light chain by type 2A protein phosphatase

Maspin is a 42kDa tumor suppressor protein that belongs to the serine protease inhibitor (serpin) family. It inhibits cell motility and invasion in vitro, and tumor growth and metastasis in nude mice; however, maspin's molecular mechanism of action has remained elusive. Maspin contains several tyrosine residues and we hypothesized that phosphorylation of maspin could play a role in its biological function. Our study reveals that maspin is phosphorylated on tyrosine moiety(ies) in normal mammary epithelial cells endogenously expressing maspin. In addition, transfection of the maspin gene, using either a stable or inducible system into maspin-deficient breast cancer cell lines, yields a protein product that is phosphorylated on tyrosine residue(s). Furthermore, recombinant maspin protein can be tyrosine-phosphorylated by the kinase domain from the epidermal growth factor receptor in vitro. These novel observations suggest that maspin, which deviates from the classical serpin, may be an important signal transduction molecule in its phosphorylated form.     

Herpes simplex virus type 1 capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular transport

Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. There is increasing evidence that the retrograde transport of herpes simplex virus type 1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Here we report that the herpes simplex virus outer capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and is sufficient to mediate retrograde transport of viral capsids in a cellular model. A library of herpes simplex virus capsid and tegument structural genes was constructed and tested for interactions with dynein subunits in a yeast two-hybrid system. A strong interaction was detected between VP26 and the homologous 14-kDa dynein light chains RP3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to RP3, Tctex1, and intact cytoplasmic dynein complexes. Recombinant herpes simplex virus capsids were constructed either with or without VP26. In pull-down assays VP26+ capsids bound to RP3; VP26-capsids did not. To investigate intracellular transport, the recombinant viral capsids were microinjected into living cells and incubated at 37 degrees C. After 1 h VP26+ capsids were observed to co-localize with RP3, Tctex1, and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, whereas VP26-capsids remained in a random distribution. We propose that VP26 mediates binding of incoming herpes simplex virus capsids to cytoplasmic dynein during cellular infection, through interactions with dynein light chains.     

Voltage-dependent anion-selective channel (VDAC) interacts with the dynein light chain Tctex1 and the heat-shock protein PBP74

The voltage-dependent anion-selective channel 1 (VDAC1), i.e. eukaryotic porin, functions as a channel in membranous structures as described for the outer mitochondrial membrane, the cell membrane, endosomes, caveolae, the sarcoplasmatic reticulum, synaptosomes, and post-synaptic density fraction.The identification of VDAC1 interacting proteins may be a promising approach for better understanding the biological context and function of the channel protein. In this study human VDAC1 was used as a bait protein in a two-hybrid screening, which is based on the Sos recruitment system (SRS). hVDAC1 interacts with the dynein light chain Tctex-1 and the heat-shock protein peptide-binding protein 74 (PBP74)/mitochondrial heat-shock protein 70 (mtHSP70)/glucose-regulated protein 75 (GRP75)/mortalin in vivo. Both interactions were confirmed by overlay-assays using recombinant partner proteins and purified hVDAC1. Indirect immunofluorescence on HeLa cells indicates a co-localisation of hVDAC1 with the dynein light chain and the PBP74. In addition, HeLa cells were transfected transiently with enhanced green fluorescent protein (EGFP)-hVDAC1 fusion proteins, which also clearly co-localise with both proteins. The functional relevance of the identified protein interactions was analysed in planar lipid bilayer (PLB) experiments. In these experiments both recombinant binding partners altered the electrophysiological properties of hVDAC1. While rTctex-1 increases the voltage-dependence of hVDAC1 slightly, the rPBP74 drastically minimises the voltage-dependence, indicating a modulation of channel properties in each case. Since the identified proteins are known to be involved in the transport or processing of proteins, the results of this study represent additional evidence of membrane-associated trafficking of the voltage-dependent anion-selective channel 1.     

