The Tctex1/Tctex2 Class of Dynein Light Chains

The Tctex1/Tctex2 family of dynein light chains associates with the intermediate chains at the base of the soluble dynein particle. These components are essential for dynein assembly and participate in specific motor-cargo interactions. To further address the role of these light chains in dynein activity, the structural and biochemical properties of several members of this polypeptide class were examined. Gel filtration chromatography and native gel electrophoresis indicate that recombinantChlamydomonas flagellar Tctex1 exists as a dimer in solution. Furthermore, yeast two-hybrid analysis suggests that this association also occurs in vivo. In contrast, both murine and Chlamydomonas Tctex2 are monomeric. To investigate protein-protein interactions involving these light chains, outer arm dynein from Chlamydomonas flagella was cross-linked using dimethylpimelimidate. Immunoblot analysis of the resulting products revealed the interaction of LC2 (Tctex2) with LC6, which is closely related to the highly conserved LC8 protein found in many enzyme systems, including dynein. Northern dot blot analysis demonstrated that Tctex1/Tctex2 family light chains are differentially expressed both in a tissue-specific and developmentally regulated manner in humans. These data provide further support for the existence of functionally distinct populations of cytoplasmic dynein with differing light chain content.


From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030-3305
The Tctex1/Tctex2 family of dynein light chains associates with the intermediate chains at the base of the soluble dynein particle. These components are essential for dynein assembly and participate in specific motorcargo interactions. To further address the role of these light chains in dynein activity, the structural and biochemical properties of several members of this polypeptide class were examined. Gel filtration chromatography and native gel electrophoresis indicate that recombinant Chlamydomonas flagellar Tctex1 exists as a dimer in solution. Furthermore, yeast two-hybrid analysis suggests that this association also occurs in vivo. In contrast, both murine and Chlamydomonas Tctex2 are monomeric. To investigate protein-protein interactions involving these light chains, outer arm dynein from Chlamydomonas flagella was cross-linked using dimethylpimelimidate. Immunoblot analysis of the resulting products revealed the interaction of LC2 (Tctex2) with LC6, which is closely related to the highly conserved LC8 protein found in many enzyme systems, including dynein. Northern dot blot analysis demonstrated that Tctex1/Tctex2 family light chains are differentially expressed both in a tissue-specific and developmentally regulated manner in humans. These data provide further support for the existence of functionally distinct populations of cytoplasmic dynein with differing light chain content.
Dyneins are molecular motors that translocate toward the minus-end of microtubules. These enzymes provide the motive force to power eukaryotic cilia and flagella and occur in the cytoplasm where they are involved in a wide variety of motile activities that are of fundamental importance to the cell. For these molecular motors to generate useful work, both microtubule motor and cargo-binding activities must be subject to precise temporal and spatial regulation. For flagellar dyneins, the ultimate cargo is another axonemal microtubule, and thus control for this process can be built directly into the flagellar-or ciliary-specific enzyme. The situation for cytoplasmic dynein is more complex, because there are many different cargoes that must be transported within a single cell. Indeed, the mechanisms by which individual cellular cargoes are attached to particular cytoplasmic dynein motors and whether this attachment is an integral property of specific enzyme subsets are of major importance for understanding motor-driven transport.
In general, dyneins are constructed around ϳ520 kDa heavy chains that consist of multiple AAA 1 (ATPases associated with cellular activities) domains (see Ref. 1 for recent review). AAA proteins represent an ancient and diverse family of ATPases ranging from bacterial metal chelatases to eukaryotic microtubule-severing proteins. The dynein HCs exhibit both ATPase and microtubule motor activities. These proteins comprise the globular head and microtubule-binding stalk domains of the complex (2,3). In addition, the N-terminal ϳ160 kDa of each HC forms a stem that interacts both with additional HCs and an intermediate chain/light chain complex to form the basal cargo-binding domain of the enzyme (4 -7). Each IC consists of a C-terminal region comprised of multiple WD-repeats (a motif of ϳ40 residues containing an invariant Trp-Asp dipeptide) and a gene-specific N-terminal region (8 -10). In flagellar dyneins, the ICs show little homology with each other in the gene-specific regions and likely play very different roles in the function of this enzyme. For example, in the Chlamydomonas outer arm, IC1 (previously termed IC78) has been shown to interact with ␣-tubulin in situ (11). This raised the possibility that ICs are responsible for direct cargo binding, although it is now clear that, at least in the flagellum, additional components are required to specify the precise attachment site (12). In contrast, Chlamydomonas IC2 (previously IC69 or IC70) apparently mediates regulatory processes that impinge on motor function (13). Cytoplasmic dynein has a single class of IC (IC74) that interacts directly with the dynactin activator of dynein-based vesicular transport (14). In mammals there are several IC74 genes, and evidence for both alternative splicing of IC74 transcripts and differential phosphorylation of the resulting proteins has been obtained (10,15). The clear similarities between flagellar and cytoplasmic dyneins raised the possibility that the various IC74 isoforms bind specific intracellular cargoes. However, considering the large number of different proteins, organelles, and other complexes that must be transported at various times during the cell cycle, it does not seem reasonable that IC74 isoforms alone can provide sufficient specificity for cargo attachment.
