Affinity chromatography demonstrates a direct binding between cytoplasmic dynein and the dynactin complex.

We used affinity chromatography to probe for a direct binding interaction between cytoplasmic dynein and dynactin. Purified cytoplasmic dynein was found to bind to an affinity column of p150, the largest polypeptide in the dynactin complex. To test the specificity of the interaction, we loaded rat brain cytosol onto the p150 affinity column and observed that cytoplasmic dynein from cytosol was specifically retained on the column. Preincubation of the p150 affinity matrix with excess exogenous dynein intermediate chain resulted in a significant reduction of dynein binding, suggesting that p150 may be interacting with dynein via this polypeptide. Therefore we constructed an affinity column of recombinant dynein intermediate chain and observed that dynactin was retained from rat brain cytosol. These results demonstrate that the native dynein and dynactin complexes are capable of direct in vitro interaction mediated by a direct binding of the dynein intermediate chain to the p150component of the dynactin complex. We have mapped the site of this interaction to the amino-terminal region of p150, which is predicted to form an α-helical coiled-coil. Regulation of the dynein-dynactin interaction may prove to be key in the control mechanism for cytoplasmic dynein-mediated vesicular transport.

We used affinity chromatography to probe for a direct binding interaction between cytoplasmic dynein and dynactin. Purified cytoplasmic dynein was found to bind to an affinity column of p150 Glued , the largest polypeptide in the dynactin complex. To test the specificity of the interaction, we loaded rat brain cytosol onto the p150 Glued affinity column and observed that cytoplasmic dynein from cytosol was specifically retained on the column. Preincubation of the p150 Glued affinity matrix with excess exogenous dynein intermediate chain resulted in a significant reduction of dynein binding, suggesting that p150 Glued may be interacting with dynein via this polypeptide. Therefore we constructed an affinity column of recombinant dynein intermediate chain and observed that dynactin was retained from rat brain cytosol. These results demonstrate that the native dynein and dynactin complexes are capable of direct in vitro interaction mediated by a direct binding of the dynein intermediate chain to the p150 Glued component of the dynactin complex. We have mapped the site of this interaction to the amino-terminal region of p150 Glued , which is predicted to form an ␣-helical coiled-coil. Regulation of the dynein-dynactin interaction may prove to be key in the control mechanism for cytoplasmic dyneinmediated vesicular transport.
Coordinated trafficking of organelles along microtubules is central to the viability of cell and is powered by the mechanochemical ATPases kinesin and cytoplasmic dynein. While the mechanisms which govern the specificity and regulation of this transport remain to be determined, there is growing evidence for the role of accessory factors in the function of the molecular motors involved. Recently, an integral membrane protein, kinectin, was found to be the essential anchor for kinesin-driven vesicle motility (1,2). Although no membrane receptor for cytoplasmic dynein has been described yet, a distinct 20 S complex, dynactin, was shown to differentially co-purify with cytoplasmic dynein from a variety of sources (3)(4)(5). Also, in an in vitro motility assay the dynactin complex was required to reconstitute dynein-mediated vesicular motility (6). These observations suggest that dynactin may interact with cytoplasmic dynein transiently or in a regulatory manner. However, the mechanism of interaction is not clearly understood.
Dynactin is a macromolecular oligomeric complex of at least 10 different polypeptides (6 -8). The two best characterized components of the dynactin complex are p150 Glued and a 45-kDa protein, centractin. cDNA cloning and amino acid sequence analysis revealed that rat p150 Glued is 32% identical to the product of the Drosophila gene Glued (5,9). It has been shown previously that the null mutation of Glued is embryonic lethal (10), thus suggesting that the p150 Glued polypeptide has a role in an essential cell function such as mitosis or vesicular transport. Centractin (also known as Arp1) 1 is a novel form of actin that shares ϳ50% identity to human ␣-actin (11,12). Analysis of the dynactin complex by immunoelectron microscopy revealed short actin-like filaments, most likely formed from the polymerization of centractin (8). Polypeptides of 135, 62, 50, 42, 37, 32, 27, and 24 kDa are also thought to be components of the dynactin complex; the 42-kDa polypeptide has been identified as conventional actin, and the 37-and 32-kDa polypeptides are the ␣ and ␤ subunits of CapZ (capping protein) (7,8).
