HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 42, 32763-32768, October 20, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/42/32763    most recent M001536200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tynan, S. H. Articles by Vallee, R. B. Search for Related Content PubMed PubMed Citation Articles by Tynan, S. H. Articles by Vallee, R. B. Social Bookmarking                      What's this?

Light Intermediate Chain 1 Defines a Functional Subfraction of Cytoplasmic Dynein Which Binds to Pericentrin^*

Sharon H. Tynan, Aruna Purohit^§, Stephen J. Doxsey^§, and Richard B. Vallee^¶

From the  Department of Cell Biology and the ^§ Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605

Received for publication, February 24, 2000, and in revised form, June 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The light intermediate chains (LICs) of cytoplasmic dynein consist of multiple isoforms, which undergo post-translational modification to produce a large number of species separable by two-dimensional electrophoresis and which we have proposed to represent at least two gene products. Recently, we demonstrated the first known function for the LICs: binding to the centrosomal protein, pericentrin, which represents a novel, non-dynactin-based cargo-binding mechanism. Here we report the cloning of rat LIC1, which is approximately 75% homologous to rat LIC2 and also contains a P-loop consensus sequence. We compared LIC1 and LIC2 for the ability to interact with pericentrin, and found that only LIC1 will bind. A functional P-loop sequence is not required for this interaction. We have mapped the interaction to the central region of both LIC1 and pericentrin. Using recombinant LICs, we found that they form homooligomers, but not heterooligomers, and exhibit mutually exclusive binding to the heavy chain. Additionally, overexpressed pericentrin is seen to interact with endogenous LIC1 exclusively. Together these results demonstrate the existence of two subclasses of cytoplasmic dynein: LIC1-containing dynein, and LIC2-containing dynein, only the former of which is involved in pericentrin association with dynein.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytoplasmic dynein is a large, multi-subunit complex (1), which functions as a molecular motor that moves cellular components toward the minus ends of microtubules and determines the distribution of many vesicular organelles (2). Cytoplasmic dynein has also been found to be involved in many aspects of mitosis, where it is found at the kinetochore, spindle poles, and cell cortex (3-7).

The cytoplasmic dynein complex is composed of four subunit classes: the heavy (HCs),¹ intermediate (ICs), light intermediate (LICs), and light chains (LCs). The dynein heavy chains are large (532 kDa) polypeptides that contain four ATPase domains and are responsible for microtubule binding and catalytic activity (2). The intermediate chains are a diverse set of subunits derived by alternative splicing from two different genes (8). The ICs have been found to be responsible for the interaction of the dynein complex with a second complex called dynactin, which is required for dynein-based motility, by directly binding to the p150^Glued dynactin subunit (8, 9). Dynactin is thought to be involved in linking dynein to various organelles in the cell; thus the intermediate chains have been proposed to have an important function in dynein targeting (7). The LCs are a diverse family of low and very low molecular weight subunits (10-12); a role in subcellular targeting has been proposed (13).

The HCs, ICs, and LCs all have homologous counterparts in flagellar and ciliary forms of dynein. The LICs, however, are unique to cytoplasmic dynein. They contain a P-loop consensus sequence of unknown function (14, 15). Two-dimensional electrophoresis of both rat and chicken LICs reveals numerous LIC species, at least some of which result from phosphorylation of the LIC polypeptides (14, 15). Based on molecular cloning of one of the rat LICs and peptide microsequencing, we proposed that there are at least two different LIC genes per organism (15). Comparison of the chicken sequence, DLC-A, to the sequence of our rat LIC2 clones and bovine LIC peptide sequences suggests that DLC-A is the chicken isoform of LIC1 and that LIC1 and LIC2 are different gene products (15). Northern blotting suggests that both LICs have a wide tissue distribution (14, 15).

Recently, we demonstrated that recombinant full-length and truncated LIC polypeptides bind to pericentrin, a structural component of the centrosome that is thought to be involved in organizing microtubule nucleating material. Pericentrin has been observed to move in a linear fashion along microtubules toward the centrosome (16). Quantitative analysis of centrosomal components during the cell cycle indicated that pericentrin, along with -tubulin, accumulates from G₁ through metaphase, at which time the centrosomal level drops dramatically (17), although somewhat different results have been reported for -tubulin in a different system (18). Using a co-overexpression and immunoprecipitation assay, we tested a number of dynein and dynactin subunits for the ability to bind to pericentrin. Of all the expression constructs used, only full-length and truncated LIC were found to co-immunoprecipitate with overexpressed pericentrin. Furthermore, when we repeated this experiment in ³⁵S-labeled cells, pericentrin and a LIC fragment were the only specific immunoprecipitated species detected, demonstrating a direct interaction. These results suggest that the LICs, like the ICs, are responsible for linking dynein to its cargo.

