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¹ Division of Molecular and Cell Biology, Institute for Medical Science
² Department of Gastroenterology, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu-machi, Tochigi 321-0293, Japan

	Abstract

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-Taxilin is a novel binding partner of the syntaxin family, which is implicated in intracellular vesicle traffic. We have here found that -taxilin interacts with the nascent polypeptide-associated complex (NAC), which is involved in transferring growing nascent polypeptide chains to appropriate co-translationally acting factors. NAC is composed of two subunits, - and ßNACs. Both these subunits bound to -taxilin through its C-terminal coiled-coil region in dose-dependent and saturable manners. The interactions of -taxilin with NAC and NAC but not with ßNAC were inhibited by syntaxin-4, indicating that -taxilin binds to NAC mainly through its interaction with NAC. When NAC was over-expressed in COS-7 cells, NAC was distributed in the cytosol and nucleus. However, co-expression of the -taxilin fragment containing the NAC-binding region eliminated the nuclear distribution of over-expressed NAC. Moreover, other taxilin family members, ß- and -taxilins, also bound to NAC and thereby affected the nuclear distribution of over-expressed NAC. Taken together with the evidence that NAC functions in the nucleus as a transcriptional coactivator, our results raise the possibility that the taxilin family is involved not only in the translational process through its interaction with NAC but also in the transcriptional process through its interaction with NAC alone.

	Introduction

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We have recently identified a novel syntaxin-binding protein and named it -taxilin, which is involved in intracellular vesicle traffic (Nogami et al. 2003a). In mammals, there is the taxilin family composed of at least three members, -, ß- and -taxilins (Nogami et al. 2004). The taxilin family shares an extraordinarily long coiled-coil region homologous to that of Uso1, a yeast tethering factor homolog of p115 (Nogami et al. 2004). - and -Taxilins are ubiquitously expressed but ß-taxilin is abundantly expressed in the skeletal muscle and heart (Fujimori et al. 2002; Nogami et al. 2004). The taxilin family interacts with syntaxin family members localized on the plasma membrane but not with those localized on the intracellular organelles (Nogami et al. 2003a). Therefore, we assume that the taxilin family is involved in the transport of the vesicles delivered to the plasma membrane.

The syntaxin family is a central player among a set of membrane proteins called soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are involved in regulation of fusion of vesicles with the target membrane (Chen & Scheller 2001). The SNARE proteins are divided into two groups such as vesicle-SNARE (v-SNARE) and target-SNARE (t-SNARE). v-SNARE, which consists of the vesicle-associated membrane protein (VAMP) family, resides on the vesicles. t-SNAREs, which consist of the syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) family, mainly reside on the target membrane. v- and t-SNAREs recognize each other and assemble into a biochemically stable trans-SNARE complex, which bridges the vesicles close to the target membrane and mediates vesicle fusion. -Taxilin binds to the syntaxin family members through their C-terminal coiled-coil region, which is involved in the SNARE complex formation (Nogami et al. 2003a). -Taxilin is not able to interact with syntaxin-1 complexed with VAMP2, SNAP-25 or Munc18 (Nogami et al. 2003b). Taken together, we assume that the taxilin family is involved in tethering of the vesicles to the target membrane before the formation of the trans-SNARE complex. However, at this time, a precise role of the taxilin family has not been well known.

The nascent polypeptide-associated complex (NAC) is an abundant cytosolic protein, which is involved in transferring growing nascent polypeptides to appropriate co-translationally acting factors through its interaction with the nascent chains on the ribosome (Rospert et al. 2002). NAC is ubiquitously expressed and the intracellular NAC concentration varies in different tissues, ranging from 3 to 10 µM (Möller et al. 1998). NAC is composed of alpha and beta subunits which form a stable heterodimeric complex (Wiedmann et al. 1994). Almost all of - and ßNACs form the heterodimeric complex in vivo, and the - and ßNACs forming a stable heterodimeric complex bind to the nascent chains on the ribosome in a functionally different manner (Beatrix et al. 2000). The importance of NAC in vivo function is emphasized by early embryonically lethal phenotypes of NAC mutants in mice and fruit flies (Deng & Behringer 1995; Markesich et al. 2000). Accumulating evidence indicates that NAC plays a role in shielding the nascent chains on the ribosome toward the cytosol and that NAC prevents inappropriate targeting of non-translating ribosome and ribosome nascent chain complex bearing signal-less nascent chains to the ER membrane by occupying the proposed membrane-attachment site on the ribosome (Lauring et al. 1995; Wang et al. 1995). Moreover, on the basis of the following observations, several groups have recently proposed that NAC is involved in the transcriptional process (Rospert et al. 2002). (1) NAC functions in the nucleus as a transcriptional coactivator for c-Jun (Moreau et al. 1998); (2) Inhibition of NAC degradation by inhibition of the proteasome pathway or glycogen synthase kinase 3ß (GSK3ß) activity results in accumulation of NAC in the nucleus and increases its coactivating potency (Quélo et al. 2004a); (3) Egd2p, a yeast homolog of NAC, is transported from the cytosol into the nucleus via the importin-dependent pathway (Franke et al. 2001).

