Interaction of Doc2 with tctex-1, a Light Chain of Cytoplasmic Dynein

Doc2 has one Munc13-interacting domain at the N-terminal region and two C2-like domains interacting with Ca2+ and phospholipid at the C-terminal region. Doc2 consists of two isoforms, Doc2α and -β. Doc2α is specifically expressed in neuronal cells and implicated in Ca2+-dependent neurotransmitter release, whereas Doc2β is ubiquitously expressed and its function is unknown. We show here that both Doc2α and -β interact with rat tctex-1, a light chain of cytoplasmic dynein, in both cell-free and intact cell systems. Overexpression of the N-terminal fragment of Doc2 containing the tctex-1-interacting domain induces changes in the intracellular localization of cation-independent mannose 6-phosphate receptor and its ligand, cathepsin D, which are transported from trans-Golgi network to late endosomes. Overexpression of the C-terminal fragment containing two C2-like domains shows the similar effect, but to a lesser extent, whereas overexpression of full-length Doc2 or the C-terminal fragment of rabphilin3 containing two C2-like domains does not show this effect. Because dynein is a minus-end-directed microtubule-based motor protein, these results suggest that Doc2, especially Doc2β, plays a role in dynein-dependent intracellular vesicle transport.

We have isolated Doc2 as a novel protein having two C2-like domains that interact with Ca 2ϩ and phospholipid (1). Doc2 consists of two isoforms, Doc2␣ and -␤ (1)(2)(3). Doc2␣ is specifically expressed in neuronal cells and localized on synaptic vesicles in nerve terminals, whereas Doc2␤ is ubiquitously expressed (1)(2)(3)(4). Both isoforms have at least one DSR 1 (1-39 aa) at the N-terminal region and two C2-like domains at the C-terminal region (1,2). Overexpression of the N-terminal fragment of Doc2␣ containing DSR or its C-terminal fragment containing the C2-like domains in PC12 cells inhibits Ca 2ϩ -dependent exocytosis (5). We have recently isolated a protein interacting with Doc2␣ and -␤ and identified it to be Munc13-1 (6). Doc2␣ and -␤ interact with Munc13-1 through the region within DSR (13-37 aa). Munc13-1 is specifically expressed in neuronal cells and localized on the presynaptic plasma membrane (7,8). Munc13-1 has two C2-like domains and one C1like domain that interacts with diacylglycerol or phorbol ester (7,8). The Doc2␣-Munc13-1 interaction is induced by the binding of diacylglycerol or phorbol ester to the C1-like domain of Munc13-1 and causes the docking of the vesicles to the plasma membrane in nerve terminals (6,9).
In contrast to Doc2␣, little is known about Doc2␤. To clarify the function of Doc2, particularly that of Doc2␤, it is important to isolate a Doc2-interacting protein(s) that is ubiquitously expressed. We have attempted here to isolate a Doc2-interacting protein(s) from a rat brain cDNA library by use of the yeast two-hybrid system and identified it to be tctex-1. tctex-1 interacted with both Doc2␣ and -␤ at the N-terminal region. tctex-1 was originally isolated as a candidate for involvement in the transmission ratio distortion of mouse t-haplotypes (10). tctex-1 has recently turned out to be a light chain of cytoplasmic dynein (11), a minus-end-directed microtubule-based motor protein (for reviews, see Refs. [12][13][14][15]. Cytoplasmic dynein interacts with a variety of structures, such as late endosomes, lysosomes, the Golgi complex, synaptic vesicles, and ER, and is implicated in transport of these organelles and vesicle transport between these organelles (12)(13)(14)(15). We describe here the Doc2-tctex-1-interaction and discuss the function of this interaction.

EXPERIMENTAL PROCEDURES
Materials and Chemicals-The N-terminal fragment (1-90 aa) of human Doc2␣ cDNA (1) was inserted into pGEX-2T vector, expressed in Escherichia coli as a GST fusion protein, and purified on a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech). Mammalian expression plasmids, pEFBOS-HA and pEFBOS-Myc, and in vitro and in vivo expression plasmids, pGEM-HA and pRSET-Flag, were generated to express fusion proteins with the N-terminal HA, Myc, or Flag epitope (5,6). The cDNA fragments encoding mouse tctex-1 (10) and human RP-3 (16) were obtained by polymerase chain reaction from mouse and human brain cDNA libraries, respectively. BHK cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C in 5% CO 2 (17). Anti-CIMPR and anticathepsin D rabbit polyclonal antibodies were prepared and purified by affinity chromatography (18). Fluorescein-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG were obtained from Jackson Immunoresearch Laboratories (West Grove, PA) and Biomeda Corp. (Foster, CA), respectively. * The investigation at Osaka University Medical School was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports, and Culture, Japan (1997,1998), by grants-in-aid for Abnormalities in Hormone Receptor Mechanisms and for Aging and Health from the Ministry of Health and Welfare, Japan (1997,1998), and by a grant from the Human Frontier Science Program (1997,1998). The costs of publication of this article were defrayed in part by the payment of page charges. This 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 /EBI Data Bank with accession number(s) AB010119.