Cytoplasmic dynein contains two light intermediate chains that are phosphorylated in a cell cycle-dependent manner (16). One of these polypeptides mediates the interaction of dynein with pericentrin (17). Thus, light intermediate chains also are involved in cargo-binding activities. Two general classes of LCs (Ͻϳ25 kDa) have been identified in dynein (18). The first comprises a diverse series of proteins that associate directly with individual HCs. These polypeptides are thought to be involved in the control of motor functions in response to various regulatory inputs. Thus far, they have been identified only in flagellar dyneins (see Ref. 18 for review).
The second LC class includes three distinct protein families (designated LC8, LC7/roadblock and Tctex1/Tctex2) that associate with the ICs at the base of the soluble dynein particle. Members of these protein families are found in both cytoplasmic and flagellar dyneins. The LC8 protein is highly conserved from Chlamydomonas to humans (19); it is not dynein-specific and is found in many other multimeric enzymes such as neuronal nitric oxide synthase (20) and myosin V (21). This protein exists as a dimer (22) and exposes two identical surfaces that appear to bind a variety of proteins (23), including dynein ICs and specific cellular cargoes such as the proapoptotic factor Bim (24) and Drosophila Swallow (25). In multicellular organisms, complete loss of LC8 function results in embryonic lethality (26). However, in Chlamydomonas, LC8-null mutants grow normally but are unable to make flagella due to disruption of intraflagellar transport (27). Multiple LC8 variants have been identified in a variety of organisms, including Drosophila, schistosomes (28), and mammals. In Chlamydomonas, flagellar outer arm dynein contains both LC8 and a homologue (LC6) that share ϳ40% sequence identity (19).
The second LC family (LC7/roadblock) is essential for both flagellar and cytoplasmic dynein function (29). Although the precise role played by these proteins remains obscure, expression of at least one member of this class is down-regulated in response to light within rat visual cortex (30).
The Tctex1 and Tctex2 proteins were originally identified in mouse testis as candidates for involvement in the non-Mendelian transmission of variant forms of mouse chromosome 17 known as the t haplotypes (31,32). Subsequently, a Tctex2 homologue was found within the Chlamydomonas outer dynein arm (33), and Tctex1 was identified in both cytoplasmic dynein and flagellar inner arm I1 (34,35). Several independent studies have implicated Tctex1 in the attachment of specific cargoes to the dynein motor (36 -39). For example, in the vertebrate photoreceptor Tctex1 binds directly to the C-terminal tail of rhodopsin whereas the related LC rp3 does not (36). Disruption of this interaction due to mutations in rhodopsin leads to retinitis pigmentosa, because rhodopsin-bearing vesicles can no longer be transported to the base of the connecting cilium for insertion into the membrane stacks. In humans, the Tctex1 gene maps at or close to the retinal cone dystrophy-1 locus (40). Intriguingly, several reports suggest that Tctex1 and its close homologue rp3 (41) are differentially expressed in fetal and adult brain (38,41). Thus, regulation of cellular LC content might provide one mechanism to control dynein-cargo interactions.
To further understand the role played by the Tctex1/Tctex2 family proteins in dynein function, we have investigated the properties of these LCs both in mammals and Chlamydomonas, because the two systems provide complementary information.
In this report, we describe the identification of additional members of the Tctex1/Tctex2 LC family and detail an analysis of LC expression patterns in various human tissues during development. Using specific antibodies, we demonstrate that Tctex2, like Tctex1, is present in both flagellar and cytoplasmic dyneins. We also show that Tctex1, but not Tctex2, is dimeric in solution and furthermore, that members of this LC class interact directly with LC8 family proteins in the Chlamydomonas outer arm.