We here attempted to isolate an -taxilin-binding protein by use of the yeast two-hybrid method, identified it to be NAC, and found that -taxilin binds to NAC mainly through its interaction with NAC. Moreover, we found that -taxilin regulates the translocation of NAC from the cytosol to the nucleus. Our results suggest new perspectives in the function of the taxilin family.

	Results

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Interaction of NAC with -taxilin

We attempted to isolate an -taxilin-interacting protein from a rat brain cDNA library using the yeast two-hybrid method with the C-terminal region of -taxilin (aa 315–497) as a bait. Among positive clones, one clone encoded full-length NAC. Then, we examined by use of a pull-down assay with GST--taxilin whether NAC interacts with -taxilin. When the Triton X-100-soluble fraction of Hela cells was subjected to SDS-PAGE followed by Western blotting with the anti-NAC antibody, one protein band, of which the mobility was similar to that of native NAC, was detected (Fig. 1A). When the fraction was incubated with GST or GST--taxilin immobilized on glutathione Sepharose beads, native NAC bound to GST--taxilin but not to GST (Fig. 1A). Then, we examined by the pull-down assay whether -taxilin directly interacts with NAC. When GST--taxilin immobilized on the beads was incubated with various concentrations of recombinant NAC, recombinant NAC bound to GST--taxilin in dose-dependent and saturable manners (Fig. 1B). About 0.17 mol of NAC maximally bound to one mol of GST--taxilin and the concentration of NAC giving a half-maximal binding to GST--taxilin was about 20 nM. Taken together, these results indicate that -taxilin directly interacts with NAC with relatively high affinity.

View larger version (22K): [in this window] [in a new window]   Figure 1  Interaction of NAC with -taxilin. (A) Interaction of -taxilin with native NAC. The Triton X-100-soluble fraction of Hela cells was incubated with GST or GST--taxilin (30 µg each). After the beads were intensively washed, proteins bound to the beads were eluted. The original sample (4% of the input) and an aliquot of the eluate (40%) were subjected to SDS-PAGE followed by Western blotting with the anti-NAC antibody. (B) Dose-dependent and saturable binding of NAC to -taxilin. GST--taxilin (5 µg) immobilized on glutathione Sepharose beads was incubated with various concentrations of recombinant NAC. After the beads were intensively washed, proteins bound to the beads were eluted. An aliquot of the eluate (20%) was subjected to SDS-PAGE followed by the Western blotting with the anti-NAC antibody. (a) Western blotting; (b) quantitation. The values were expressed as means ± SEM of four independent experiments. The results shown are representative of four independent experiments.

Interaction of ßNAC with -taxilin

It has been revealed that NAC forms a stable heterodimeric complex with ßNAC (Wiedmann et al. 1994; Beatrix et al. 2000). Therefore, it is important to examine whether NAC complexed with ßNAC binds to -taxilin. When the Triton X-100-soluble fraction of Hela cells was subjected to SDS-PAGE followed by Western blotting with the anti-ßNAC antibody, two protein bands were detected (Fig. 2A). The mobilities of the upper and lower protein bands were similar to those of native ß2- and ß1NACs, respectively (Beatrix et al. 2000). ß2NAC is a splicing variant of ß1NAC (Beatrix et al. 2000). When the fraction was incubated with GST or GST--taxilin immobilized on glutathione Sepharose beads, both of native ßNACs were detected in the eluted fraction from GST--taxilin-immobilized beads but not in that from GST-immobilized beads (Fig. 2A). -, ß1- and ß2NACs bound to GST--taxilin at a molar ratio of about 1 : 1: 1. Since it has been revealed that almost all of - and ßNACs are present as a stable heterodimeric complex in bovine brain cytosol (Beatrix et al. 2000), the result suggests that NAC complexed with ßNAC binds to -taxilin. However, since the amounts of ß1- and ß2NACs bound to GST--taxilin are two-fold larger than that of NAC bound to GST--taxilin, it is possible that ßNAC also directly binds to -taxilin. Then, to solve the issue, the following experiment was performed. When GST--taxilin immobilized on the beads was incubated with various concentrations of recombinant ß1NAC, recombinant ß1NAC bound to GST--taxilin in dose-dependent and saturable manners (Fig. 2B). About 0.2 mol of ß1NAC maximally bound to one mol of GST--taxilin and the concentration of ß1NAC giving a half-maximal binding to GST--taxilin was about 3 µM. Taken together, these results indicate that -taxilin directly interacts with ßNAC with relatively low affinity.