In this study, we report the cDNA cloning and sequencing of rat LIC1, establishing the existence of two LIC genes, and we compare the ability of both LIC1 and LIC2 to bind to pericentrin. We find that LIC1, but not LIC2, is capable of this interaction, and we have identified the central portions of both LIC1 and pericentrin as the interacting regions. To determine whether a distinct pericentrin-binding form of the dynein complex exists, we examined the LIC content of dynein and have found that the LICs form homooligomers, but not heterooligomers, and that in triple overexpression studies, HC can bind to either LIC1 or LIC2, but not both. Furthermore, endogenous LIC1, but not LIC2, co-immunoprecipitates with overexpressed pericentrin. These results indicate that the LICs specify different subtypes of dynein, only one of which interacts with pericentrin.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Cloning and Sequencing-- A rat brain cDNA library in the Lambda ZAP II vector (Stratagene, La Jolla, CA) was screened by plaque hybridization as described previously (15) with probes random-primed (DECAprime II kit, Ambion, Inc., Austin, TX) from rat LIC2 cDNA (15). Isolated cDNA clones were sequenced (Sequenase, version 2.0, Amersham Pharmacia Biotech) and compared with LIC peptide and LIC2 cDNA sequence (15). We identified one LIC1 cDNA and used probes random-primed from it to isolate additional LIC1 cDNAs from the library. The 5'-end of the cDNA was obtained using the Marathon cDNA amplification kit with rat brain mRNA (CLONTECH, Palo Alto, CA). The entire LIC1 cDNA sequence was assembled and analyzed using the GCG DNA analysis programs, including BESTFIT and PILEUP. The National Center for Biotechnology Information (NCBI) data bases were scanned using BLAST (19).

Mammalian Expression Constructs-- LIC1 and LIC2 mammalian expression constructs (see Fig. 3) were made by removing the -gal sequence from pCMV  (CLONTECH) with NotI (New England BioLabs, Beverly, MA) digestion and inserting LIC1 and LIC2 full-length or partial cDNAs. The LIC cDNAs had 3'-end NotI sites and coding sequence for epitope tags: myc (MEQKLISEEDLN), HA (YPYPVPDYA), or FLAG (DYKDDDDK) added by PCR. NotI restriction sites were added to the 5'-ends by either PCR or cloning into pARK (gift from Dr. Melissa Gee), which has NotI sites flanking the MCS. LIC1 P-loop point mutations were made using the Chameleon double-stranded, site-directed mutagenesis kit (Stratagene). To produce an amino-terminal fragment of pericentrin (pHAI/pericentrin 1-575), we cut the full-length HA-pericentrin construct (20) with PaeR7I creating a clone extending from the start codon (nucleotide 295) to nucleotide 2019. A truncated central domain of pericentrin containing nucleotides 2587-4317 of the full-length pericentrin was constructed (pHAI/pericentrin 764-1341) by PCR amplification using clone lambda Pc1.2 (21), a 5'-primer with a 5'-EcoRI restriction enzyme site (5'-GCGAATTCCTGAAACGCCAACAT3-') and a 3'-primer with a 3'-PaeR7I site (5'-CGATTTCCTCTGCTTTATCC3-'). The PCR product was digested with EcoRI and PaeR7I and ligated into the pHAI plasmid digested with EcoRI/PaeR7I. A third truncated form of pericentrin coding for the carboxyl terminus (pHAI/pericentrin 1341-1920) was constructed by digesting lambda pc1.2 with XbaI. The restriction fragment was cut by PaeR7I and inserted into pHAI to yield a clone containing nucleotides 4317-6309, with a stop codon at nucleotide 6054.

