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.     

The roadblock light chain binds a novel region of the cytoplasmic Dynein intermediate chain

Cytoplasmic dynein is the major minus-end directed microtubule-based motor in eukaryotic cells. It is composed of a number of different subunits including three light chain families: Tctex1, LC8, and roadblock. The incorporation of the roadblock light chains into the cytoplasmic dynein complex had not been determined. There are two roadblock genes in mammals, ROBL-1 and ROBL-2. We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein. This was determined with three complementary approaches. A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from the IC74-1 and IC74-2 genes in vivo. This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay. The roadblock-binding domain on the intermediate chain was mapped to an approximately 72 residue region. The binding domain is downstream of each of the two alternative splice sites in the intermediate chains. This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform. In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain.     

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.     

Identification and developmental regulation of a neuron-specific subunit of cytoplasmic dynein.

Cytoplasmic dynein is the microtubule minus-end-directed motor for the retrograde axonal transport of membranous organelles. Because of its similarity to the intermediate chains of flagellar dynein, the 74-kDa intermediate chain (IC74) subunit of dynein is thought to be involved in binding dynein to its membranous organelle cargo. Previously, we identified six isoforms of the IC74 cytoplasmic dynein subunit in the brain. We further demonstrated that cultured glia and neurons expressed different dynein IC74 isoforms and phospho-isoforms. Two isoforms were observed when dynein from glia was analyzed. When dynein from cultured neurons was analyzed, six IC74 isoforms were observed, although the relative amounts of the dynein isoforms from cultured neurons differed from those found in dynein from brain. To better understand the role of the neuronal IC74 isoforms and identify neuron-specific IC74 dynein subunits, the expression of the IC74 protein isoforms and mRNAs of various tissues were compared. As a result of this comparison, the identity of each of the isoform spots observed on two-dimensional gels was correlated with the products of each of the IC74 mRNAs. We also found that between the fifteenth day of gestation (E15) and the fifth day after birth (P5), the relative expression of the IC74 protein isoforms changes, demonstrating that the expression of IC74 isoforms is developmentally regulated in brain. During this time period, there is relatively little change in the abundance of the various IC74 mRNAs. The E15 to P5 time period is one of rapid process extension and initial pattern formation in the rat brain. This result indicates that the changes in neuronal IC74 isoforms coincide with neuronal differentiation, in particular the extension of processes. This suggests a role for the neuronal IC74 isoforms in the establishment or regulation of retrograde axonal transport.     

The 78,000 M(r) intermediate chain of Chlamydomonas outer arm dynein isa WD-repeat protein required for arm assembly

We have isolated and sequenced a full-length cDNA clone encoding the 78,000 Mr intermediate chain (IC78) of the Chlamydomonas outer arm dynein. This protein previously was shown to be located at the base of the solubilized dynein particle and to interact with alpha tubulin in situ, suggesting that it may be involved in binding the outer arm to the doublet microtubule. The sequence predicts a polypeptide of 683 amino acids having a mass of 76.5 kD. Sequence comparison indicates that IC78 is homologous to the 69,000 M(r) intermediate chain (IC69) of Chlamydomonas outer arm dynein and to the 74,000 M(r) intermediate chain (IC74) of cytoplasmic dynein. The similarity between the chains is greatest in their COOH-terminal halves; the NH(2)-terminal halves are highly divergent. The COOH-terminal half of IC78 contains six short imperfect repeats, termed WD repeats, that are thought to be involved in protein-protein interactions. Although not previously reported, these repeated elements also are present in IC69 and IC74. Using the IC78 cDNA as a probe, we screened a group of slow-swimming insertional mutants and identified one which has a large insertion in the IC78 gene and seven in which the IC78 gene is completely deleted. Electron microscopy of three of these IC78 mutants revealed that each is missing the outer arm, indicating that IC78 is essential for arm assembly or attachment to the outer doublet. Restriction fragment length polymorphism mapping places the IC78 gene on the left arm of chromosome XII/XIII, at or near the mutation oda9, which also causes loss of the outer arm. Mutants with defects in the IC78 gene do not complement the oda9 mutation in stable diploids, strongly suggesting that ODA9 is the structural gene for IC78.     