EXPERIMENTAL PROCEDURES
Purification of Axonemes and Dynein-Wild-type Chlamydomonas were deflagellated using dibucaine and isolated by standard methods (42). Membranes were removed with 1% Nonidet P-40. Dynein was extracted from the resulting axonemes by treatment with 0.6 M NaCl. Subsequently, the ␣␤ subparticle of the outer arm was purified by centrifugation through a 5-20% sucrose density gradient (43).
Cytoplasmic dynein was obtained from rat brain, liver, kidney, spleen, and testis homogenates by immunoprecipitation using monoclonal antibody 74-1, which reacts specifically with IC74 (44). These samples were kindly provided by Drs. M. Salata and K. Pfister (University of Virginia).
Cross-linking with Dimethylpimelimidate-Protein⅐protein interactions within Chlamydomonas axonemes and purified dynein were detected by cross-linking with dimethylpimelimidate (DMP) (45). Samples were exchanged into 100 mM triethanolamine, pH 8.2, and treated with 0 -10 mM DMP for 60 min at room temperature. The cross-linker was dissolved in methanol and added directly to the sample to achieve a final solvent concentration of 10% (v/v). Reactions were terminated by addition of gel sample buffer.
Preparation of Recombinant Proteins and Antibodies-The entire coding region for murine Tctex2 was cloned into the pMal-c2 vector, expressed as a C-terminal fusion with maltose-binding protein (MBP) and purified by amylose affinity chromatography. The LC and fusion partner were separated by digestion with Factor Xa. The entire fusion protein was used as the immunogen for preparation of rabbit polyclonal antibody R7714. The serum was blot-purified against the isolated LC (46), and the resulting antibody preparation was used at a dilution of 1/50 for immunoblot analysis. The Chlamydomonas LC6 fusion protein and corresponding rabbit antibody R4928 were prepared in a similar manner. Purification of rp3, Tctex1, and Chlamydomonas LC2 fusion proteins and the preparation of antibodies R5205 and R5391 that recognize Tctex1 and LC2, respectively, have been described previously (33,34,47).
Native and Denaturing Polyacrylamide Gel Electrophoresis-Denatured proteins were separated using 8 and 12.5% acrylamide slab gels and 5-15% acrylamide gradient gels containing SDS. Gels were stained with Coomassie Blue or were blotted to nitrocellulose in 10 mM NaHCO 3 , 3 mM Na 2 CO 3 , 0.01% SDS, 20% methanol. Blots were probed with affinity-purified antibodies, and reactivity was assessed using a peroxidase-conjugated secondary antibody and a chemiluminescent detection system (ECL, Amersham Pharmacia Biotech).
Native gel electrophoresis was used to determine the solution molecular weight of recombinant polypeptides (22,48). Proteins were electrophoresed in gels of different acrylamide concentration and the negative slope of 100 (log [R F ϫ 100]) used to determine the retardation coefficient (K R ). Standard proteins used were jack bean urease (545-kDa hexamer and 272-kDa trimer), bovine serum albumin (132-kDa dimer and 66-kDa monomer), ovalbumin (45 kDa), bovine carbonic anhydrase (29 kDa), and ␣-lactalbumin (14.2 kDa). A plot of log K R versus log M r for these proteins yielded a standard curve from which the native mass of the test samples was determined.
Circular Dichroism Spectroscopy-Recombinant Chlamydomonas Tctex1 was prepared at 47.8 M. The circular dichroism spectrum was measured in the far UV range between 190 and 280 nm using a Jasco J-715 spectropolarimeter. The signal at 222 nm was converted to mean residue ellipticity [] 222 in mdeg.cm 2 .dmol Ϫ1 using [] 222 ϭ 100 ϫ CD signal/n (number of residues) ϫ l (path length ϭ 0.1 cm) ϫ C ([protein] in mM). The approximate helical content of Tctex1 was determined using a value of [] 222 ϭ Ϫ32,600 mdeg.cm 2 .dmol Ϫ1 for a completely helical protein (49).
Yeast Two-hybrid Screen-The full-length murine Tctex1 cDNA was cloned into the pAS2-1 vector resulting in the in-frame fusion of Tctex1 with the DNA-binding domain of the yeast GAL4 protein. A mouse brain cDNA library (9-to 12-week male BALB/c) containing 3.5 ϫ 10 6 independent clones (CLONTECH Laboratories Inc.) was cloned into pACT2 to produce fusions between the encoded proteins and the DNA activation domain of GAL4. The yeast strain (Y190) used for the screening assay contained both HIS3 and lacZ reporter genes under control of a GAL4-responsive upstream activation site. Lack of autonomous activation by the Tctex1/DNA-binding domain fusion was demonstrated by plating cells transformed with the bait plasmid alone on media lacking histidine. For the assay, bait and library plasmids were transformed simultaneously into yeast and positive interactions initially identified by growth of His ϩ cells on media lacking histidine. Putative positives were then further tested by assaying colonies for ␤-galactosidase activity. Following confirmation of the specificity of the interaction, the Tctex1-binding partners were identified by sequence analysis.