Antibodies-- The LIC1 and LIC2 cDNAs were put into the NdeI site of pET 15b (Novagen, Inc., Madison, WI) expression vector and expressed in Escherichia coli strain BL21DE3. The bacteria were lysed by French press, and the cell debris was pelleted. The bacterial cytoplasm was passed over a nickel affinity column (Novagen), washed, and eluted with imidazole (Sigma). The eluates were dialyzed into Dulbecco's phosphate-buffered saline and concentrated in Slide-A-Lyzer dialysis cassettes (Pierce, Rockford, IL). The recovered proteins were conjugated to preactivated keyhole limpet hemocyanin (Pierce), mixed with Freund's complete or incomplete adjuvant (Pierce) and injected into New Zealand White rabbits (Millbrook Farms, Amherst, MA). After the initial injections and two more boosts, blood was collected and the sera were tested against COS-7 cell extract on immunoblots (7).

Anti-myc polyclonal antibody used was described previously (22); anti-HA polyclonal and monoclonal antibodies were purchased from BAbCO (Richmond, CA); and anti-FLAG M2 monoclonal antibody and affinity resin were purchased from Sigma. Secondary horseradish peroxidase-conjugated donkey anti-mouse and anti-rabbit antibodies were purchased from Jackson Immunoresearch Labs, Inc. (West Grove, PA).

Co-immunoprecipitation Assays-- COS-7 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal calf serum and penicillin/streptomycin (Life Technologies, Inc.). The cells were transfected (LipofectAMINE, Life Technologies, Inc.) for 6-12 h with 1-4 µg of DNA. Transfections were individually optimized for each construct and combinations of constructs. 30-48 h after transfection, the cells were harvested by washing the monolayer twice with Dulbecco's phosphate-buffered saline and scraping into modified RIPA buffer (100 mM NaCl, 1 mM EGTA, 50 mM Tris, pH 8.0, 1 mM Pefabloc SC (Roche Molecular Biochemicals), and 2 µg/ml leupeptin and pepstatin). The cells were placed in a microcentrifuge tube on ice for 20 min and then spun in a microcentrifuge at maximum speed for 10 min. The resulting extract was used for all co-immunoprecipitation experiments with protein G (Amersham Pharmacia Biotech)- or protein A (Pierce)-Sepharose beads and the appropriate antibody. Immunoprecipitations were incubated overnight at 4 °C with gentle agitation. The beads were then washed with modified RIPA buffer five times at room temperature and eluted into 2× SDS-polyacrylamide gel electrophoresis sample buffer at 100 °C for 5 min. The entire eluate and a sample of the supernatant were used for immunoblotting. Co-transfection efficiency was determined by double indirect immunofluorescence microscopy and varied considerably between combinations of constructs. When extracts from singly transfected cultures were mixed, co-precipitation was never observed, indicating that interactions between co-expressed proteins occurred in vivo.

Co-immunoprecipitation of overexpressed pericentrin and endogenous LICs was performed essentially as described above, with the following change: the beads were eluted with 2× SDS-polyacrylamide gel electrophoresis sample buffer, which did not contain -mercaptoethanol, at 100 °C for 5 min.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

LIC1 cDNA Cloning and Sequencing-- Bovine LIC peptide sequences generated in our earlier study (15) included one LIC1 (indicated as LIC 57/59 in the previous manuscript) peptide sequence that was unique but corresponded to part of the deduced sequence from chicken DLC-A, suggesting the existence of a second LIC gene in rat. To test this possibility, we isolated additional LIC cDNAs from a rat brain cDNA library; we identified a novel cDNA clone, which we termed LIC1. Two different mRNAs were represented among LIC1 clones obtained; they differed only in their polyadenylation site, consistent with what was reported for chicken DLC-A (14). The complete cDNA sequence of rat LIC1 has been deposited (GenBank^TM accession number AF181992). A line diagram of the two rat LICs is shown in Fig. 1A. Using BESTFIT, we find rat LIC1 to be 65% identical (71% similar) to rat LIC2 and 81% identical (90.8% similar) to DLC-A. The sequences are closely related throughout the entire length, with the greatest divergence being at the ends. Fig. 1A shows the location of the peptide sequences that we reported previously (15). Two of the LIC2-derived peptides do not have homologous sequences in LIC1 (Fig. 1A, line 1); one of these peptides is at the amino terminus, and the other is within the relatively divergent portion of the carboxyl terminus (amino acids 400 to the end). The rat LIC1 sequence contains regions corresponding to all of the peptide sequences we previously obtained from the LIC1 doublet, including the peptide that was found only in DLC-A (Fig. 1A, line 3), thus confirming our hypothesis that DLC-A is the chicken isoform of LIC1. Both of these sequences contain a P-loop consensus (Fig. 1A, open boxes at left) sequence, which has been found to be highly conserved among LICs (see "Discussion"). We note that a LIC1 antiserum cross-reacted with LIC2, consistent with the high degree of sequence conservation; surprisingly, an anti-LIC2 serum was monospecific (Fig. 1B).