Structure and dynamics of the homodimeric dynein light chain km23

km23 (96 residues, 11 kDa) is the mammalian ortholog of Drosophila roadblock, the founding member of LC7/robl/km23 class of dynein light chains. km23 has been shown to be serine-phosphorylated following TGFbeta receptor activation and to bind the dynein intermediate chain in response to such phosphorylation. Here, we report the three-dimensional solution structure of km23, which is shown to be that of a homodimer, similar to that observed for the heterodimeric complex formed between p14 and MP1, two distantly related members of the MglB/robl superfamily, but distinct from the LC8 and Tctex-1 classes of dynein light chains, which also adopt homodimeric structures. The conserved surface residues of km23, including three serine residues, are located predominantly on a single face of the molecule. Adjacent to this face is a large cleft formed by the incomplete overlap of loops from opposite monomers. As shown by NMR relaxation data collected at two fields, several cleft residues are flexible on the ns-ps and ms-mus timescales. Based on these observations, we propose that the patch of conserved residues on the central face of the molecule corresponds to the site at which km23 binds the dynein intermediate chain and that the flexible cleft formed between the overlap of loops from the two monomers corresponds to the site at which km23 binds other partners, such as the TGFbeta type II receptor or Smad2.     

The crystal structure of mouse Nup35 reveals atypical RNP motifs and novel homodimerization of the RRM domain

The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.     

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.     

Structure of the PIN/LC8 dimer with a bound peptide.

The structure of the protein known both as neuronal nitric oxide synthase inhibitory protein, PIN (protein inhibitor of nNOS), and also as the 8 kDa dynein light chain (LC8) has been solved by X-ray diffraction. Two PIN/LC8 monomers related by a two-fold axis form a rectangular dimer. Two pairs of alpha-helices cover opposite faces, and each pair of helices packs against a beta-sheet with five antiparallel beta-strands. Each five-stranded beta-sheet contains four strands from one monomer and a fifth strand from the other monomer. A 13-residue peptide from nNOS is bound to the dimer in a deep hydrophobic groove as a sixth antiparallel beta-strand. The structure provides key insights into dimerization of and peptide binding by the multifunctional PIN/LC8 protein.     

Characterization of the human dynein light chain Rp3 and its use as a non-viral gene delivery vector

Dynein light chains mediate the interaction between the cargo and the dynein motor complex during retrograde microtubule-mediated transport in eukaryotic cells. In this study, we expressed and characterized the recombinant human dynein light chain Rp3 and developed a modified variant harboring an N-terminal DNA-binding domain (Rp3-Db). Our approach aimed to explore the retrograde cell machinery based on dynein to enhance plasmid DNA (pDNA) traffic along the cytosol toward the nucleus. In the context of non-viral gene delivery, Rp3-Db is expected to simultaneously interact with DNA and dynein, thereby enabling a more rapid and efficient transport of the genetic material across the cytoplasm. We successfully purified recombinant Rp3 and obtained a low-resolution structural model using small-angle X-ray scattering. Additionally, we observed that Rp3 is a homodimer under reducing conditions and remains stable over a broad pH range. The ability of Rp3 to interact with the dynein intermediate chain in vitro was also observed, indicating that the recombinant Rp3 is correctly folded and functional. Finally, Rp3-Db was successfully expressed and purified and exhibited the ability to interact with pDNA and mediate the transfection of cultured HeLa cells. Rp3-Db was also capable of interacting in vitro with dynein intermediate chains, indicating that the addition of the N-terminal DNA-binding domain does not compromise its function. The transfection level observed for Rp3-Db is far superior than that reported for protamine and is comparable to that of the cationic lipid Lipofectamine^TM. This report presents an initial characterization of a non-viral delivery vector based on the dynein light chain Rp3 and demonstrates the potential use of modified human light chains as gene delivery vectors.     