View larger version (22K): [in this window] [in a new window]   Figure 2  Interaction of ßNAC with -taxilin. (A) Interaction of -taxilin with native ßNAC. The Triton X-100-soluble fraction of Hela cells was incubated with GST or GST--taxilin (30 µg each). After the beads were intensively washed, proteins bound to the beads were eluted. The original sample (4% of the input) and an aliquot of the eluate (40%) were subjected to SDS-PAGE followed by Western blotting with the anti-ßNAC antibody. The arrows and arrowheads indicate ß2- and ß1NACs, respectively. (B) Dose-dependent and saturable binding of ß1NAC to -taxilin. GST--taxilin (5 µg) immobilized on glutathione Sepharose beads was incubated with various concentrations of recombinant ß1NAC. After the beads were intensively washed, proteins bound to the beads were eluted. An aliquot of the eluate (20%) was subjected to SDS-PAGE followed by the Western blotting with the anti-ßNAC antibody. (a) Western blotting; (b) quantitation. The values were expressed as means ± SEM of four independent experiments. The results shown are representative of four independent experiments.

Inhibition of the interaction of NAC with -taxilin by syntaxin-4

The binding regions of -taxilin to - and ß1NACs were determined by the yeast two-hybrid method. The C-terminal half of the coiled-coil region of -taxilin (aa 315–497) was responsible for its interaction with NAC (Fig. 3Ab). On the other hand, the same region was also responsible for the interaction of -taxilin with ß1NAC (Fig. 3Ac). This region of -taxilin overlapped with the syntaxin-binding region (Nogami et al. 2003a). Therefore, it is possible that syntaxin affects the interactions of -taxilin with - and ßNACs. Then, we examined by use of the pull-down assay whether the interactions of -taxilin with - and ß1NACs are affected by syntaxin-4, which more favorably interacts with -taxilin among syntaxin family members (Nogami et al. 2003b). First, to prepare NAC complexed with GST--taxilin, GST--taxilin immobilized on glutathione Sepharose beads was incubated with NAC. Second, the complex was incubated with various concentrations of syntaxin-4C. Syntaxin–4C inhibited the interaction of NAC with GST--taxilin in a dose-dependent manner through its interaction with GST--taxilin (Fig. 3Ba). The inhibitory effect of syntaxin–4C on the interaction of NAC with GST--taxilin was eliminated by the pretreatment of syntaxin-4C with trypsin (data not shown). The same experiments were performed by use of ß1NAC instead of NAC. Even though ß1NAC binds to the same region of -taxilin as NAC, syntaxin-4C did not affect the interaction of ß1NAC with GST--taxilin (Fig. 3Bb). Syntaxin-4C did not interact with either - or ß1NAC (data not shown). These results indicate that syntaxin and NAC but not ßNAC mutually interact with -taxilin and that - and ßNACs bind to -taxilin in a different manner.

View larger version (39K): [in this window] [in a new window]   Figure 3  Inhibition of the interaction of NAC with -taxilin by syntaxin-4. (A) Binding regions of -taxilin to - and ß1NACs. A pGBKT7 vector harboring the cDNA fragment of the indicated truncated forms of -taxilin and a pGAD424 vector harboring the cDNA fragment encoding - or ß1NAC were cointroduced into yeast reporter strain AH109. Equal amount of the indicated transformants was spotted on to the indicated plates, and incubated for 3 days at 30 °C. (a) Schematic representation of -taxilin and its truncated forms; (b) binding region of -taxilin to NAC; (c) binding region of -taxilin to ß1NAC. (B) Effect of syntaxin–4 on the interactions of -taxilin with - and ß1NACs. First, to prepare - and ß1NACs complexed with GST--taxilin, GST--taxilin (5 µg) immobilized on glutathione Sepharose beads was incubated with NAC (0.5 µM) and ß1NAC (10 µM), respectively. Second, the beads were incubated with various concentrations of syntaxin-4C treated with or without tryptic digestion. After the beads were intensively washed, proteins bound to the beads were eluted. An aliquot of the eluate (20%) was subjected to SDS-PAGE followed by the Western blotting with the anti-NAC antibody for NAC, with the anti-ßNAC antibody for ß1NAC, and with the anti-syntaxin-4 antibody for syntaxin-4C. (a) Effect of syntaxin–4 on the interaction of -taxilin with NAC. (upper panel) Western blotting; (lower panel) quantitation. () NAC; (•) syntaxin-4C. (b) Effect of syntaxin–4 on the interaction of -taxilin with ß1NAC. (upper panel) Western blotting; (lower panel) quantitation. () ß1NAC; (•) syntaxin-4C. The values were expressed as means ± SEM of four independent experiments. The results shown are representative of four independent experiments.