View larger version (7K): [in this window] [in a new window]   Fig. 1.   A, line diagram of rat LIC1 and LIC2. Open boxes at left represent conserved P-loop sequences. Open boxes labeled A and B within LIC2 represent alternative splice regions. The short black lines between the LICs represent the original peptide sequences we previously reported (15). The two peptides indicated on line 1 are found in LIC2, exclusively. The peptides on line 2 are found in both LIC sequences, and the peptide on line 3 is LIC1-specific. The bracket on line 4 indicates the pericentrin binding region of LIC1. B, COS-7 cell extract was immunoblotted with antiserum raised against rat LIC1 (lane 1) and LIC2 (lane 2).

We note that during the course of these experiments we also obtained cDNA clones that indicated there are four alternative LIC2 isoforms in rat (Fig. 1A, open boxes labeled A and B); the human LIC2 isoforms have all appeared in GenBank^TM (23). We have obtained no comparable pattern in our LIC1 cDNAs.

LIC1 Specifically Binds to Pericentrin-- Because rat LIC1 and LIC2 are very highly homologous to each other, we were interested in potential functional similarities or differences between them. Our initial analysis of pericentrin binding made use of LIC1, but the relative ability of different LICs to interact with pericentrin was not explored (20). Fig. 2 shows co-immunoprecipitation assays in which HA-pericentrin was overexpressed with LIC1-myc or LIC2-myc. Anti-HA immunoprecipitates of pericentrin were probed with anti-myc to detect the LICs. Only LIC1, and not LIC2, was found to co-immunoprecipitate with pericentrin.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Comparison of cytoplasmic dynein LIC1 and LIC2 co-immunoprecipitation with pericentrin. LIC1-myc and LIC2-myc were overexpressed alone () or with HA-pericentrin (+) in COS-7 cells. Resulting cell extracts were used for anti-HA immunoprecipitations. Precipitates (left) and supernatants (right) were immunoblotted and probed with anti-myc to detect the LICs.

All LICs sequenced to date contain a P-loop consensus sequence, indicating potential ATPase activity. Because this P-loop is well conserved from human to Caenorhabditis elegans (see "Discussion"), it is likely that it plays a functional role in LIC activities. To test for any effect of ATPase activity on the LIC1/pericentrin interaction, we constructed two different full-length LIC1 constructs with point mutations in the P-loop sequence. Because it is difficult to predict the effects on binding and/or nucleotide hydrolysis in response to point mutations (24), we tested a mutation of the invariant lysine (K80) to glutamic acid (E) and a mutation of the threonine (T81) to alanine. Both of these point mutants were used in the co-immunoprecipitation assay with pericentrin. We were unable to detect any binding differences between these mutants and wild type LIC1 (Fig. 3, and data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Cytoplasmic dynein LIC1 fragments bind to pericentrin. Various truncation constructs of LIC1 were co-expressed with HA-pericentrin. The gray box represents the P-loop sequence; the X within the boxes (LIC1TA and LIC1KE) represents point mutations within the P-loop sequence. On the right + indicates positive co-immunoprecipitation with HA-pericentrin, and  indicates negative co-immunoprecipitation. The black bar summarizes the deduced pericentrin binding region. Data for several immunoprecipitations are shown in Figs. 2 and 4.