Multivalency in the Assembly of Intrinsically Disordered Dynein Intermediate Chain

Dynein light chains are thought to increase binding efficiency of dynein intermediate chain to both dynein heavy chain and dynactin, but their exact role is not clear. Isothermal titration calorimetry and x-ray crystallography reported herein indicate that multivalency effects underlie efficient dynein assembly and regulation. For a ternary complex of a 60-amino acid segment of dynein intermediate chain (IC) bound to two homodimeric dynein light chains Tctex1 and LC8, there is a 50-fold affinity enhancement for the second light chain binding. For a designed IC construct containing two LC8 sites, observed the 1000-fold enhancement reflects a remarkably pure entropic chelate effect of a magnitude commensurate with theoretical predictions. The lower enhancement in wild-type IC is attributed to unfavorable free energy changes associated with incremental interactions of IC with Tctex1. Our results show assembled dynein IC as an elongated, flexible polybivalent duplex, and suggest that polybivalency is an important general mechanism for constructing stable yet reversible and functionally versatile complexes.     

Transient Tertiary Structures of Disordered Dynein Intermediate Chain Regulate its Interactions with Multiple Partners

The N-terminal domain of dynein intermediate chain (N–IC) is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and regulation of motor activity. It is a prototypical intrinsically disordered protein (IDP), serving as a primarily disordered polybivalent molecular scaffold for numerous binding partners, including three dimeric dynein light chains and coiled coil domains of dynein partners dynactin p150^Glued and NudE. At the very N-terminus, a 40 amino acid single alpha helix (SAH) forms the major binding site for both p150^Glued and NudE, while a shorter nascent helix (H2) separated from SAH by a disordered linker, is necessary for tight binding to dynactin p150^Glued but not to NudE. Here we demonstrate that transient tertiary interactions in this highly dynamic protein underlie the differences in its interactions with p150^Glued and NudE. NMR paramagnetic relaxation enhancement experiments and restrained molecular dynamics simulations identify interactions between the two non-contiguous SAH and H2 helical regions, the extent of which correlates with the length and stability of H2, showing clearly that tertiary and secondary structure formation are coupled in IDPs. These interactions are significantly attenuated when N–IC is bound to NudE, suggesting that NudE binding shifts the conformational ensemble to one that is more extended and with less structure in H2. While the intrinsic disorder and flexibility in N–IC modulate its ability to serve as a binding platform for numerous partners, deviations of this protein from random-coil behavior provide a process for regulating these binding interactions and potentially the dynein motor.     

Tumor suppressor REIC/Dkk-3 interacts with the dynein light chain, Tctex-1

REIC/Dkk-3 is a member of the Dickkopf family proteins known as Wnt-antagonists, and REIC/Dkk-3 expression is downregulated in a broad range of cancer types. REIC/Dkk-3 acts as a tumor suppressor in multiple cancer cell lines by inducing apoptosis through endoplasmic reticulum (ER) stress signaling. However, the intracellular interaction partners of REIC/Dkk-3 have not been fully elucidated. By employing yeast two-hybrid screening, we identified the human dynein light chain, Tctex-1, as a novel interaction partner of REIC/Dkk-3. We further disclosed that the interaction involves the 136–157 amino acid region of REIC/Dkk-3 by using the mammalian two-hybrid system. Interestingly, this binding region of REIC/Dkk-3 with Tctex-1 contains an amino acid sequence motif [-E-X-G-R-R-X-H-] which was previously reported as the Tctex-1 binding domain of dynein intermediate chain (DIC). Immunocytochemistry demonstrated that both REIC/Dkk-3 and Tctex-1 were localized around the ER of human fibroblasts, and the similar distribution pattern of the proteins suggests that their interaction occurs around the ER. This is the first study showing the interaction of a Dickkopf family protein with a dynein motor complex protein. The link between REIC/Dkk-3 and Tctex-1 may be of significance for understanding the molecular functions of the proteins in ER stress signaling and intracellular dynein motor dynamics, respectively.     