A number of genetic studies have provided indirect evidence for an in vivo interaction between cytoplasmic dynein and dynactin in that mutations in components of either of the complexes give rise to similar phenotypes. In Neurospora crassa, mutant alleles which result in curled hyphal growth and altered nuclear distribution have been shown to encode polypeptides with homology to the cytoplasmic dynein heavy chain (DHC), p150 Glued and centractin (13). In S. cerevisiae, mutations in a putative centractin homologue result in defects in spindle orientation and nuclear migration which are similar to the phenotype observed in dynein heavy chain mutants (14 -16, 26). In Drosophila, it has been reported that certain Dhc64C (cytoplasmic dynein heavy chain) mutations can act as dominant suppressors or enhancers of the Glued phenotype (36). Taken together, these observations suggest that dynein and dynactin interact in vivo or affect the same cellular processes.
In order to investigate the hypothesis that there is a direct biochemical interaction between cytoplasmic dynein and the dynactin complex, we constructed affinity columns of p150 Glued or dynein intermediate chain (DIC), then passed over preparations of purified dynein or of whole rat brain cytosol and looked for specific retention of cytoplasmic dynein on a p150 Glued affinity column or of the dynactin complex on a DIC affinity column. The results indicate that the cytoplasmic dynein complex interacts with the dynactin complex via a direct binding of the dynein intermediate chain to p150 Glued component of the dynactin complex. This direct binding between cytoplasmic dynein and dynactin provides evidence in support of involvement of an accessory factor in dynein function. These results also suggest that modulation of the dynein-dynactin interaction in vivo may be a key step in the mechanism of regulation of cytoplasmic dynein-mediated organelle trafficking within the cell.

MATERIALS AND METHODS
Cytosol and Dynein Preparation from Rat Brain-Brain cytosol was prepared by the high speed centrifugation (150,000 ϫ g, 45 min) of brain homogenate and clarified using a 0.45-m filter. A conservative estimate of dynein in the cytosol, assuming that roughly 50% purifies by the method of Paschal et al. (18) is ϳ20 g/ml. Cytoplasmic dynein from rat brain was purified essentially as described by Paschal et al. (17,18), using a 1:1 ratio (w/v) of brain to homogenization buffer (50 mM Na-PIPES, 50 mM Na-HEPES, 1 mM EDTA, 2 mM MgCl 2 , pH 6.9). The 20 S peak fractions from the sucrose density gradients which comprised the final step of the purification contained both the dynein and dynactin complexes (5). These fractions were kept on ice and used within 3 days. The sucrose in the peak fractions from the gradients was removed using a gel filtration column (PD-10; Pharmacia Biotech Inc.) before use.
Expression and Purification of Ligand-cDNAs encoding amino acids 133-899 of rat p150 Glued (5) and full-length rat DIC (19) were subcloned into the bacterial expression vectors pET-15B and pET-14B, respectively (Novagen). The resulting constructs are predicted to produce recombinant proteins with 6 histidine residues fused in frame to the amino termini when expressed in Escherichia coli. Following induction of the transformed bacterial cultures with isopropyl-1-thio-␤-D-galactopyranoside, the cells were harvested and resuspended and lysed in buffer B (0.1 M NaH 2 PO 4 , 0.01 M Tris, pH 8.0) containing 8 M urea. The resulting lysate was centrifuged (18 K, SS-34, Sorvall) to remove cell debris, and the supernatant was loaded on a Ni ϩ2 column (Qiagen). The column was extensively washed in buffer B containing 20 mM imidazole, pH 6.3, with decreasing concentrations of urea to allow proper refolding of proteins and was then eluted with 500 mM imidazole in buffer B. The eluate was dialyzed against Sepharose 4B coupling buffer (100 mM NaHCO 3 , 500 mM NaCl, pH 8.0).