Inhibition of the interaction of NAC with -taxilin by syntaxin-4

We examined by use of the pull-down assay whether the interaction of -taxilin with NAC is affected by syntaxin-4. Since we failed to prepare NAC complexed with ß1NAC by use of recombinant - and ß1NACs, the Triton X-100-soluble fraction of Hela cells was used for resources of NAC complexed with ßNAC. First, to prepare NAC complexed with GST--taxilin, GST--taxilin immobilized on glutathione Sepharose beads was incubated with the Triton X-100-soluble fraction of Hela cells. Second, the complex was incubated with various concentrations of syntaxin-4C. ß1NAC was dissociated from GST--taxilin by syntaxin-4C in a dose-dependent manner, being in parallel with dissociation of NAC from GST--taxilin (Fig. 4). The inhibitory effect of syntaxin–4C on the interactions of GST--taxilin with - and ß1NACs was eliminated by the pretreatment of syntaxin-4C with trypsin (data not shown). Since syntaxin-4C affects the interaction of GST--taxilin with NAC but not with ß1NAC as described above, the result indicates that NAC complexed with ß1NAC is dissociated from GST--taxilin by syntaxin-4C and suggests that NAC complexed with ß1NAC binds to -taxilin mainly through its interaction with NAC. On the other hand, syntaxin-4C did not affect the interaction of ß2NAC with -taxilin (Fig. 4). It has been assumed that ß2NAC as well as ß1NAC forms a stable heterodimeric complex with NAC in vivo (Beatrix et al. 2000). However, taken together with the result that -, ß1- and ß2NACs present in the Triton X-100-soluble fraction of Hela cells bound to GST--taxilin at a molar ratio of about 1 : 1: 1, these results suggest that ß2NAC free of NAC present in the Triton X-100-soluble fraction of Hela cells binds to GST--taxilin and that biochemical property of ß2NAC is different from that of ß1NAC.

View larger version (31K): [in this window] [in a new window]   Figure 4  Inhibition of the interaction of NAC with -taxilin by syntaxin-4. First, to prepare NAC complexed with GST--taxilin, GST--taxilin (30 µg) immobilized on glutathione Sepharose beads was incubated with the Triton X-100-soluble fraction of Hela cells. Second, the beads were incubated with various concentrations of syntaxin-4C treated with or without tryptic digestion. After the beads were intensively washed, proteins bound to the beads were eluted. An aliquot of the eluate (40% for NAC, and ß1- and ß2NACs; 3% for syntaxin-4C) was subjected to SDS-PAGE followed by the Western blotting with the anti-NAC antibody for NAC, with the anti-ßNAC antibody for ß1- and ß2NACs, and with the anti-syntaxin-4 antibody for syntaxin-4C. (A) Western blotting; (B) quantitation. (•) NAC; () ß1NAC; () ß2NAC; () syntaxin-4C. The values were expressed as means ± SEM of four independent experiments. The results shown are representative of four independent experiments.

Interaction of - and ßNACs with taxilin family members

We have previously revealed that in mammals, the taxilin family consists of at least three members, namely -, ß- and -taxilins (Nogami et al. 2004). Then, we examined by use of the yeast two-hybrid method whether - and ß1NACs interact with ß- and -taxilins. NAC interacted with both of ß- and -taxilins (Fig. 5Ba). On the other hand, ß1NAC interacted with -taxilin but not with ß-taxilin (Fig. 5Bb). Although an exact reason why ß1NAC interacted with -taxilin but not with ß-taxilin, is not known, the following reasons are thought: The primary structure of ß-taxilin is less similar to those of - and -taxilins (Nogami et al. 2004). - and ß1NACs bind to -taxilin in a different manner as described above.