Structure and dynamics of LC8 complexes with KXTQT-motif peptides: swallow and dynein intermediate chain compete for a common site

The dynein light chain LC8 is an integral subunit of the cytoplasmic dynein motor complex that binds directly to and promotes assembly of the dynein intermediate chain (IC). LC8 interacts also with a variety of putative dynein cargo molecules such as Bim, a proapoptotic Bcl2 family protein, which have the KXTQT recognition sequence and neuronal nitric oxide synthase (nNOS), which has the GIQVD fingerprint but shares the same binding grooves at the LC8 dimer interface. The work reported here investigates the interaction of LC8 with IC and a putative cargo, Swallow, which share the KXTQT recognition sequence, and addresses the apparent paradox of how LC8, as part of dynein, mediates binding to cargo. The structures of Drosophila LC8 bound to peptides from IC and Swallow solved by X-ray diffraction show that the IC and Swallow peptides bind in the same grooves at the dimer interface. Differences in flexibility between bound and free LC8 were evaluated from hydrogen isotope exchange experiments using heteronuclear NMR spectroscopy. Peptide binding causes an increase in protection from exchange primarily in residues that interact directly with the peptide, such as the beta-strand intertwined at the interface and the N-terminal end of helix alpha2. There is considerably more protection upon Swallow binding, consistent with tighter binding relative to IC. Comparison with the LC8/nNOS complex shows how both the GIQVD and KXTQT fingerprints are recognized in the same groove. The similar structures of LC8/IC and LC8/Swa and the tighter binding of Swallow call into question the role for LC8 as a cargo adaptor protein, and suggest that binding of LC8 to Swallow serves another function, possibly that of a dimerization engine, which is independent of its role in dynein.     

Is outer arm dynein intermediate chain 1 multifunctional?

The outer arm dynein of sea urchin sperm axoneme contains three intermediate chains (IC1, IC2, and IC3; M(r) 128,000, 98,000, and 74,000, respectively). IC2 and IC3 are members of the WD family; the WD motif is responsible for a protein-protein interaction. We describe here the molecular cloning of IC1. IC1 has a unique primary structure, the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic acid residues. Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be a multifunctional protein. The thioredoxin-related part of IC1 is more closely related to those of two redox-active Chlamydomonas light chains than thioredoxin. Antibodies were prepared against the N-terminal and middle domains of IC1 expressed as His-tagged proteins in bacteria. These antibodies cross-reacted with some dynein polypeptides (potential homologues of IC1) from distantly related species. We propose here that the three intermediate chains are the basic core units of sperm outer arm dynein because of their ubiquitous existence. The recombinant thioredoxin-related part of IC1 and outer arm dyneins from sea urchin and distantly related species were specifically bound to and eluted from a phenylarsine oxide affinity column with 2-mercaptoethanol, indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction.     

Structure of Tctex-1 and its interaction with cytoplasmic dynein intermediate chain

The minus-ended microtubule motor cytoplasmic dynein contains a number of low molecular weight light chains including the 14-kDa Tctex-1. The assembly of Tctex-1 in the dynein complex and its function are largely unknown. Using partially deuterated, (15)N,(13)C-labeled protein samples and transverse relaxation-optimized NMR spectroscopic techniques, the secondary structure and overall topology of Tctex-1 were determined based on the backbone nuclear Overhauser effect pattern and the chemical shift values of the protein. The data showed that Tctex-1 adopts a structure remarkably similar to that of the 8-kDa light chain of the motor complex (DLC8), although the two light chains share no amino acid sequence homology. We further demonstrated that Tctex-1 binds directly to the intermediate chain (DIC) of dynein. The Tctex-1 binding site on DIC was mapped to a 19-residue fragment immediately following the second alternative splicing site of DIC. Titration of Tctex-1 with a peptide derived from DIC, which contains a consensus sequence R/KR/KXXR/K found in various Tctex-1 target proteins, indicated that Tctex-1 binds to its targets in a manner similar to that of DLC8. The experimental results presented in this study suggest that Tctex-1 is likely to be a specific cargo adaptor for the dynein motor complex.     

Cautionary tail: the presence of an N-terminal tag on dynein light-chain Roadblock/LC7 affects its interaction with a functional partner

As part of structural investigations of components of the molecular motor, dynein, we prepared the light chain, Robl1_mouse, with and without an N-terminal His-tag. We found that the His-tag introduced a spurious binding site for a second protein, IC74. We propose a molecular mechanism for functional interference by the His-tag.     