Construction of Affinity Columns-Activated CH-Sepharose 4B (Pharmacia) beads were swollen and washed in 1 mM HCl followed by washes in coupling buffer (100 mM NaHCO 3 , 500 mM NaCl, pH 8.0). The beads were mixed gently with the ligand at a minimum protein concentration of 0.2 mg/ml overnight at 4°C. Excess active groups on the beads were blocked with 100 mM Tris-HCl for 2 h at room temperature. The beads were extensively washed in PHEM buffer (50 mM Na-PIPES, 50 mM Na-HEPES, 1 mM EDTA, 2 mM MgCl 2 , pH 6.9) and packed in 1-ml Econo columns (Bio-Rad), to yield 0.5-ml settled bed volumes. The amount of bound ligand was determined using the Bradford assay (Bio-Rad) before and after cross-linking to the Sepharose beads. Typically, 0.25 mg of ligand was cross-linked to 0.5 ml of beads.
Affinity Chromatography-The 20 S peak fractions resulting from the sucrose gradient purification of the ATP extract of microtubules, which contained both dynein and dynactin, or whole brain cytosol, was loaded on the appropriate affinity column. The columns were washed extensively with either 50 or 100 mM NaCl (as noted) in HP buffer (50 mM HEPES, 50 mM PIPES, pH 6.9). The columns were eluted with a step gradient of successive 1-ml volumes of 0.5 M and 1.0 M NaCl. The proteins in the fractions collected were precipitated either with 25% trichloroacetic acid on ice overnight or with 10 volumes of ice-cold methanol. The resulting precipitates were resolved by 7% SDS-PAGE (20) and Coomassie Brilliant Blue-stained or transferred onto Immobilon-P (Millipore) and probed with the indicated antibodies that were detected using the Renaissance Western blot chemiluminescence reagent (DuPont NEN). Due to a high concentration of total proteins in high speed supernatant, 20 S sucrose peak, and flow-through column samples, lane 1 in all gels was loaded with 5 l of high speed supernatant or 20 S fraction and lane 2 was loaded with 5 l of the flow-through column samples. The remainder of the lanes were loaded with 25 l out of a total of 50 l of trichloroacetic acid or methanol precipitate from the eluted samples or the final washes.
Antibody Production-Polyclonal rabbit antibodies to p150 Glued and centractin were produced on a custom basis (Cocalico Biological, Inc.) using bacterially expressed polypeptides. The detailed description of the antibody production is reported elsewhere. 2 Briefly, an antibody to centractin was raised against an immunogen polypeptide corresponding to the carboxyl-terminal half of human centractin (27), and an antibody to p150 Glued was raised against a recombinant polypeptide which included amino acids 133-899 from the rat cDNA clone (5). The polyclonal antibodies to p150 Glued and centractin were affinity-purified from rabbit serum against the immunogen polypeptide immobilized on activated CH-Sepharose 4B (Pharmacia). The DIC antibody used here was a mouse monoclonal IgG, a generous gift of K. K. Pfister (5). The DHC antibody is a rabbit polyclonal generously provided by E. Vaisberg (21).

RESULTS
Purified Cytoplasmic Dynein Binds to p150 Glued -Although several genetic studies have suggested an in vivo interaction between cytoplasmic dynein and the dynactin complex, evidence showing a direct biochemical interaction between the two has been lacking. In order to investigate a direct binding of cytoplasmic dynein to the dynactin complex, we constructed an affinity column on which an amino-terminal portion (amino acids 133-899) of p150 Glued , the largest polypeptide in the dynactin complex, was covalently linked to activated CH-Sepharose 4B beads. The 20 S peak fraction resulting from sucrose gradient purification of ATP extracts from a microtubule affinity preparation from rat brain, which is enriched in both cytoplasmic dynein and dynactin, was passed through the p150 Glued affinity column (Fig. 1). Following extensive buffer washes, the column was eluted with 0.5 M and 1.0 M NaCl. The resulting fractions were resolved by SDS-PAGE, electroblotted, and then probed for dynein binding to p150 Glued using mouse monoclonal antibodies to DIC. Results in Fig. 1 indicate that the 20 S sucrose fraction contains DIC (lane 1), which when loaded on to the p150 Glued affinity column (lanes 2-5), binds to the matrix. The DIC was eluted from the column with a step gradient of 0.5 M and 1.0 NaCl (lanes 4 and 5). A BSA column which was constructed identically did not retain any DIC (lanes 6 -9). Compared with the amount of dynein loaded (lane 1), there is no DIC present in the flow-through sample from the p150 Glued affinity column (lane 2), indicating that all dynein present in the loaded sample bound to the column. Although lane 1 in Fig. 1 shows two bands at ϳ74 kDa, consistent with the previous observation of Paschal et al. (19), indicating multiple isoforms of dynein intermediate chain, the resolution of these multiple forms on SDS-PAGE is variable (34).