Rab6 family proteins interact with the dynein light chain protein DYNLRB1

The small GTPase Rab6 is a key regulator in the retrograde transfer from endosomes via the Golgi to the ER. Three isoforms of Rab6 have been identified, the ubiquitously expressed Rab6A and Rab6A', and the brain specific Rab6B. Recent studies have shown that Rab6A' is the major isoform regulating this retrograde transport. Cytoplasmic dynein is the main motor protein complex for this transport. Dynein consists of two heavy chains, two intermediate chains, four light intermediate chains and several light chains, called roadblock/LC7 proteins or DYNLRB proteins. In mammalian cells two light chain isoforms have been identified, DYNLRB1 and DYNLRB2. We here show with yeast-two-hybrid, co-immunoprecipitation and pull down studies that DYNLRB1 specifically interacts with all three Rab6 isoforms and co-localises at the Golgi. This is the first example of a direct interaction between Rab6 isoforms and the dynein complex. Pull down experiments showed further preferred association of DYNLRB1 with GTP-bound Rab6A and interestingly GDP-bound Rab6A' and Rab6B. In addition DYNLRB1 was found in the Golgi apparatus where it co-localises with EYFP-Rab6 isoforms. DYNLRB is a putative modulator of the intrinsic GTPase activity of GTP-binding proteins. In vitro we were not able to reproduce this effect on Rab6 GTPase activity.     

The light chain composition of chicken brain myosin-Va: calmodulin, myosin-II essential light chains, and 8-kDa dynein light chain/PIN

Class V myosins are a ubiquitously expressed family of actin-based molecular motors. Biochemical studies on myosin-Va from chick brain indicate that this myosin is a two-headed motor with multiple calmodulin light chains associated with the regulatory or neck domain of each heavy chain, a feature consistent with the regulatory effects of Ca(2+) on this myosin. In this study, the identity of three additional low molecular weight proteins of 23-,17-, and 10 kDa associated with myosin-Va is established. The 23- and 17-kDa subunits are both members of the myosin-II essential light chain gene family, encoded by the chicken L23 and L17 light chain genes, respectively. The 10-kDa subunit is a protein originally identified as a light chain (DLC8) of flagellar and axonemal dynein. The 10-kDa subunit is associated with the tail domain of myosin-Va.     

DYNLL/LC8: a light chain subunit of the dynein motor complex and beyond

The LC8 family members of dynein light chains (DYNLL1 and DYNLL2 in vertebrates) are highly conserved ubiquitous eukaryotic homodimer proteins that interact, besides dynein and myosin 5a motor proteins, with a large (and still incomplete) number of proteins involved in diverse biological functions. Despite an earlier suggestion that LC8 light chains function as cargo adapters of the above molecular motors, they are now recognized as regulatory hub proteins that interact with short linear motifs located in intrinsically disordered protein segments. The most prominent LC8 function is to promote dimerization of their binding partners that are often scaffold proteins of various complexes, including the intermediate chains of the dynein motor complex. Structural and functional aspects of this intriguing hub protein will be highlighted in this minireview.     

Proteomic identification of brain proteins that interact with dynein light chain LC8

Cytoplasmic dynein is a large minus end-directed microtubule motor that translocates cargos towards the minus end of microtubules. Light chain 8 of the dynein machinery (LC8) has been reported to interact with a large variety of proteins that possess K/RSTQT or GIQVD motifs in their sequence, hence permitting their transport in a retrograde manner. Yeast two-hybrid analysis has revealed that in brain, LC8 associates directly with several proteins such as neuronal nitric oxide synthase, guanylate kinase domain-associated protein and gephyrin. In this work, we report the identification of over 40 polypeptides, by means of a proteomic approach, that interact with LC8 either directly or indirectly. Many of the neuronal proteins that we identified cluster at the post-synaptic terminal, and some of them such as phosphofructokinase, lactate dehydrogenase or aldolase are directly involved in glutamate metabolism. Other pool of proteins identified displayed the LC8 consensus binding motif. Finally, recombinant LC8 was produced and a library of overlapping dodecapeptides (pepscan) was employed to map the LC8 binding site of some of the proteins that were previously identified using the proteomic approach, hence confirming binding to the consensus binding sites.     