To determine whether specific regions within LIC1 and pericentrin were important in their interaction, we evaluated a series of truncation mutants of both LIC1 and pericentrin in our co-immunoprecipitation assay. Fig. 3 shows LIC1 truncations in a summary of the results of co-immunoprecipitation assays using full-length HA-pericentrin (data for several of these truncations are shown in Figs. 2 and 4). This series of truncations shows that the region of LIC1 between amino acids 140 and 236 is important in the interaction. Mutants that contain any part of this region were found to interact with HA-pericentrin in the assay, whereas mutants that do not contain any of this region did not interact.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Cytoplasmic dynein LIC1 coimmunoprecipitations with pericentrin fragments. A, three pericentrin fragments were constructed for co-immunoprecipitation. The amino acids included in each fragment are listed at the right; all fragments had amino-terminal HA tags. B, LIC constructs listed at right were co-overexpressed with pericentrin constructs listed at the top. Pericentrin fragments were immunoprecipitated with anti-HA. Anti-myc was used to probe immunoblots of the precipitations for LIC1-myc fragments.

Fig. 4 shows three fragments of pericentrin, the amino-terminal, central, and carboxyl-terminal portions. The full-length LIC1 would not co-express with all of the pericentrin fragments, so amino- and carboxyl-terminal truncated LIC1 fragments were used. We found that both bind to the central portion of pericentrin, amino acids 764-1341.

The LICs Specify Different Subtypes of Dynein-- In light of the finding that pericentrin binds to LIC1, but not LIC2, we tested the ability of LICs to homooligomerize or heterooligomerize. We overexpressed LIC1-HA with LIC1-myc or LIC2-myc (Fig. 5A). When LIC1-HA was immunoprecipitated with anti-HA, only LIC1-myc was found to co-precipitate, indicating that LIC1 forms homooligomers, but not heterooligomers. Fig. 5B shows that oligomers will also form from LIC2-myc and LIC2-FLAG. Homooligomers also occurred when the myc-tagged constructs had point mutations in the P-loop sequences (data not shown), indicating that a functional P-loop is not required for LIC self-association. It is not possible for us to tell from these experiments how many LIC molecules form a homooligomer.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Cytoplasmic dynein LICs form homooligomers but not heterooligomers. Supernatants from immunoprecipitations are shown at the right to verify expression of LIC fragments. A, LIC1-HA overexpressed in combination with LIC1-myc and LIC2-myc (indicated at the top) was immunoprecipitated with anti-HA and immunoblots were probed with anti-myc. Only LIC1-myc co-precipitated with LIC1-HA. B, LIC2-myc, which did not co-precipitate with LIC1-HA, does co-precipitate with LIC2-FLAG.

We used triple overexpression to determine whether LIC1 and LIC2 homooligomers were present in the same or different dynein complexes (Fig. 6). LIC1-HA and LIC2-myc were overexpressed along with C1140-myc, a dynein heavy chain construct that binds to the LICs (see accompanying article (25)). Fig. 6A shows the triple overexpression extract immunoprecipitated with both anti-HA and anti-LIC2. When probed with anti-myc, C1140-myc was found in both immunoprecipitates, but LIC2-myc was only found in anti-LIC2 and not in anti-HA precipitations. Fig. 6B shows the same blots reprobed with anti-HA to localize LIC1-HA. LIC1-HA is found only in anti-HA and not in anti-LIC2 immunoprecipitations. This experiment demonstrates that each LIC independently associates with HC; LIC1 and LIC2 do not bind to HC simultaneously.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Cytoplasmic dynein HC does not bind to LIC1 and LIC2 simultaneously. A, extracts overexpressing the HC carboxyl terminus (HC-C1140-myc), LIC2-myc, and LIC1-HA were immunoprecipitated with anti-HA (left lanes) and anti-LIC2 (right lanes); cell extracts are shown in the center lanes. Immunoblots were probed with anti-myc. B, the same blots from A were reprobed with anti-HA. In the right-hand section, there is a nonspecific smudge that appeared upon overexposure of the blot; no specific LIC1 band was seen in that panel.

Finally, we tested our hypothesis that there are two different dynein populations by looking at the endogenous LICs that co-precipitate with overexpressed pericentrin. Our previous work has shown that overexpressed pericentrin will precipitate endogenous dynein HC and IC. LIC content has not been demonstrated due to technical difficulties caused by the similar molecular weights of the LICs and the antibody heavy chain. To avoid this problem, we eluted anti-HA precipitations with sample buffer lacking -mercaptoethanol (Fig. 7), which kept the antibody intact, so that it did not interfere with the LICs. As predicted, endogenous LIC1 co-precipitated with pericentrin, but LIC2 was not detected, demonstrating the existence of a subpopulation of dynein, which contains only LIC1.