Computational Methods-Members of the Tctex1/Tctex2 protein family were identified in the nonredundant and EST data bases at NCBI using BLAST. Multiple sequence alignment was prepared using CLUSTALW, and the output was processed using BOXSHADE. The phylogenetic analysis was performed using the Phylip suite of programs. Specifically, distances were calculated with PROTDIST and FITCH, and the unrooted tree was drawn using DRAWTREE. Secondary structure was predicted using PHD.

RESULTS
Phylogeny of the Tctex1/Tctex2 LC Family-Previously, we identified several dynein components belonging to the Tctex1/ Tctex2 light chain family. These included Tctex1 (in cytoplasmic dynein and flagellar inner arm I1 (35)), its close homologue rp3 (in cytoplasmic dynein (47)), and the more distantly related Tctex2 (in flagellar outer arm dynein (33)). Recent analysis of the nonredundant and EST data bases at NCBI using BLAST has allowed us to identify several additional members of this class of dynein component of both mammalian and nematode origin. A ClustalW sequence comparison of the currently identified members of this family is shown in Fig. 1a. These proteins share most identity in the C-terminal regions and have gene-specific N-terminal sections that show varying degrees of similarity with each other. The phylogenetic relationships within this class of polypeptide were calculated using the Phylip suite of programs and are shown in Fig. 1b. This analysis demonstrates that the Tctex1 and Tctex2 light chains define two major subgroups of this dynein LC class and reveals that Tctex2-related proteins derive from many sources other than testis, suggesting that they may not be flagella-specific.
Structural and Solution Properties of Tctex1 and Tctex2-To further address the role played by these proteins in both flagellar and cytoplasmic dynein function, the structural and solution properties of the Tctex1/Tctex2 LC class were examined. A secondary structure analysis of Chlamydomonas Tctex1 using PHD (50) indicates that the N-terminal half of the protein is likely formed from two ␣ helices both of which are predicted with high reliability values. The C-terminal section of Tctex1 appears to consist of several ␤ strands, although the prediction of the precise boundaries of these elements is significantly less reliable. 2 To assess the general validity of this structure prediction, the circular dichroism spectrum of Tctex1 in the far UV was measured. The spectrum shows clear evidence for a mixture of helix and sheet structures as predicted by the program PHD. From the signal at 222 nm, the mean residue ellipticity [] 222 was calculated. Assuming [] 222 ϭ Ϫ32,600 mdeg.cm 2 .dmol Ϫ1 for a completely helical protein (49), 25.6% of residues within Tctex1 are in a helical conformation. This value agrees very well with the secondary structure prediction of 29.8%. Tctex1 Is Dimeric in Solution-Following denaturing electrophoresis, most recombinant Tctex1 migrates with M r ϭ 13,000, which is completely consistent with the calculated molecular mass of 12,805 Da (includes a single additional N-terminal His residue) (Fig. 2, right inset). However, an extra band at M r ϳ 27,000 also is evident. This band is recognized by antibody R5205 and therefore likely represents a Tctex1 dimer. To test whether native Tctex1 is indeed dimeric, the solution molecular weight of the recombinant protein was determined by gel filtration chromatography (Fig. 2). The major Tctex1 peak has a mass of ϳ31.5 kDa, strongly suggesting that this protein is dimeric in solution. No peak corresponding to the Tctex1 monomer was observed under native conditions even at high dilution, suggesting a very high affinity between monomers.
To confirm that Tctex1 is dimeric, the molecular mass was also determined by native gel electrophoresis using the method of Hedrick and Smith (22,48). This analysis yielded an estimate of 22 kDa, supporting the dimeric nature of this protein (Table I). To assess whether other members of this protein family exist as oligomers, the solution molecular weight of MBP/Tctex2 and MBP/LC2 fusion proteins also were determined (Table I). The native molecular weight of the control protein MBP/lacZ was very close to the calculated value, indicating that MBP itself does not dimerize (see also Ref. 22). Both MBP/Tctex2 and MBP/LC2 yielded molecular weight estimates that strongly suggest these proteins are monomeric.