View larger version (32K): [in this window] [in a new window]   Figure 5  Interactions of - and ß1NACs with taxilin family members. A pGBKT7 vector harboring the cDNA fragments of ß- or -taxilin and a pGAD424 vector harboring the cDNA fragment encoding - or ß1NAC were cointroduced into yeast reporter strain AH109. Equal amount of the indicated transformants was spotted on to the indicated plates, and incubated for 3 days at 30 °C. (A) Schematic representation of the taxilin family members. (B) Interactions of - and ß1NACs with the taxilin family members. (a) Interactions of NAC with the taxilin family members; (b) interactions of ß1NAC with the taxilin family members. The results shown are representative of four independent experiments.

Effect of taxilin family members on the subcellular distribution of NAC

When HA-tagged NAC was expressed in COS-7 cells, HA-tagged NAC was stained not only in the cytosol but also in the nucleus (Fig. 6A). Since it has been recently revealed that a part of NAC translocates from the cytosol to the nucleus and subsequently regulates transcription (Moreau et al. 1998; Yotov et al. 1998), the nuclear distribution of HA-tagged NAC assumes to be meaningful. When HA-tagged NAC was co-expressed with Myc-tagged full-length -taxilin, HA-tagged NAC was stained only in the cytosol but not in the nucleus (Fig. 6Ba). Moreover, a part of HA-tagged NAC was co-localized with Myc-tagged full-length -taxilin (Fig. 6Ba). When HA-tagged NAC were coexpressed with Myc-tagged -taxilinN containing the NAC-binding region, similar results were obtained (Fig. 6Bb). However, when HA-tagged NAC were co-expressed with Myc-tagged -taxilinC lacking the NAC-binding region, HA-tagged NAC was stained not only in the cytosol but also in the nucleus and was not co-localized with Myc-tagged -taxilinC (Fig. 6Bc). These results suggest that -taxilin inhibits the translocation of NAC from the cytosol to the nucleus through its interaction with NAC. To confirm that -taxilin interacts with NAC in COS-7 cells, an immunoprecipitation assay was performed. When each Myc-tagged protein was immunoprecipitated with the anti-Myc antibody from the Triton X-100-soluble fractions of the cells co-expressing HA-tagged NAC and Myc-tagged full-length -taxilin, Myc-tagged -taxilinN, or Myc-tagged -taxilinC, HA-tagged NAC was co-immunoprecipitated with Myc-tagged full-length -taxilin and Myc-tagged -taxilinN but not with Myc-tagged -taxilinC (Fig. 6C). The same experiments were performed by use of Myc-tagged ß-taxilin or Myc-tagged -taxilin instead of Myc-tagged full-length -taxilin. Both of Myc-tagged ß-taxilin and Myc-tagged -taxilin also inhibited the translocation of NAC from the cytosol to the nucleus (Fig. 7A). A part of HA-tagged NAC was co-localized with Myc-tagged ß-taxilin and Myc-tagged -taxilin (Fig. 7A). When each Myc-tagged protein was immunoprecipitated from the Triton X-100-soluble fractions of the cells co-expressing HA-tagged NAC and Myc-tagged ß-taxilin or Myc-tagged -taxilin, HA-tagged NAC was co-immunoprecipitated with Myc-tagged ß-taxilin and Myc-tagged -taxilin (Fig. 7B). We attempted to examine the effect of the taxilin family members on the subcellular distribution of endogenous NAC. However, our antibody against NAC did not work in immunocytochemistry. These results suggest that the taxilin family regulates the translocation of NAC from the cytosol to the nucleus through its interaction with NAC.

View larger version (62K): [in this window] [in a new window]   Figure 6  Effect of -taxilin on the subcellular distribution of NAC. (A) Subcellular distribution of NAC in COS-7 cells. COS-7 cells transiently expressing HA-tagged NAC on gelatin-coated coverslips were stained with the anti-HA monoclonal antibody for HA-tagged NAC and with TOPRO-3 for the nucleus. DIC: differential interference contrast. Bar, 15 µm (B) Effect of -taxilin on the subcellular distribution of NAC. COS-7 cells transiently co-expressing HA-tagged NAC and Myc-tagged full-length -taxilin (a), Myc-tagged -taxilinN (b), or Myc-tagged -taxilinC (c) on gelatin-coated coverslips were triply stained with the anti-Myc polyclonal antibody for Myc-tagged proteins, with the anti-HA monoclonal antibody for HA-tagged NAC, and with TOPRO-3 for the nucleus. Bar, 15 µm. (C) Co-immunoprecipitation of NAC with -taxilin. A Myc-tagged protein was immunoprecipitated with the mouse immunoglobulin or with the anti-Myc monoclonal antibody (5 µg each) from the Triton X-100-soluble fraction of COS-7 cells co-expressing HA-tagged NAC and the indicated Myc-tagged forms of -taxilin. The original sample (5% of the input) and an aliquot of the eluate (40%) were subjected to SDS-PAGE followed by Western blotting with the anti-HA monoclonal antibody for HA-tagged NAC (upper panel) or with the anti-Myc polyclonal antibody for Myc-tagged proteins (lower panel). (a) Myc-tagged full-length -taxilin; (b) Myc-tagged -taxilinN; (c) Myc-tagged -taxilinC. The results shown are representative of four independent experiments.