Two-hybrid Screening-The N-terminal fragment (1-90 aa) of human Doc2␣ cDNA (1) was inserted into pBTM116 (pLexA-Doc2␣N) (6). The yeast reporter strain L40 was transformed with pLexA-Doc2␣N and a rat brain cDNA library constructed in pGAD10 (CLONTECH). Library plasmids from positive clones were analyzed by transformation tests and DNA sequencing. The clones containing the putative fulllength coding region of tctex-1 were isolated by screening a rat brain cDNA library. The cDNA fragments encoding mouse tctex-1 (10) and human RP-3 (16) were inserted into pGAD10. The cDNA fragments encoding several Doc2␣ and -␤ deletion mutants were inserted into pBTM116 (6). After co-transformation into yeast strain L40, ␤-galactosidase activity was measured by liquid and filter assays (19,20).
Assay for Doc2-tctex-1 Interaction in a Cell-free System-The cDNA fragment of tctex-1 or RP-3, which was inserted into pRSET-Flag, was translated in vitro using a TNT T7-coupled reticulocyte lysate system (Promega, Madison, WI). Two g of GST-Doc2␣ (1-90 aa) was immobilized onto 20 l of glutathione-Sepharose 4B beads. The immobilized beads were added to 500 l of Buffer A (150 mM NaCl, 50 mM HEPES/ NaOH, pH 7.4, and 1 mM EGTA) containing the in vitro translated product, and gently mixed at 4°C for 4 h. The beads were washed four times with Buffer A, and bound proteins were eluted by addition of 100 l of Buffer A containing 20 mM glutathione. The eluates were subjected to SDS-PAGE followed by autoradiography.
Assay for Doc2-tctex-1 Interaction in an Intact Cell System-HeLa cells were plated at a density of 1 ϫ 10 5 cells/60-mm dish and incubated for 18 h. The cells were infected for 30 min with T7 RNA polymerase recombinant vaccinia virus (LO-T7) and then co-transfected with 2 g of pGEM-HA encoding Doc2␣ and 2 g of pRSET-Flag encoding tctex-1, by use of a LipofectAMINE reagent (Life Technologies, Inc.). At 5 h after the transfection, the cell lysate was prepared and subjected to immunoprecipitation with 3 g of the anti-HA monoclonal antibody bound to 20 l of protein A-Sepharose beads (6). Comparable amounts of the pellets were subjected to SDS-PAGE, followed by immunoblot analysis with the biotinylated mouse anti-Flag antibody (Eastman Kodak, New Haven, CT).
Effect of Overexpression of the Doc2 Mutants on the Intracellular Localization of CIMPR and Cathepsin D in BHK Cells-BHK cells were transfected with 3 g of the pEF-BOS-HA or -Myc encoding the indicated protein by use of a SuperFect reagent (Qiagen, Chatsworth, CA). At 12 h after the transfection, the cells were detached using an EDTA/ trypsin solution, seeded onto 35-mm grid dishes, and further incubated for 12 h. The cells were then fixed, co-stained with the anti-HA or -Myc antibody and the anti-CIMPR or -cathepsin D antibody, and analyzed by confocal microscopy (LSM-GB200; Olympus, Tokyo, Japan).
Other Procedures-Northern blot analysis was performed using the Northern blot sheet of poly(A) ϩ RNA from various rat tissues (CLON-TECH). The rat tctex-1 cDNA containing the whole open reading frame was used as a probe. SDS-PAGE and immunoblotting were performed as described (21,22). Protein concentrations were determined with bovine serum albumin as a reference protein as described (23).

RESULTS AND DISCUSSION
We first attempted to isolate a Doc2-interacting protein which is ubiquitously expressed by use of the yeast two-hybrid system with the N-terminal region (1-90 aa) of Doc2␣ as a bait from a rat brain cDNA library. Screening of 1 ϫ 10 6 transformants yielded nine independent positive clones that interacted with Doc2␣. The seven positive clones encoded Munc13-1 as described (6). The remaining two positive clones had the same cDNA inserts with 0.75 and 0.73 kb, respectively. Using the cDNA with 0.75 kb as a probe, we isolated the cDNA encoding the whole open reading frame from a rat brain cDNA library. The nucleotide sequence analysis of the cloned cDNA revealed that the encoded protein consisted of 113 aa and showed a calculated M r of 12,438 (GenBank TM accession number AB010119) (Fig. 1A). The deduced aa sequence of this molecule had 96% identity with mouse tctex-1 (GenBank TM accession number M25825) (10) and 52% identity with human RP-3 (Gen-Bank TM accession number U02556) (16) (Fig. 1B). RP-3 was isolated as a candidate gene of X-linked retinitis pigmentosa type 3 (16). Therefore, we concluded that the cloned cDNA is rat tctex-1 cDNA. The rat tctex-1 mRNA level was examined in various rat tissues by Northern blot analysis. The tctex-1 mRNA appeared as a single band of approximately 0.8 kb and was ubiquitously expressed, although it was abundantly expressed in testis (data not shown). These results suggest that tctex-1 is a Doc2-interacting protein that is ubiquitously expressed.