Cytosolic Cytoplasmic Dynein Binds to p150 Glued -To further test the specificity of the interaction, we asked whether a p150 Glued column could specifically retain cytoplasmic dynein from whole brain cytosol. Rat brain cytosol was loaded on p150 Glued affinity column as well as a control BSA column. The columns were washed extensively with 25 mM NaCl (Fig. 2), then eluted with a step gradient of 0.5 M and 1.0 M NaCl. The fractions were resolved by SDS-PAGE and analyzed by Coomassie Brilliant Blue-staining (Fig. 2). The Coomassie Brilliant Blue-stained gel of the eluates shows that out of the whole cytosol, proteins of molecular mass Ͼ300, 75, and 50 -58 kDa which co-migrate with components of the cytoplasmic dynein complex (DHC, DIC, and LICs), purified according to published methods (18), are specifically retained by the p150 Glued affinity column, whereas they are not retained by the BSA control column.
The identities of the polypeptides specifically retained from cytosol by the p150 Glued column were verified by immunoblotting with antibodies specific for the dynein intermediate chain (Fig. 3A) and heavy chain (Fig. 3B). Both the dynein heavy chain and intermediate chain were retained specifically by the p150 Glued column and not by the control BSA column. Some cross-reactivity of the DIC monoclonal antibody to native p150 Glued present in the cytosol is evident by the presence of the doublet at approximately 150/135 kDa in lanes 1 and 2 in Fig. 3A as was observed previously (5).
Comparison of the immunoblots shown in Fig. 3, A and B, indicates that comparatively more DIC than DHC was eluted from the p150 Glued affinity column with 0.5 M NaCl. It is likely that this is artifactual, due to the higher sensitivity of the anti-DIC antibody as compared with the DHC antibody. Alternatively, it is possible that there may be a limited pool of free dynein intermediate chain in the cytosol, as was observed previously by Paschal et al. (7). This free DIC may bind to dynactin with a lower affinity than does the intact dynein holoenzyme, thus eluting at a lower ionic strength from the affinity column.
The Dynein Intermediate Chain Mediates the Dynein-Dynactin Binding-Potentially, the interaction between dynein and dynactin could be mediated by the binding of p150 Glued to the DHC, DIC, or to the dynein LICs. However, by analogy to the intermediate chain of flagellar outer arm dynein which may mediate the ATP-insensitive binding to A subfiber microtubule (22), the DIC of cytoplasmic dynein may localize to the base of the dynein and mediate the interaction of motor with its cargo. This binding of cytoplasmic dynein to the organelle may be direct (23) or may be mediated by a vesicle bound receptor. Dynactin may function to link dynein to the membrane, as p150 Glued has been suggested to localize to organelles and vesicles within the cell, based on its punctate staining pattern by immunocytochemistry (6, 7). Also, Fath et al. (24) have reported that p150 Glued co-purifies with isolated Golgi-enriched preparations from intestinal epithelial cells.
To test whether the dynein-dynactin interaction is mediated by an interaction between p150 Glued and DIC we prepared p150 Glued affinity beads as before but preincubated the matrix with 2.5 M excess of bacterially expressed DIC before loading the cytosol onto the column (Fig. 4). A control column was pretreated with 25 M excess BSA. If DIC mediates the dyneindynactin interaction, then excess DIC should block the binding sites on p150 Glued , and therefore dynein should no longer bind to the column. When the salt eluates from DIC-treated or BSA-treated p150 Glued affinity columns were probed for DHC, we observed a significant reduction (Ͼ80%, as judged by comparative densitometry) in the observed binding of DHC to the DIC-treated column, whereas the BSA-treated p150 Glued column showed high levels of binding (compare lanes 4 and 5 with  lanes 8 and 9). This result suggests that DIC mediates the binding of cytoplasmic dynein to the p150 Glued component of the dynactin complex.