Characterisation of a family of Schistosoma japonicum proteins related to dynein light chains.

Dynein light chains (DLC) are components of dynein, an enzyme complex involved in various aspects of microtubule-based motility. We report here the molecular cloning and sequencing of cDNAs encoding a family of DLC-like polypeptides (SjcDLC1-5) from the human bloodfluke Schistosoma japonicum with open reading frames of 87-104 amino acids and deduced molecular masses ranging from 10.5 to 12.3 kDa. Two-dimensional Western blot analysis confirmed the presence of several S. japonicum DLC isoforms with differing pI values and molecular sizes. We also describe the molecular characterisation, genomic organisation and expression of clone SjcDLC1, and the immunological characterisation and localisation of its encoded protein. Northern blot analysis of adult worm RNA indicated SjcDLC1 is encoded by a single message of approximately 650 bp and Southern analysis suggested one SjcDLC1 gene exists in the S. japonicum genome. Immunolocalisation studies demonstrated that the SjcDLC1 protein is present in the tegument of the adult and cercarial stages of S. japonicum. SjcDLC1 and the other SjcDLC may function in the transport of specialised organelles, comprising membranous and discoid bodies, through the tegument to the schistosome-unique heptalaminate tegumental membrane at the external surface of the adult worm. As a consequence, they may provide novel targets for anti-schistosome vaccine and/or drug development.     

Localization of calmodulin and dynein light chain LC8 in flagellar radial spokes

Genetic and in vitro analyses have revealed that radial spokes play a crucial role in regulation of ciliary and flagellar motility, including control of waveform. However, the mechanisms of regulation are not understood. Here, we developed a novel procedure to isolate intact radial spokes as a step toward understanding the mechanism by which these complexes regulate dynein activity. The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes. Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14. Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8. Analyses of flagellar mutants and chemical cross-linking studies demonstrated calmodulin and LC8 form a complex located in the radial spoke stalk. We postulate that calmodulin, located in the radial spoke stalk, plays a role in calcium control of flagellar bending.     

The Sireviruses, a plant-specific lineage of the Ty1/copia retrotransposons, interact with a family of proteins related to dynein light chain 8

Plant genomes are rich in long terminal repeat retrotransposons, and here we describe a plant-specific lineage of Ty1/copia elements called the Sireviruses. The Sireviruses vary greatly in their genomic organization, and many have acquired additional coding information in the form of an envelope-like open reading frame and an extended gag gene. Two-hybrid screens were conducted with the novel domain of Gag (the Gag extension) encoded by a representative Sirevirus from maize (Zea mays) called Hopie. The Hopie Gag extension interacts with a protein related to dynein light chain 8 (LC8). LC8 also interacts with the Gag extension from a Hopie homolog from rice (Oryza sativa). Amino acid motifs were identified in both Hopie Gag and LC8 that are responsible for the interaction. Two amino acids critical for Gag recognition map within the predicted LC8-binding cleft. Two-hybrid screens were also conducted with the Gag extension encoded by the soybean (Glycine max) SIRE1 element, and an interaction was found with light chain 6 (LC6), a member of the LC8 protein family. LC8 and LC6 proteins are components of the dynein microtubule motor, with LC8 being a versatile adapter that can bind many unrelated cellular proteins and viruses. Plant LC8 and LC6 genes are abundant and divergent, yet flowering plants do not encode other components of the dynein motor. Although, to our knowledge, no cellular roles for plant LC8 family members have been proposed, we hypothesize that binding of LC8 proteins to Gag aids in the movement of retrotransposon virus-like particles within the plant cell or possibly induces important conformational changes in the Gag protein.     