Further evidence for the dimerization of Tctex1 was obtained from a yeast two-hybrid screen of a mouse brain library using murine Tctex1 as the bait. The overall goal of this screen was to identify potential dynein-cargo interactions mediated by Tc-tex1. A number of novel interactions between Tctex1 and other proteins were identified. Intriguingly, ϳ10% of the confirmed clones recovered from the screen were found to encode Tctex1 itself, suggesting that the Tctex1-Tctex1 interaction can occur in vivo. Clones encoding other members of this dynein LC family were not obtained from this screen even though at least one, rp3, is expressed at much higher levels in brain than Tctex1 (see below).
Interaction of Tctex2 with an LC8-related Protein-The Tctex1 and Tctex2 LCs associate with the dynein ICs and several additional LCs at the base of the soluble dynein particle (47). In Chlamydomonas flagellar outer arm dynein, LC2 (Tctex2) is essential for the assembly of the dynein particle into the axoneme (51). To further investigate interactions involving this LC class, Chlamydomonas axonemes were treated for 60 min with 0 -10 mM DMP (Fig. 3a), which cross-links primary amines with a final linker length of 9.2 Å. Reactions were terminated by addition of gel sample buffer, and the samples were electrophoresed. Following Coomassie Blue staining, cross-linking was evident as a smearing of individual bands and an increase in the background staining in samples treated with the highest concentrations of DMP (Fig. 3b). To identify interactions involving LC2, both purified dynein and isolated axoneme samples were treated with DMP, blotted to nitrocellulose, and probed with antibodies that react with individual dynein components. In both dynein and axoneme samples, a single major cross-linked product of M r 32,000 was observed, suggesting that LC2 (M r ϭ 20,000 (52); actual mass ϭ 15,883 Da) had become cross-linked to a protein of ϳ14 kDa (Fig. 4, a  and b). Examination of identical samples with antibody R4928 revealed that this band also contained LC6 (mass ϭ 13,857 Da), an LC that is closely related to the highly conserved LC8 protein found in many different enzyme systems (19). Antibodies against other dynein LCs did not detect this band. These data suggest that LC2 and LC6 interact directly in situ. The minor band migrating slightly more slowly than the LC2-LC6 2 The proposed arrangement of secondary structural elements within complex recognized by antibodies R4928 and R5391 likely represents a variant of reduced charge generated by modification of additional Lys residues. Similar charge modification products were observed previously following treatment of LC8 with amine-selective cross-linking reagents (22).
In the purified dynein sample, an additional minor DMP product of M r ϳ 100,000 containing both LC2 and LC6 was observed. This band is of the appropriate size to represent attachment of the LC2-LC6 complex to a dynein IC. The R4928 antibody against LC6 also detected one DMP-generated band of Tctex1 (chltctex1; AF039437), human rp3 (humanrp3; U02556), Chlamydomonas LC2 (chlLC2; U89649), Caenorhabditis elegans ORF (d1009-5), C. elegans EST (C48724), C. elegans ORF (t05c12-5). b, following alignment using ClustalW, distances were calculated with PROTDIST and FITCH from the Phylip suite of programs, and the tree was constructed using DRAWTREE. The unrooted tree reveals two major subclasses of this LC family centered on the Tctex1 and Tctex2 proteins.
M r 29,000 that did not contain either LC2 or any other dynein LC. Given that there are two copies of LC6 per dynein particle (52) and that the related LC8 protein is dimeric (22), this band most probably derives from direct LC6-LC6 cross-linking. The calculated mass of the LC6 dimer (27,714 Da) is consistent with this interpretation.
Inner arm I1 contains both Tctex1 and LC8 (35). However, in contrast to LC2 (Tctex2) and LC6 in the outer arm, no products containing Tctex1 and LC8 were observed in DMP-cross-linked axoneme samples (data not shown).