View larger version (67K): [in this window] [in a new window]   Fig. 7.   Overexpressed pericentrin co-precipitates endogenous LIC1 but not LIC2. Extracts overexpressing pericentrin were immunoprecipitated with anti-HA or beads alone (left panel). Immunoblots were probed with pan-anti-LIC antibody to determine the LIC content of co-precipitating dynein. Supernatants are shown on the right for comparison.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have identified a second LIC gene in rat, confirming our earlier hypothesis (15) and further accounting for the considerable diversity in LIC forms observed in purified cytoplasmic dynein complexes. We have also demonstrated that LIC1 is specific for pericentrin binding and that the LICs form homooligomers, but not heterooligomers. Through triple overexpression studies, we have shown that the two LIC isoforms cannot bind to HC simultaneously. These results together provide evidence for the existence of functionally distinct cytoplasmic dynein complexes that differ by LIC content.

LIC Sequences-- From comparisons of rat LIC2 and chicken DLC-A sequences we previously hypothesized the existence of two different but related LIC genes in rat (15). The amino acid sequences of LIC1 and LIC2 differ throughout the entire length, and the DNA sequences are only 68% identical, with no large (>30 nucleotides) stretches of complete identity, supporting the idea that they are derived from different genes. LIC1 and LIC2 amino acid sequences account for all 11 peptide sequences that we obtained from bovine cytoplasmic dynein LICs (15), suggesting that there are no additional LIC genes.

BLAST searches of the C. elegans genome, which is nearly complete, with either LIC sequence yield only one match. The gene product is an LIC that is equally homologous to both LIC1 and LIC2. These results suggest that C. elegans has only one LIC gene. Because the full range of LIC functions is not known in any species, it remains to be seen whether a single LIC in C. elegans can carry out all LIC functions or if the diversity provided by two separate vertebrate LICs allows for additional roles for this class of subunit. Despite relatively low homology between rat and worm sequences, the C. elegans LIC has a conserved P-loop sequence, further suggesting that the LICs are functional ATPases. No sequences homologous to LIC have been found in the S. cerevisiae genome, apparently indicating that LIC is not involved in the limited functions attributed to yeast dynein. It is also interesting to note that the LICs have not appeared among nuclear migration mutants of Aspergillus nidulans (the nud mutants) (26, 27) and Neurospora crassa (the ropy mutants) (28). All other dynein subunit classes and some dynactin subunits have been identified in the screening of these mutants, suggesting that the LICs may not be involved in nuclear migration.

LIC1/Pericentrin Interaction-- The isoform specificity of pericentrin binding that we describe here adds strong support for the specificity of the LIC-pericentrin interaction. The interaction site (amino acid 140-236 of LIC1) is bracketed in Fig. 1A. This region shows a high degree of identity between LIC1 and LIC2 except for amino acids 201-219, which appears to be isoform-specific. This region may account for the binding specificity of LIC1.

The P-loop sequence of LIC1 is located amino-terminal to the interaction region. Mutations in the P-loop do not appear to have any obvious effect on the LIC1/pericentrin interaction. Some fragments of LIC1 bind more efficiently to pericentrin than full-length LIC1 does; for example, LIC1N174 and LIC1C173 each contain part of the interacting region, and each co-immunoprecipitate with pericentrin more efficiently than full-length LIC1. The most likely explanation for this observation is that when LIC1 is truncated, regulatory elements are removed, allowing for maximum binding between LIC1 and pericentrin. Conversely, the pericentrin fragment that binds to LIC1 does not bind any better than full-length pericentrin, suggesting that any regulation of the interaction occurs with LIC1, rather than pericentrin. We would expect the LIC1/pericentrin interaction to be highly regulated to allow for the slow accumulation and rapid dissociation of pericentrin at the centrosome.

-Tubulin has been found to co-immunoprecipitate with and accumulate at the centrosome with pericentrin (17). -Tubulin does not associate with the central portion of pericentrin,² in contrast to LIC1 (Fig. 4). This suggests that pericentrin may be capable of associating with LIC1 (and thus dynein) and the -tubulin containing -tubulin ring complex at the same time. This would allow for the -TURC complex to be transported to the centrosome at the same time as pericentrin, without any components of the complex directly binding to dynein.