View larger version (67K): [in this window] [in a new window]   Figure 7  Effect of taxilin family members on the subcellular distribution of NAC. (A) Effect of ß- and -taxilins on the subcellular distribution of NAC. COS-7 cells transiently co-expressing HA-tagged NAC and Myc-tagged ß-taxilin (a) or Myc-tagged -taxilin (b) on gelatin-coated coverslips were triply stained with the anti-Myc polyclonal antibody for Myc-tagged proteins, with the anti-HA monoclonal antibody for HA-tagged NAC, and with TOPRO-3 for the nucleus. Bar, 15 µm. (B) Co-immunoprecipitations of NAC with ß- and -taxilins. A Myc-tagged protein was immunoprecipitated with the mouse immunoglobulin or with the anti-Myc monoclonal antibody (5 µg each) from the Triton X-100-soluble fraction of COS-7 cells co-expressing HA-tagged NAC and Myc-tagged ß-taxilin or Myc-tagged -taxilin. The original sample (5% of the input) and an aliquot of the eluate (40%) were subjected to SDS-PAGE followed by Western blotting with the anti-HA monoclonal antibody for HA-tagged NAC (upper panel) or with the anti-Myc polyclonal antibody for Myc-tagged proteins (lower panel). (a) Myc-tagged ß-taxilin; (b) Myc-tagged -taxilin. The results shown are representative of four independent experiments.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

- and ßNACs are highly conserved proteins from yeast to human (Shi et al. 1995). NAC is thought to first contact nascent polypeptide chains emerging from the ribosome (Wiedmann et al. 1994; Beatrix et al. 2000). On the basis of the following lines of evidence, it has been assumed that NAC prevents inappropriate interactions of the nascent chains with other cytosolic proteins and thereby transfers growing nascent polypeptides to appropriate co-translationally acting factors. Depleting NAC from translating ribosome leads to an inappropriate interaction of the nascent chains lacking signal peptides with the signal recognition particle, which interacts with signal peptides of the nascent chains on the ribosome during targeting to the ER membrane (Wiedmann et al. 1994). In the absence of NAC, ribosome translating non-secretory polypeptides interacts with translocation sites on the ER membrane and subsequently the signal-less chains are translocated into the ER lumen (Lauring et al. 1995). In this study, a physiological role of the interaction of -taxilin with NAC could not be clearly determined. However, it has been recently revealed that in Drosophila, NAC is required for correct localization of oskar mRNA to the posterior pole and that loss of NAC activity disrupts the localization of oskar mRNA and subsequently affects Oskar protein accumulation (Braat et al. 2004). Therefore, it is thought that after NAC binds to the nascent Oskar peptide emerging from the ribosome and subsequently oskar mRNA on the ribosome is translocated to the posterior pole, translational elongation of Oskar peptide occurs in the posterior pole. Taken together, our results suggest that the taxilin family is involved in the translational process through its interaction with NAC and raise one possibility that the taxilin family is involved in the NAC-mediated transport of the translating ribosome, which is delivered to the restricted regions of the plasma membrane. If so, it is possible that since the amount of the translating ribosome which is localized on the restricted regions is thought to be restricted, the interaction of endogenous -taxilin with endogenous NAC was hardly detected by use of an immunoprecipitation assay (data not shown).