Tctex2 Is a Component of Cytoplasmic Dynein-Tctex2, unlike Tctex1, has been described previously only in flagellar outer arm dynein (33). To further examine whether Tctex2 is indeed flagellar-specific, murine Tctex2 was expressed as a C-terminal fusion with maltose-binding protein (Fig. 5a) and used as the immunogen for antiserum production in rabbit R7714. Tctex2 was separated from the fusion partner by digestion with Factor Xa (Fig. 5a), and the purified light chain used to obtain a Tctex2-specific antibody fraction from R7714 serum FIG. 2. Tctex1 is a dimer in solution. To determine the native molecular weight and oligomerization status of this class of LC, recombinant Chlamydomonas Tctex1 was subjected to gel filtration chromatography on a Superose 6 column. A single major peak with a molecular mass of 31.5 kDa was observed. Some aggregated protein of very high molecular weight also is evident in the chromatogram. The left inset shows the column calibration using serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa) as standards. Right inset, electrophoretic analysis on a 12.5% acrylamide gel of the Tctex1 preparation prior to (left lane) and following (right lane) concentration in a Centricon-10 unit. Equal volumes of the two preparations were loaded. Some Tctex1 protein migrates as a dimer even in the presence of SDS. These data suggest that Tctex1 (monomer molecular mass ϭ 12.8 kDa) exists as a dimer in solution.   4. DMP cross-linking reveals the interaction of the Tctex1/Tctex2 and LC8 protein families in outer arm dynein. a, purified Chlamydomonas outer arm dynein was treated with the indicated concentrations of DMP. Following electrophoresis and blotting to nitrocellulose, the samples were probed with antibodies R5391 and R4928 that are specific for LC2 (Tctex2) and LC6 (a homologue of LC8), respectively. After treatment with DMP, both antibodies detect the same major product of M r 32,000, indicating that LC2 and LC6 have been cross-linked to each other. The minor product at M r ϳ 100,000 likely derives from cross-linking of the LC2⅐LC6 complex to one dynein IC. b, isolated Chlamydomonas flagellar axonemes were treated with 10 mM DMP and probed with the R5391 and R4928 antibodies. Both antibodies detect the M r 32,000 band observed in a, indicating that the interaction between LC2 and LC6 also occurs in situ.
by blot affinity. The specificity of antibody R7714 was assessed by immunoblot analysis against recombinant members of the Tctex1/Tctex2 family, including the Chlamydomonas Tctex2 homologue (LC2) and mammalian Tctex1, Tctex2, and rp3 (Fig.  5b). Immunoreactivity was observed only against mammalian Tctex2, indicating that the antibody is indeed highly specific and can readily distinguish between various members of this LC class. Tctex2 is expressed at high levels in mammalian testis (32) (and see below). Therefore, the specificity of the R7714 antibody was further assessed by immunoblot analysis against a whole testis homogenate (Fig. 5c). Both R7714 and R5205 (versus Tctex1) antibodies revealed single immunoreactive bands in testis; with R5205 a very minor band at M r ϭ 61,000 also was detected following very prolonged exposure.
To determine whether Tctex2 occurs in cytoplasmic as well as flagellar dynein, the cytoplasmic isozyme was purified by immunoprecipitation from rat brain, kidney, liver, spleen, and testis extracts using monoclonal antibody 74-1 (44). Purified dynein from all these tissues has previously been shown to contain both the Tctex1 and rp3 LCs (47). In contrast, immunoblot analysis using the R7714 antibody against Tctex2 detected immunoreactive bands only in cytoplasmic dynein samples derived from kidney and spleen but not in those from brain, liver, and testis (Fig. 5d). The lack of signal in the testis cytoplasmic dynein sample suggests that the immunoprecipitates obtained using the 74-1 monoclonal antibody contain only cytoplasmic dynein and are not contaminated with detectable amounts of sperm flagellar dyneins. These data suggest that Tctex2, like Tctex1, is present in subsets of both flagellar and cytoplasmic dyneins. Although the R7714 antibody did not react with other members of this protein family on immunoblots, it does remain possible that the protein detected in cytoplasmic dynein samples by R7714 is closely related, but not identical, to Tctex2.