Significance of Dynein Subtypes-- Our findings that only LIC1 binds to pericentrin and that LIC1 and LIC2 binding to HC is mutually exclusive suggest that a substantial subfraction of dynein is incapable of binding to pericentrin. In other work (see accompanying manuscript), we have demonstrated that the LIC1 and LIC2 binding sites on the HC are identical, further supporting our contention that the LICs bind to the HC, and thus the complex, mutually exclusively to define functional subfractions of dynein. LIC1-containing dynein can bind to pericentrin, whereas LIC2 dynein cannot, and there may be LIC-less dynein that also cannot. This adds diversity to the pool of dynein in the cell. The subcellular targeting of dynein may be simplified by the existence of dynein subtypes, each of which is responsible for a given set of dynein functions. Pericentrin binding is likely to be one of several functions of LIC1, because there appears to be considerably more LIC1 than pericentrin in cells. This suggests that other mechanisms are still required for the specific binding of dynein to various cellular components, but alternative LIC subunit content provides some amount of specificity.

We have speculated that the LICs may be responsible for a non-dynactin-based targeting mechanism for dynein, perhaps specific for soluble proteins rather than membrane-bound organelles (20). In this view, The LICs would function independently of dynactin, with each having different targeting specificities. Dynactin-mediated targeting of dynein would, presumably, involve either a subfraction of dynein, which lacks LICs or in which the LICs have been inactivated. However, given that p50 overexpression has been observed to disrupt the accumulation of pericentrin at centrosomes (16), it is also quite possible that dynactin and the LICs function in concert in certain situations to support both binding and motility. Further work is necessary to determine the actual relationship between LIC- and dynactin-mediated functions.

	ACKNOWLEDGEMENT

We thank Dr. Elizabeth Luna for her assistance in using 5' rapid amplification of cDNA ends to complete the 5'-end of the LIC1 cDNA.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants GM47434 (to R. B. V.) and GM51994 (to S. J. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF181992.

^¶ To whom correspondence should be addressed: Dept. of Cell Biology, University of Massachusetts Medical Center, 377 Plantation St., Worcester, MA 01605. Tel.: 508-856-8504; Fax: 508-856-8987; E-mail: Richard.vallee@umassmed.edu.

Published, JBC Papers in Press, July 11, 2000, DOI 10.1074/jbc.M001536200

² W. Zimmerman and S. Doxsey, unpublished results.

	ABBREVIATIONS

The abbreviations used are: HC, heavy chain subunit; IC, intermediate chain subunit; LIC, light intermediate chain subunit; LC, light chain subunit; PCR, polymerase chain reaction; HA, hemagglutinin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. Ha, K. W.-H. Lo, K. R. Myers, T. M. Carr, M. K. Humsi, B. A. Rasoul, R. A. Segal, and K. K. Pfister A neuron-specific cytoplasmic dynein isoform preferentially transports TrkB signaling endosomes J. Cell Biol., October 21, 2008; 181(6): 1027 - 1039. [Abstract] [Full Text] [PDF]

				K. W.-H. Lo, J. M. Kogoy, B. A. Rasoul, S. M. King, and K. K. Pfister Interaction of the DYNLT (TCTEX1/RP3) Light Chains and the Intermediate Chains Reveals Novel Intersubunit Regulation during Assembly of the Dynein Complex J. Biol. Chem., December 21, 2007; 282(51): 36871 - 36878. [Abstract] [Full Text] [PDF]

				S. A. Stehman, Y. Chen, R. J. McKenney, and R. B. Vallee NudE and NudEL are required for mitotic progression and are involved in dynein recruitment to kinetochores J. Cell Biol., August 9, 2007; 178(4): 583 - 594. [Abstract] [Full Text] [PDF]

				J. C. Williams, P. L. Roulhac, A. G. Roy, R. B. Vallee, M. C. Fitzgerald, and W. A. Hendrickson Structural and thermodynamic characterization of a cytoplasmic dynein light chain intermediate chain complex PNAS, June 12, 2007; 104(24): 10028 - 10033. [Abstract] [Full Text] [PDF]

				M. Haghnia, V. Cavalli, S. B. Shah, K. Schimmelpfeng, R. Brusch, G. Yang, C. Herrera, A. Pilling, and L. S.B. Goldstein Dynactin Is Required for Coordinated Bidirectional Motility, but Not for Dynein Membrane Attachment Mol. Biol. Cell, June 1, 2007; 18(6): 2081 - 2089. [Abstract] [Full Text] [PDF]