It has been recently revealed that NAC is involved in the transcriptional process (Rospert et al. 2002). NAC is translocated from the cytosol to the nucleus and subsequently functions as a transcriptional coactivator for c-Jun (Moreau et al. 1998). The proteasome pathway is involved in regulation of the nuclear localization of NAC. Inhibition of NAC degradation by inhibition of the proteasome pathway results in the nuclear accumulation of NAC and subsequently increases its co-activating potency (Quélo et al. 2004a). The NAC degradation on the proteasome is regulated by GSK3ß, and GSK3ß-dependent phosphorylation of NAC at threonine 159 results in degradation of the phosphorylated form of NAC on the proteasome (Quélo et al. 2004a). Moreover, it has been revealed that the nuclear localization of NAC is induced upon cell adhesion, which is followed by integrin-linked kinase (ILK) activation (Quélo et al. 2004b). It is thought that activation of ILK induces phosphorylation of GSK3ß and thereby inhibits activity of GSK3ß. Taken together, it has been proposed that accumulation of NAC free of ßNAC or other NAC-binding proteins induces the nuclear localization of NAC. In this study, we have found that over-expressed HA-tagged NAC is localized not only in the cytosol but also in the nucleus, consisting with the evidence that over-expressed Egd2p, a yeast homolog of NAC, is distributed in the cytosol and nucleus (Franke et al. 2001). The nuclear localization of HA-tagged NAC is thought to result from accumulation of HA-tagged NAC free of ßNAC or other NAC-binding proteins. Moreover, we have here found that the nuclear localization of HA-tagged NAC is inhibited by coexpression of full-length -taxilin and its truncated form containing the NAC-binding region. Similar results were obtained by co-expression of ß- or -taxilin instead of -taxilin. Taken together, our results suggest that the taxilin family regulates the activity of NAC as a transcriptional coactivator.

In this study, we have shown interactions of the taxilin family with - and ßNACs. Our present results suggest involvement of the taxilin family in the transcriptional and translational processes. However, further studies are necessary for our understanding the whole picture of the role of the taxilin family in the transcriptional and translational processes.

	Experimental procedures

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Materials and chemicals

GST-syntaxin-4C (1–273 amino acids) and --taxilin were prepared as previously described (Nakano et al. 2001; Nogami et al. 2003a). GST-NAC and -ß1NAC was purified from Escherichia coli according to the manufacturer's instructions. To obtain a recombinant protein without GST, a purified GST-fusion protein was treated with a PreScission protease (Amersham Biosciences Ltd, Buckinghamshire, UK) and further purified according to the manufacturer's instructions. An anti-syntaxin-4 antibody was prepared as previously described (Nakano et al. 2001). pcDNA-Myc--taxilin (full-length), pcDNA-Myc--taxilinC (aa 1–206), pcDNA-Myc--taxilinN (aa 192–546), pcDNA-Myc-ß-taxilin (full-length), and pcDNA-Myc--taxilin (full-length) were constructed as previously described (Nogami et al. 2003a, 2004). The cDNA fragment encoding full-length ß1NAC was cloned from a Hela cell cDNA library (Marathon-ready^TM. cDNA, BD Biosciences Clontech, Palo Alto, CA, USA) by PCR and the recombinant sequences were determined to be free of PCR errors by DNA sequence analysis. Other materials were obtained from commercial sources.

Generation of antibodies

A rabbit polyclonal antibody against His₆-tagged -taxilin was newly generated according to standard procedures (Sambrook et al. 1989). Rabbit polyclonal antibodies against the C-terminal peptides of - or ßNAC (Arg-Ala-Leu-Lys-Asn-Asn-Ser-Asn-Asp-Ile-Val-Asn-Ala-Ile-Met-Glu-Leu-Thr-Met for NAC; Glu-Asn-Phe-Asp-Glu-Ala-Ser-Lys-Asn-Glu-Ala-Asn for ßNAC) were generated according to standard procedures (Sambrook et al. 1989). Each antiserum was affinity purified by using the immunogen recombinant protein or peptide coupled to an NHS-activated column (Amersham Biosciences Ltd) according to standard procedures (Sambrook et al. 1989).

Plasmid construction

Standard recombinant DNA techniques were used to construct the following expression constructs: pGBKT7--taxilin (full-length), pGBKT7--taxilin-1 (aa 1–206), pGBKT7--taxilin-2 (aa 192–546), pGBKT7--taxilin-3 (aa 170–338), pGBKT7--taxilin-4 (aa 315–497), pGBKT7--taxilin-5 (aa 498–546), pGBKT7-ß-taxilin (full-length), pGBKT7--taxilin (full-length), pGAD424-HA-NAC (full-length), pcDNA-HA-NAC (full-length), pGEX6p-NAC (full-length), pGAD424-HA-ß1NAC (full-length), pcDNA-HA-ß1NAC (full-length), and pGEX6p-ß1NAC (full-length). The constructions were done by inserting the fragments generated by PCR or by restrict digestion into the vectors. The entire PCR products were sequenced and the structures of all plasmids were confirmed by restriction analysis.