Tissue-specific Differential Expression-In addition to the high level expression of Tctex2 in testis (32), previous studies have suggested that Tctex1 and rp3 are differentially expressed during brain development (38,41). Because these LCs have different binding affinities for at least one cytoplasmic dynein cargo (36), differential LC expression may act as a mechanism to regulate the interaction of specific cellular cargoes with the cytoplasmic dynein motor. Therefore, to further investigate the tissue-specific distribution of the Tctex1/Tctex2 family, poly(A) ϩ RNA from a wide variety of adult and fetal human tissues was examined by Northern dot blot analysis. The multiple tissue mRNA array was probed sequentially with FIG. 6. Tissue-specific expression of Tctex1/Tctex2 family proteins in humans. Northern dot blot analysis of poly(A) ϩ RNA from various human tissues. The array was obtained from CLONTECH and sequentially probed with the following cDNAs: rp3, Tctex1, Tctex2, AI421187, and AI492091. The latter EST derived from B-lymphocytes and is essentially identical to the kidney-derived EST AW612564 used to construct the phylogenetic tree shown in Fig. 1b. FIG. 5. Tctex2 is present in mammalian cytoplasmic dynein. a, murine Tctex2 was expressed as a C-terminal fusion with maltosebinding protein and purified by amylose affinity chromatography. The resulting fusion protein was used as the immunogen for preparation of antiserum R7714. Following digestion with Factor Xa, the Tctex2 light chain was separated from the fusion partner. In the uncut sample, a band of M r ϳ 130,000 corresponding to MBP/Tctex2 dimer is evident. The intensity of this band is diminished following Factor Xa digestion. b, polyclonal rabbit antiserum R7714 was raised against murine Tctex2 expressed as a C-terminal fusion with maltose-binding protein. Following blot purification versus recombinant Tctex2, the specificity of the antibody was assessed by immunoblot analysis of several recombinant dynein LCs, including human rp3, Chlamydomonas LC2 (a Tctex2 homologue), Chlamydomonas Tctex1, and murine Tctex2. c, approximately 150 g/lane testis homogenate were electrophoresed in a 5-15% acrylamide gradient gel. Total protein is shown in the left lane (Coomassie Blue stain). Identical samples were blotted to nitrocellulose and probed with blot-purified antibodies R5205 and R7714 versus Tctex1 and Tctex2. Both antibodies are highly specific and recognize single bands of the appropriate M r (14,000 and 22,000, respectively). d, cytoplasmic dynein was immunoprecipitated from rat brain, kidney, liver, spleen, and testis using monoclonal antibody 74-1 (44). Following electrophoresis and immunoblotting using blot-purified R7714, bands of M r ϳ 20,000 were detected in cytoplasmic dynein samples from kidney and spleen but not in those from brain, liver, and testis. human clones encoding rp3, Tctex1, Tctex2, and the AI421187 (from glioblastoma) and AI492091 (from B-lymphocyte, essentially identical to the kidney-derived AW612564) ESTs (Fig. 6). All five probes revealed tissue-specific alterations in expression level. For example, rp3 was heavily expressed in adult brain, kidney, and liver but not in bone marrow or ovary. In contrast, the close homologue Tctex1 was readily detected in many tissues, including bone marrow but was less evident in adult brain. Tctex2 and related ESTs exhibited a more restricted distribution. Minor amounts of Tctex2 were detected in most samples with enhanced levels found only in testis, adult liver, and fetal thymus. All LCs showed differential expression between certain adult and fetal tissues, suggesting that the levels of these proteins are controlled in a developmentally regulated manner. Interestingly, not all LCs were regulated in the same fashion. For instance in liver, Tctex2, rp3, and AI421187 were significantly up-regulated in the adult tissue whereas both Tctex1 and AI492091 showed little alteration. In contrast, both Tctex2 and AI492091 were up-regulated in fetal thymus whereas the other proteins were not.

DISCUSSION
Members of the Tctex1/Tctex2 class of dynein LCs associate with the ICs and several additional LCs at the base of the soluble motor particle. Genetic studies in Chlamydomonas have revealed that strains lacking the Tctex2 homologue LC2 fail to assemble outer dynein arms and thus have compromised motility (53). Furthermore, there is now considerable support for the hypothesis that these proteins mediate specific cytoplasmic dynein-cargo interactions, including those with rhodopsin (36), Fyn kinase (37,38), and the vesicle-associated protein Doc-2 (39). To gain further insight into the role of this LC class in dynein function we report here on the structure and expression of these polypeptides and on the protein-protein interactions in which they are involved.
Models for protein-protein associations within the IC-LC complex of both cytoplasmic dynein and the flagellar outer arm are shown in Fig. 7. Both sets of ICs interact with HCs, the LC8 dimer, a member of the LC7/roadblock family and cargo (␣-tubulin or dynactin). The major difference is that the outer arm ICs associate with monomeric Tctex2 and the LC6 dimer, whereas most cytoplasmic dynein ICs bind the Tctex1 dimer as apparently is also the case for inner arm I1. It will be of interest to determine whether a single cytoplasmic dynein particle can directly bind both the Tctex1 dimer and a Tctex2 monomer, whether their association is mutually exclusive or if interaction of Tctex2 requires accessory proteins such as LC6.