				H.-S. Kim, M. Takahashi, K. Matsuo, and Y. Ono Recruitment of CG-NAP to the Golgi apparatus through interaction with dynein-dynactin complex Genes Cells, March 1, 2007; 12(3): 421 - 434. [Abstract] [Full Text] [PDF]

				K. W.-H. Lo, H.-M. Kan, and K. K. Pfister Identification of a Novel Region of the Cytoplasmic Dynein Intermediate Chain Important for Dimerization in the Absence of the Light Chains J. Biol. Chem., April 7, 2006; 281(14): 9552 - 9559. [Abstract] [Full Text] [PDF]

				S. Li, C. E. Oakley, G. Chen, X. Han, B. R. Oakley, and X. Xiang Cytoplasmic Dynein's Mitotic Spindle Pole Localization Requires a Functional Anaphase-promoting Complex, {gamma}-Tubulin, and NUDF/LIS1 in Aspergillus nidulans Mol. Biol. Cell, August 1, 2005; 16(8): 3591 - 3605. [Abstract] [Full Text] [PDF]

				W.-L. Lee, M. A. Kaiser, and J. A. Cooper The offloading model for dynein function: differential function of motor subunits J. Cell Biol., January 17, 2005; 168(2): 201 - 207. [Abstract] [Full Text] [PDF]

				Y. Hou, G. J. Pazour, and G. B. Witman A Dynein Light Intermediate Chain, D1bLIC, Is Required for Retrograde Intraflagellar Transport Mol. Biol. Cell, October 1, 2004; 15(10): 4382 - 4394. [Abstract] [Full Text] [PDF]

				W. C. Zimmerman, J. Sillibourne, J. Rosa, and S. J. Doxsey Mitosis-specific Anchoring of {gamma} Tubulin Complexes by Pericentrin Controls Spindle Organization and Mitotic Entry Mol. Biol. Cell, August 1, 2004; 15(8): 3642 - 3657. [Abstract] [Full Text] [PDF]

				M.-g. Li, M. Serr, E. A. Newman, and T. S. Hays The Drosophila tctex-1 Light Chain Is Dispensable for Essential Cytoplasmic Dynein Functions but Is Required during Spermatid Differentiation Mol. Biol. Cell, July 1, 2004; 15(7): 3005 - 3014. [Abstract] [Full Text] [PDF]

				D. Chen, A. Purohit, E. Halilovic, S. J. Doxsey, and A. C. Newton Centrosomal Anchoring of Protein Kinase C {beta}II by Pericentrin Controls Microtubule Organization, Spindle Function, and Cytokinesis J. Biol. Chem., February 6, 2004; 279(6): 4829 - 4839. [Abstract] [Full Text] [PDF]

				C. A. Perrone, D. Tritschler, P. Taulman, R. Bower, B. K. Yoder, and M. E. Porter A Novel Dynein Light Intermediate Chain Colocalizes with the Retrograde Motor for Intraflagellar Transport at Sites of Axoneme Assembly in Chlamydomonas and Mammalian Cells Mol. Biol. Cell, May 1, 2003; 14(5): 2041 - 2056. [Abstract] [Full Text] [PDF]

				J. C. Schafer, C. J. Haycraft, J. H. Thomas, B. K. Yoder, and P. Swoboda XBX-1 Encodes a Dynein Light Intermediate Chain Required for Retrograde Intraflagellar Transport and Cilia Assembly in Caenorhabditis elegans Mol. Biol. Cell, May 1, 2003; 14(5): 2057 - 2070. [Abstract] [Full Text] [PDF]

				J. Zhang, S. Li, R. Fischer, and X. Xiang Accumulation of Cytoplasmic Dynein and Dynactin at Microtubule Plus Ends in Aspergillus nidulans Is Kinesin Dependent Mol. Biol. Cell, April 1, 2003; 14(4): 1479 - 1488. [Abstract] [Full Text] [PDF]

				K. L. M. Boylan and T. S. Hays The Gene for the Intermediate Chain Subunit of Cytoplasmic Dynein Is Essential in Drosophila Genetics, November 1, 2002; 162(3): 1211 - 1220. [Abstract] [Full Text] [PDF]