Intrinsic disorder in dynein intermediate chain modulates its interactions with NudE and dynactin

The functional diversity of cytoplasmic dynein is in part attributed to multiple interactions between noncatalytic dynein subunits and an array of regulatory proteins. This study focuses on the interaction between the dynein intermediate chain subunit (IC) and a dynein regulator protein (NudE). We use isothermal titration calorimetry and NMR spectroscopy to map their interacting sections to their respective N-terminal domains, which are predicted to form dimeric coiled-coils. Interestingly, the specific residues within IC that interact with NudE are a subset of the bi-segmental binding region reported for p150(Glued), a subunit of the dynein activator protein dynactin. Although the IC binding domains of both NudE and p150(Glued) form dimeric coiled-coils and bind IC at a common site, we observe distinct binding modes for each regulatory protein: 1) NudE binds region 1 of the bi-segmental binding footprint of p150(Glued), whereas p150(Glued) requires regions 1 and 2 to match the binding affinity of NudE with region 1 alone. 2) Compared with unbound IC, NudE-bound IC shows a slight increase in flexibility in region 2, in contrast to the increase in ordered structure observed for p150(Glued)-bound IC (Morgan, J. L., Song, Y., and Barbar, E. (2011) J. Biol. Chem. 286, 39349-39359). 3) Although NudE has a higher affinity for the common binding segment on IC, when all three proteins are in solution, IC preferentially binds p150(Glued). These results underscore the importance of a bi-segmental binding region of IC and disorder in region 2 and flanking linkers in selecting which regulatory protein binds IC.     

Structural dynamics and multiregion interactions in dynein-dynactin recognition

Cytoplasmic dynein is a 1.2-MDa multisubunit motor protein complex that, together with its activator dynactin, is responsible for the majority of minus end microtubule-based motility. Dynactin targets dynein to specific cellular locations, links dynein to cargo, and increases dynein processivity. These two macromolecular complexes are connected by a direct interaction between dynactin's largest subunit, p150(Glued), and dynein intermediate chain (IC) subunit. Here, we demonstrate using NMR spectroscopy and isothermal titration calorimetry that the binding footprint of p150(Glued) on IC involves two noncontiguous recognition regions, and both are required for full binding affinity. In apo-IC, the helical structure of region 1, the nascent helix of region 2, and the disorder in the rest of the chain are determined from coupling constants, amide-amide sequential NOEs, secondary chemical shifts, and various dynamics measurements. When bound to p150(Glued), different patterns of spectral exchange broadening suggest that region 1 forms a coiled-coil and region 2 a packed stable helix, with the intervening residues remaining disordered. In the 150-kDa complex of p150(Glued), IC, and two light chains, the noninterface segments remain disordered. The multiregion IC binding interface, the partial disorder of region 2 and its potential for post-translational modification, and the modulation of the length of the longer linker by alternative splicing may provide a basis for elegant and multifaceted regulation of binding between IC and p150(Glued). The long disordered linker between the p150(Glued) binding segments and the dynein light chain consensus sequences could also provide an attractive recognition platform for diverse cargoes.     

Mutually exclusive cytoplasmic dynein regulation by NudE-Lis1 and dynactin

Cytoplasmic dynein is responsible for a wide range of cellular roles. How this single motor protein performs so many functions has remained a major outstanding question for many years. Part of the answer is thought to lie in the diversity of dynein regulators, but how the effects of these factors are coordinated in vivo remains unexplored. We previously found NudE to bind dynein through its light chain 8 (LC8) and intermediate chain (IC) subunits (1), the latter of which also mediates the dynein-dynactin interaction (2). We report here that NudE and dynactin bind to a common region within the IC, and compete for this site. We find LC8 to bind to a novel sequence within NudE, without detectably affecting the dynein-NudE interaction. We further find that commonly used dynein inhibitory reagents have broad effects on the interaction of dynein with its regulatory factors. Together these results reveal an unanticipated mechanism for preventing dual regulation of individual dynein molecules, and identify the IC as a nexus for regulatory interactions within the dynein complex.