The Dynactin Complex Binds to Dynein Intermediate Chain-Although we demonstrated that exogenous DIC blocks dynein binding to the p150 Glued column, we cannot exclude the possibility that dynein-dynactin binding may also be mediated by components other than DIC within the cytoplasmic dynein complex. To test the hypothesis that p150 Glued may directly bind to DIC, we constructed affinity columns of bacterially expressed DIC. Whole brain cytosol was loaded onto an affinity column of bacterially expressed DIC. The column was washed extensively and then eluted with 0.5 and 1.0 M NaCl. The resulting fractions were resolved by SDS-PAGE and probed for the binding of dynactin complex to DIC by Western blot using antibodies to p150 Glued and centractin. Because p150 Glued and centractin are bona fide components of the dynactin complex, the presence of both components would suggest that the intact dynactin com- plex is binding to the DIC column. As shown in Fig. 5, both p150 Glued (A) and centractin (B) were found to be retained by the DIC affinity column (lanes 4 and 5). In previous work we have determined that centractin binds directly to p150 Glued (27). Thus the retention of centractin by the DIC column is most likely mediated by its association with p150 Glued . Neither the p150 Glued or centractin polypeptides of the dynactin complex were found to be retained on a similarly constructed BSA control column (lanes 8 and 9).
We have found that if we increase the stringency of our affinity chromatography by increasing the ionic strength of the column wash buffer to 200 mM NaCl, the 135-kDa band recognized by our anti-p150 Glued antibody is not retained by the DIC column. The 135-kDa band corresponds to an alternatively spliced form of p150 Glued . 2 This observation indicates a weaker interaction of 135-kDa species with DIC and suggests that isoform diversity may modulate the affinity of cytoplasmic dynein and dynactin within the cell.
Not all of the p150 Glued or centractin in the cytosol was observed to bind to the DIC affinity column. The capacity of the column may have been exceeded. Nonetheless, a significant fraction of p150 Glued and centractin in brain cytosol was retained by the DIC affinity column. This simultaneous retention of both p150 Glued and centractin by the DIC column suggests that the dynactin complex as a whole binds directly to DIC. DISCUSSION Cytoplasmic dynein and dynactin have been suggested to interact in vitro and in vivo. Such an interaction is important, since this might provide the basis for the functional regulation of cytoplasmic dynein. Dynactin is a 20 S oligomeric complex that variably co-purifies with cytoplasmic dynein (5)(6)(7)25). However, the basis for this apparent co-purification has been unclear, since it might result from either a direct interaction or because the two complexes share similar physical properties such as microtubule affinity and size. A functional link between the two complexes has been suggested by in vitro assays in which the reconstitution of microtubule-based vesicular motility mediated by cytoplasmic dynein required a fraction that contained dynactin (6). While it has been observed that antibodies to dynein failed to co-precipitate components of dynactin complex, and vice versa (7), a direct in vivo interaction has been suggested by genetic evidence where mutations in components of either the dynein or the dynactin complex give rise to similar phenotypes (13-16, 26).
In our experiments we investigated the ability of columnbound polypeptides to interact with native protein complexes.
The results presented in this paper clearly demonstrate that native cytoplasmic dynein binds to column-immobilized p150 Glued and that the dynactin complex binds to columnimmobilized DIC. We also show that this interaction is mediated by the direct binding of the DIC to p150 Glued , since we could effectively block dynein binding to the p150 Glued column by excess exogenous DIC. A biochemical interaction between polypeptides of cytoplasmic dynein and the dynactin complex has also been observed by Vaughan and Vallee, 3 using the solid-phase blot overlay method.
We have mapped the DIC binding domain to the aminoterminal half of p150 Glued , between amino acids 133 and 899. This region is predicted to form an extended ␣-helical coiled coil (5). Recent results from our laboratory demonstrate that the p150 Glued component of the dynactin complex binds to both the microtubule and to centractin (27), an actin-related protein which is a major stoichiometric component of the dynactin complex (6 -8, 11, 12). Taken together, these data suggest that p150 Glued is a multifunctional polypeptide with at least three interacting domains as depicted in Fig. 6A.
One perplexing observation is that although dynein and the dynactin complex biochemically interact, as we have demonstrated here, they do not co-precipitate when antibodies to components of either complex are used (7). However, this apparent discrepancy may result from the blocking of the sites of interaction by the immunoprecipitating antibodies. Recently Waterman-Storer et al. 4 observed that two distinct polyclonal antibodies to p150 Glued blocked the interaction between dynein and dynactin. This lends support to the idea that antibodies used previously to co-precipitate dynein and dynactin interfered with the dynein-dynactin interaction and hence led to the failure to co-precipitate.