Three members of the LC8/DYNLL family are required for outer arm dynein motor function

The highly conserved LC8/DYNLL family proteins were originally identified in axonemal dyneins and subsequently found to function in multiple enzyme systems. Genomic analysis uncovered a third member (LC10) of this protein class in Chlamydomonas. The LC10 protein is extracted from flagellar axonemes with 0.6 M NaCl and cofractionates with the outer dynein arm in sucrose density gradients. Furthermore, LC10 is specifically missing only from axonemes of those strains that fail to assemble outer dynein arms. Previously, the oda12-1 insertional allele was shown to lack the Tctex2-related dynein light chain LC2. The LC10 gene is located approximately 2 kb from that of LC2 and is also completely missing from this mutant but not from oda12-2, which lacks only the 3' end of the LC2 gene. Although oda12-1 cells assemble outer arms that lack only LC2 and LC10, this strain exhibits a flagellar beat frequency that is consistently less than that observed for strains that fail to assemble the entire outer arm and docking complex (e.g., oda1). These results support a key regulatory role for the intermediate chain/light chain complex that is an integral and highly conserved feature of all oligomeric dynein motors.     

Affinity, avidity, and kinetics of target sequence binding to LC8 dynein light chain isoforms

LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with K(d) values of 9 and 40 μM, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy- and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: K(d) values of 37 and 3.5 nM for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent K(d) value (3 μM). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network.     

Potential role for phosphorylation in differential regulation of the assembly of dynein light chains

The homodimeric light chains LC8 and Tctex-1 are integral parts of the microtubule motor cytoplasmic dynein, as they directly associate with dynein intermediate chain IC and various cellular cargoes. These light chains appear to regulate assembly of the dynein complex by binding to and promoting dimerization of IC. In addition, both LC8 and Tctex-1 play roles in signaling, apoptosis, and neuronal development that are independent of their function in dynein, but it is unclear how these various activities are modulated. Both light chains undergo specific phosphorylation, and here we present biochemical and NMR analyses of phosphomimetic mutants that indicate how phosphorylation may regulate light chain function. For both LC8 and Tctex-1, phosphorylation promotes dissociation from IC while retaining their binding activity with other non-dynein proteins. Although LC8 and Tctex-1 are homologs having a common fold, their reduced affinity for IC upon phosphorylation arises by different mechanisms. In the case of Tctex-1, phosphorylation directly masks the IC binding site at the dimer interface, whereas for LC8, phosphorylation dissociates the dimer and indirectly eliminates the binding site. This modulation of the monomer-dimer equilibrium by phosphorylation provides a novel mechanism for discrimination among LC8 binding partners.     

Solution structure of isoform 1 of Roadblock/LC7, a light chain in the dynein complex

Roadblock/LC7 is a member of a class of dynein light chains involved in regulating the function of the dynein complex. We have determined the three-dimensional structure of isoform 1 of the mouse Roadblock/LC7 cytoplasmic dynein light chain (robl1_mouse) by NMR spectroscopy. In contrast to a previously reported NMR structure of the human homolog with 96% sequence identity (PDB 1TGQ), which showed the protein as a monomer, our results indicate clearly that robl1 exists as a symmetric homodimer. The two beta3-strands pair with each other and form a continuous ten-stranded beta-sheet. The 25-residue alpha2-helix from one subunit packs antiparallel to that of the other subunit on the face of the beta-sheet. Zipper-like hydrophobic contacts between the two helices serve to stabilize the dimer. Through an NMR titration experiment, we localized the site on robl1_mouse that interacts with the 40 residue peptide spanning residues 243 through 282 of IC74-1_rat. These results provide physical evidence for a symmetrical interaction between dimeric robl1 and the two molecules of IC74-1 in the dynein complex.     

LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization

LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1. Thus, our results imply a potential cellular interference between DYNLL1 and ATMIN functions.