Identification of additional mammalian Tctex2-related proteins is of considerable interest, because this LC subclass had previously been reported only in the flagellar outer arm (33). Although there is significant similarity between these proteins, Tctex2 and its close relatives include N-terminal extensions of varying lengths that may mediate gene-specific functions. Several of these novel homologues are derived from cells/tissues that do not contain cilia/flagella raising the possibility that members of this LC group also may occur in the cytoplasm. Examination of cytoplasmic dynein samples revealed the presence of Tctex2 in kidney and spleen but not in brain, liver, or testis. Because Tctex2 is heavily expressed in testis where it is a component of flagellar dynein, this suggests that the cytoplasmic dynein immunoprecipitates were not contaminated with detectable amounts of the flagellar enzyme. These data support the hypothesis that Tctex2 proteins, like Tctex1, are present in both flagellar and cytoplasmic dyneins, although their tissue distribution is considerably more restricted.
Both Tctex1 and Tctex2 were originally described as testisspecific proteins, although that interpretation has now been revised. Previous studies indicated that Tctex1 and its close homologue rp3 are differentially expressed during brain development (38,41) with Tctex1 mRNA being more prominent in fetal than adult brain whereas rp3 followed the opposite pattern. The Northern analysis presented here confirms that rp3 is greatly up-regulated in the adult tissue whereas Tctex1 is present in much lower amounts. Although, intriguingly, both proteins may be readily detected in cytoplasmic dynein samples derived from adult rat brain (47). Tctex1 especially was present in high levels in many tissues, whereas rp3 expression FIG. 7. Model for protein-protein interactions within dynein IC⅐LC complexes. These models depict known protein-protein interactions within the IC⅐LC complexes from Chlamydomonas outer arm dynein and mammalian cytoplasmic dynein. Although certain similarities are evident, the models also suggest that a major difference is in the binding of either a Tctex1 dimer or monomeric LC2 (Tctex2) combined with the presence of, and interaction with, the LC8 homologue LC6. Although flagellar dyneins contain either Tctex1 or Tctex2, it is unclear whether binding of Tctex1 and Tctex2 to cytoplasmic dynein is mutually exclusive. The LC8 dimer structure derives from Protein Data Bank accession number 1B1W (23).
was considerably more restricted. Tctex2 and its related homologues also were found in lesser amounts, and indeed Tctex2 and the glioblastoma-derived EST were present at high levels in only a few tissues, with clear differences between certain fetal and adult samples. These expression studies suggest that cytoplasmic dynein derived from various tissues differs in terms of LC content, supporting the notion that the resulting dynein subtypes may exhibit distinct cargo-binding activities.
Murine Tctex1 and Tctex2 are encoded within a region of chromosome 17 known as the t complex (31,32). Variant forms of this chromosomal region (the t haplotypes) are transmitted in a non-Mendelian fashion by heterozygous males due to expression of mutant responder and distorter proteins that lead to defects in sperm bearing the wild-type version of the chromosome (reviewed in Ref. 54). Homozygosity for t haplotypes results in either embryonic lethality due to recessive lethal factors (for identical t haplotypes; t x /t x ) or male sterility (with complementing t haplotypes; t x /t y ) caused by a series of sterility factors. Tctex1 and Tctex2 are candidates for the proximal and central distorter/sterility factors based on gene location, testis-enriched expression and the presence of t haplotypeencoded mutations that might affect protein function (31,32). The distorters and sterility factors were previously thought to be identical, because homozygosity for distorters results in infertility. However, recent high resolution mapping of the t complex region indicates that at least the proximal distorter and sterility factors are distinct genetic entities (55).
Tctex1 was initially found in cytoplasmic dynein (34) and subsequently in flagellar inner arm I1 (35). Analysis of Chlamydomonas flagella also revealed the presence of Tctex2 as an LC within the outer arm (33). This protein is encoded at the locus ODA12, and null mutants are unable to assemble outer arms and consequently swim slowly (53). These observations gave rise to the hypothesis that defects in flagellar dyneins provide the underlying biological basis for the transmission ratio distortion phenomenon (33,35). This hypothesis has recently received considerable support from the observation that the strongest distorter maps at the same location as an axonemal dynein HC encoded at the Hybrid Sterility-6 locus (56). Furthermore, the responder has now been identified as a mutant form of a protein kinase required for sperm motility (57), suggesting that differential phosphorylation of dynein proteins may be involved in production of sperm with defective motility. Intriguingly, a Tctex2 homologue in both sea urchin and chum salmon sperm is phosphorylated during the activation of flagellar motility (58), although it is not yet clear whether this phosphorylation event is required for sperm activation or is a consequence of it.
In this report, we have identified additional members of the Tctex1/Tctex2 family of dynein LCs and found that they are differentially expressed in both a tissue-specific and developmentally regulated manner. These observations lend further support to the hypothesis that LC expression patterns might affect the cargo-binding capabilities of cytoplasmic dynein.