The Doc2-tctex-1 interaction was confirmed by the binding of in vitro translated, [ 35 S]methionine-labeled tctex-1 to the GSTtagged N-terminal region of Doc2␣ (1-90 aa) (Fig. 2B). In vitro translated RP-3 did not bind to the GST-tagged N-terminal region of Doc2␣. The Doc2-tctex-1 interaction was furthermore confirmed by co-immunoprecipitation from cultured HeLa cells of Flag-tagged full-length tctex-1 and HA-tagged full-length Doc2␣ (Fig. 2C). Flag-tagged full-length RP-3 was not co-immunoprecipitated with HA-tagged full-length Doc2␣ (data not shown). These results indicate that tctex-1 interacts with Doc2 in both cell-free and intact cell systems.
It has been shown that dynein is involved in the vesicle transport from early to late endosomes (24). CIMPR and its ligand, cathepsin D, are transported from trans-Golgi network to early endosomes and then from early to late endosomes in BHK cells (25). We finally examined whether the Doc2-tctex-1 interaction is involved in this dynein-dependent vesicle transport. When the intracellular localization of CIMPR in BHK cells was analyzed by immunocytochemistry using the anti- Doc2-Dynein Interaction 30066 CIMPR antibody, CIMPR was distributed in a compact juxtanuclear region corresponding to late endosomes (Fig. 3A). Overexpression of the N-terminal fragment of Doc2␣ containing the tctex-1-binding region induced the dispersion of CI-MPR. The severity of this change varied somewhat among transfected cells, usually as a result of variation in expression levels of Doc2␣. Overexpression of the C-terminal fragment containing C2-like domains showed the similar effect, but to a lesser extent. In contrast, overexpression of full-length Doc2␣ did not affect the intracellular localization of CIMPR (data not shown). To determine the specificity of the effect of the Doc2 mutants, we used the similar mutants of rabphilin3, which is a downstream target of the Rab3 small G protein subfamily implicated in Ca 2ϩ -dependent exocytosis and has two C2-like domains (26), and full-length RP-3, which does not interact with Doc2 in both cell-free and intact cell systems. Overexpression of the N-terminal fragment of rabphilin3 containing Rab3binding domain, its C-terminal fragment containing C2-like domains (data not shown) (27), or full-length RP-3 (Fig. 3A) showed no effect. As to the effect of overexpression of the Doc2␣ fragments on the localization of cathepsin D, large dot-like structures were scattered throughout the cytoplasm in the untransfected cells (Fig. 3B). This staining indicates that mature cathepsin D is localized in lysosomes. Overexpression of the N-terminal fragment of Doc2␣ caused the disappearance of large dot-like structures. Overexpression of its C-terminal fragment showed the similar effect, but to a lesser extent. In contrast, overexpression of full-length Doc2␣, the N-terminal fragment of rabphilin3, its C-terminal fragment (data not shown), or full-length RP-3 (Fig. 3B) did not affect the intracellular localization of cathepsin D. These results suggest that Doc2 is involved at least in the vesicle transport from early to late endosomes in cooperation with dynein in BHK cells.
Cytoplasmic dynein consists of two heavy chains, three in- termediate chains, four light intermediate chains, and three light chains (11)(12)(13)(14)(15). Of these subunits, the heavy chains have ATPase activity and interact with microtubules (12)(13)(14)(15). The intermediate chains and dynactin are suggested to interact with cargos (13)(14)(15). The function of the light chains has been unknown, but our present results indicate that one of the light chains with a molecular mass of 14 kDa interacts with Doc2. The intracellular localization of Doc2␤ is unknown in nonneuronal cells, but our previous result, that Doc2␣ is localized on synaptic vesicles in neuronal cells (1), suggests that Doc2␤ is associated with some specific vesicles in nonneuronal cells. Taken together, Doc2 may link dynein to its cargos through interaction with tctex-1. Doc2 probably associates with cargos through its C-terminal region because overexpression of the C-terminal fragment of Doc2␣ shows a similar effect of overexpression of its N-terminal fragment containing the tctex-1interacting domain.
Cytoplasmic dynein is implicated in a broad range of cellular functions in addition to the vesicle transport from early to late endosomes (12)(13)(14)(15): chromosome segregation, spindle formation, nuclear migration, the distribution of organelles (Golgi complex, late endosomes, and lysosomes), the retrograde organelle transport in axon, and the transport of intermediate compartment from ER to Golgi complex. Doc2 may be involved in these dynein-dependent functions. Further study is necessary to establish the physiological role of the Doc2-tctex-1 system.