Yeast two-hybrid

Yeast reporter strain AH109 was transformed with pGBKT7--taxilin-4. A rat brain cDNA library was introduced into the transformant according to the manufacturer's instructions (BD Biosciences Clontech). Library plasmids from positive clones were analyzed by transformation tests and DNA sequencing. BLAST searches were conducted using the NCBI on-line service. To examine the interactions of - and ßNACs with the taxilin family members, a pGBKT7 vector harboring the indicated cDNA fragments and a pGAD424 vector harboring the cDNA fragment encoding - or ß1NAC were cointroduced into yeast reporter strain AH109. Equal amount of the indicated transformants was spotted on to synthetic media lacking leucine, tryptophan, adenine, and histidine with 2% glucose (–LWAH) plate for the examination of the interaction between co-expressed proteins or on to synthetic media lacking leucine and tryptophan with 2% glucose (–LW) plate for the examination of plasmid maintenance, and incubated for 3 days at 30 °C.

Pull-down assay

The following procedures were done at 0–4 °C. The indicated GST fusion proteins (5 or 30 µg) were immobilized on 20 µL of glutathione Sepharose beads (Amersham Biosciences Ltd), equilibrated with Buffer A (20 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM dithiothreitol (DTT), 150 mM NaCl, and 1% Triton X-100). The beads were incubated with the indicated samples in Buffer A for 3 h. After the beads were intensively washed with Buffer A, proteins bound to the beads were eluted with the Laemmli's sample buffer. The indicated amounts of the eluted fraction were subjected to SDS-PAGE followed by Western blotting with the indicated antibodies.

Cell culture, transfection and immunofluorescence microscopy

Hela and COS-7 cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air in Dulbecco's modified Eagle's medium containing 10% foetal calf serum (Invitrogen Corp., Carlsbad, CA, USA), 100 units/mL penicillin, and 100 µg/mL streptomycin. Where indicated, COS-7 cells were cultured on gelatin-coated coverslips. The cells were transiently transfected with the indicated plasmids using the Lipofectamine 2000 transfection reagent (Invitrogen Corp.) according to the manufacturer's instructions. Indirect immunofluorescence microscopy was performed as previously described (Nogami et al. 2003a). Briefly, the cells were fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. After being soaked in 10% normal goat serum and 1% bovine serum albumin (BSA) in PBS for 1 h, the cells were treated with the first antibody in 10% normal goat serum and 1% BSA in PBS for 1 h. The cells were washed with PBS three times, followed by incubation with the second antibody in 10% normal goat serum and 1% BSA in PBS for 1 h. After that, the cells were washed with PBS three times, followed by incubation with TOPRO-3 (Invitrogen Corp.) in 1% BSA in PBS for 10 min. After being washed with PBS three times, the cells were examined by use of an FV500 confocal laser scanning microscope (Olympus, Tokyo, Japan).

Preparation of the Triton X-100-soluble fraction and immunoprecipitation

Preparation of the Triton X-100-soluble fraction of the cells and immunoprecipitation were performed as previously described (Nogami et al. 2003a). Briefly, the cells harvested from a 100-mm dish were lyzed on ice for 1 h with 2.4 mL of Buffer A containing 10 µMp-amidinophenylmethansulfonyl fluoride and 10 µM leupeptin. The lysate was centrifuged at 100 000 g for 1 h at 4 °C and the supernatant was used as a Triton X-100-soluble fraction. To immunoprecipitate a Myc-tagged protein, the indicated Triton X-100-soluble fractions were incubated with the anti-Myc monoclonal antibody coupled to protein A-Sepharose beads and proteins bound to the beads were eluted with Laemmli's sample buffer.

Other procedures

SDS-PAGE was performed as described (Laemmli 1970). Protein concentrations were determined with BSA as a reference protein as described (Bradford 1976). Western blotting was performed using the ECL-Plus immunoblotting detection system (Amersham Biosciences Ltd) according to the manufacturer's instructions. The intensity of the protein bands was measured with LAS-1000 Plus (Fujifilm, Tokyo, Japan). The amounts of each protein were determined by measuring the intensity of each purified protein as a standard in a linear range.

	Acknowledgements

 
We are grateful to H. Hirata, H. Kaneko, and T. Namatame for technical assistance. We wish to thank Laboratory Animal Research Center and Laboratory of Analytical Instruments, Dokkyo University School of Medicine, for the use of their facilities. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science & Technology, Japan (2003, 2004).

	Footnotes

 
Communicated by: Yoshimi Takai

^* Correspondence: E-mail: hiro-sh{at}dokkyomed.ac.jp

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Received: 7 January 2005
Accepted: 3 February 2005

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