The observation that the binding of cytoplasmic dynein to   (19) have therefore predicted that the cytoplasmic DIC may function in an analogous manner to attach cytoplasmic dynein to organelles or kinetochores. Since p150 Glued is localized to the membranous structures in the cytoplasm, it is possible that the dynactin complex on the surface of membranous structures targets the binding of the dynein motor via the interaction between p150 Glued and DIC.
It has been demonstrated recently that p150 Glued binds to microtubules independently of its association with cytoplasmic dynein (27). This microtubule-binding motif shares homology with a similar motif in the microtubule-organelle linking protein CLIP-170 (27,28). CLIP-170 has been proposed to act as a docking protein for the binding of membranous vesicles to the microtubule (28). By analogy with CLIP-170, we speculate that p150 Glued may function to target organelles and vesicles to the microtubule and subsequently allow cytoplasmic dynein to bind. Fig. 6B Fig. 6B, centractin may be a structural link to the organelle, potentially via the cortical cytoskeleton. Now that our results establish a direct binding between the cytoplasmic dynein and dynactin complexes through DIC and p150 Glued , it will be important to determine how the interactions among dynein, dynactin, the microtubule, and the cellular cargo are regulated if we are to understand the molecular mechanism of dynein-dynactin function. How p150 Glued that is bound to the microtubule in an ATP-insensitive manner would facilitate dynein-based motility if it is simultaneously bound to both dynein and microtubule is an important issue and raises the possibility that interaction of dynactin to either the microtubule or DIC is transient and regulatory. Phosphorylation of p150 Glued may regulate dynein function by altering the affinity of the polypeptide for either the microtubule or the DIC. Farshori and Holzbaur 5 have shown that p150 Glued is differentially phosphorylated in response to cellular effectors that have been reported to increase cellular vesicle transport (29). CLIP-170 has been shown to dissociate from microtubules upon phosphorylation (30) and by analogy the interaction of p150 Glued to microtubules may also be regulated in a similar manner to allow transport of organelles along microtubules by cytoplasmic dynein. Alternatively, phosphorylation of p150 Glued may regulate its binding to dynein. In this context it is interesting to note that Lin and Collins (31) and Lin et al. (32) have demonstrated that okadaic acid (an inhibitor of phosphatases 1 and 2a) causes redistribution of cytoplasmic dynein from lysosomes to the cytosol. On the basis of our current results, this redistribution could be due to phosphorylation of p150 Glued which induces the dissociation of DIC from p150 Glued and therefore the release of cytoplasmic dynein from the organelles.
Only very recently have genetic and biochemical studies provided useful insight into the interaction between cytoplasmic dynein and its proposed activator, the dynactin complex, although we do not as yet understand the mechanism by which dynactin regulates dynein activity. In this paper we have described a system where the dynein-dynactin interaction can be observed in vitro and have identified the components involved in this interaction. These results now provide insight into the cellular basis for the lethal defect observed in the Glued mutation in Drosophila, as dynactin may be an essential component of the retrograde transport mechanism.  (27) have defined several domains of interaction on p150 Glued . There is a microtubule-binding domain at the amino terminus of p150 Glued (28) which is homologous to the microtubule-binding domain of CLIP-170 (28), and near the carboxyl terminus there is a highly charged cluster of amino acids that mediates p150 Glued binding to centractin. This study has identified an additional domain in the amino-terminal half of p150 Glued that binds DIC. B, a model for the biochemical interaction among DIC, p150 Glued , centractin, and microtubule has been depicted. Immunoelectron microscopy analysis of chick dynactin preparations (8) revealed a characteristic tilt of p150 Glued with respect to the centractin filament as shown, as well as the immunolocalization of many of the polypeptides within the dynactin complex. Dynein heavy chains are depicted as two globular heads interacting with microtubule and contain the catalytic ATPase domains (33). Organelles that might constitute minus end targeted cargo for cytoplasmic dynein have been omitted, since their exact location in the context of dynein-dynactin is not yet established.
Note that this static model shows several simultaneous biochemical interactions; however, in vivo these interactions are likely to be dynamic and closely regulated.