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Genes to Cells (2008) 13, 905-914. doi:10.1111/j.1365-2443.2008.01215.x
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N-terminal region of ZW10 serves not only as a determinant for localization but also as a link with dynein function

 Mamiko Inoue{dagger}, Kohei Arasakia{dagger}, Akihiro Ueda, Takehiro Aoki and Mitsuo Tagaya*

School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
ZW10 interacts with dynamitin, a subunit of the dynein accessory complex dynactin, and functions in termination of the spindle checkpoint during mitosis and in membrane transport between the endoplasmic reticulum (ER) and Golgi apparatus during interphase. Its associations with kinetochores and ER membranes are mediated by Zwint-1 and RINT-1, respectively. A previous yeast two-hybrid study showed that the C-terminal region of ZW10 interacts with dynamitin, and part of this region has been used as an inhibitor of ZW10 function. In the present study, we reinvestigated the interaction between ZW10 and dynamitin, and showed that the N-terminal region of ZW10 is the major binding site for dynamitin and, like full-length ZW10, could potentially move along microtubules to the centrosomal area in a dynein-dynactin-dependent manner. Competitive binding experiments demonstrated that dynamitin and RINT-1 occupy the same N-terminal region of ZW10 in a mutually exclusive fashion. Consistent with this, over-expression of RINT-1 interfered with the dynein-dynactin-mediated movement of ZW10 to the centrosomal area. Given that the N-terminal region of ZW10 also interacts with Zwint-1, this region may be important for switching partners; one partner is a determinant for localization (kinetochore and ER) and the other links ZW10 to dynein function.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Cytoplasmic dynein is required for the microtubule minus end-directed movement of intracellular architectures. It is a large motor complex composed of two motor subunits with additional subunits, some of which bind cargo molecules (Höök & Vallee 2006). Dynein–cargo interactions are also mediated by a mutisubunit dynein accessory protein complex, dynactin (Kamal & Goldstein 2002; Karcher et al. 2002; Schroer 2004). In interphase, dynein plays a major role in transport of membrane cargos (Caviston & Holzbaur 2006). Dynein mediates the transport of vesicles from the endoplasmic reticulum (ER) to the Golgi apparatus and from the plasma membrane to cell interior. The perinuclear localization of the Golgi apparatus, the movement and morphology of mitochondria, and the motility of endosomes and lysosomes are also maintained by dynein activity. At the onset of mitosis, membrane trafficking is arrested and dynein participates in segregation of chromosome, mitotic spindle organization and orientation, and the removal of spindle checkpoint proteins from the kinetochore (McIntosh et al. 2002; Karess 2005).

ZW10 is a kinetochore-associated protein that interacts with dynamitin (Starr et al. 1997, 1998), a subunit of dynactin (Schroer 2004). The outer kinetochore protein ZW10 is linked to the inner kinetochore Mis12 complex via Zwint-1 (Cheeseman et al. 2004; Obuse et al. 2004; Wang et al. 2004; Kops et al. 2005). ZW10 forms a complex with ROD and Zwilch and acts as a dynein-dynactin receptor on kinetochores (Chan et al. 2000; Williams et al. 2003). After microtubule capture, the ZW10–ROD–Zwilch complex on kinetochores is transported away by dynein-dynactin along kinetochore microtubules (Basto et al. 2004), leading to the termination of the spindle checkpoint (Howell et al. 2001; Wojcik et al. 2001).

In addition to its role in mitosis, ZW10 plays a role during interphase. We previously reported that ZW10 is associated with ER membranes and implicated in membrane transport between the ER and the Golgi apparatus (Hirose et al. 2004). The association of ZW10 with ER membranes is mediated by RINT-1, a Rad50-interacting protein (Xiao et al. 2001), which binds to ER-associated SNAP receptors, syntaxin 18, p31 (Use1p) and BNIP1 (Hatsuzawa et al. 2000; Nakajima et al. 2004; Arasaki et al. 2006). Binding experiments revealed that the respective N-terminal regions of ZW10 and RINT-1 interact with each other (Hirose et al. 2004; Arasaki et al. 2006).

Recently, Varma et al. (2006) extensively investigated the role of ZW10 in interphase cytoplasmic dynein function. They found that depletion of ZW10 decreases the frequency of the minus end-directed movement of Golgi, endosomal and lysosomal markers. In addition, injection of an antibody against a C-terminal peptide and expression of the dominant-negative C-terminal fragment, which contains part of the dynamitin-interacting region (Starr et al. 1998), were found to cause Golgi dispersal and a substantial loss of centrosome-centered microtubule organization. Based on these results, Varma et al. (2006) concluded that ZW10 plays a role in anchoring dynein to membranous organelles.

In the present study, we reinvestigated the interaction of ZW10 with dynamitin. We found that dynamitin and RINT-1 bind to the N-terminal region of ZW10 in a mutually exclusive manner. Based on the results obtained with other data, the role of the N-terminal region of ZW10 is discussed.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
N-terminal RINT-1-interacting region of ZW10 is the major binding site for dynamitin

A previous study showed that the C-terminal region (amino acids (aa) 468–779) of human ZW10 (HZW10) weakly interacts with dynamitin in a yeast two-hybrid system (Starr et al. 1998). To detect this interaction in cultured cells, three truncated HZW10 mutants (N: aa 1–316, M: aa 257–537 and C: aa 468–779) fused with glutathione S-transferase (GST) were co-expressed with FLAG-dynamitin in 293T cells, and pull-down experiments were conducted. As shown in Fig. 1A, FLAG-dynamitin was pulled down with GST-HZW10 N (lane 6), as well as with the full-length construct (lane 5), whereas little, if any, association was observed between HZW10 C and dynamitin (lane 8), raising the question of the involvement of the C-terminal region of ZW10 in the interaction with dynamitin. Consistent with our previous finding (Hirose et al. 2004), endogenous RINT-1 was pulled down with GST-HZW10 N (Fig. 1B, lane 4), but not with GST-HZW10 C (lane 6).


Figure 1
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  		Figure 1  RINT-1 and dynamitin occupy the same N-terminal 170 aa of HZW10. (A and B) 293T cells were transfected with 1 µg of the plasmid encoding the indicated GST-HZW10 constructs plus (A) or minus (B) 50 ng of the plasmid encoding FLAG-dynamitin. After 24 h, cell lysates were prepared, and the GST-tagged proteins were pulled down. The pulled down proteins were analyzed by immunoblotting with antibodies against GST and FLAG (A) or RINT-1 (B). 4.3% of the lysates was also analyzed. (C and D) Experiments were conducted as described in (A) and (B) using the indicated constructs. (E) The two-hybrid interactions of HZW10 constructs with RINT-1 and dynamitin were analyzed by β-galactosidase filter assay. The color was developed for 22 h at 30 °C.

 
The N-terminal 316 aa of HZW10 contains three putative coiled-coil regions (aa 13–38, aa 53–83 and aa 107–130) according to the Lupas algorithm with the 21-residue window (Lupas et al. 1991). To define in detail the region of HZW10 that interacts with dynamitin, the N-terminal region was further divided into fragments (HZW10 N1 (aa 1–55), N2 (aa 1–170), N3 (aa 170–316) and N4 (aa 45–170)). HZW10 N2 and N3 contained all the three and no coiled-coil regions, respectively. HZW10 N1 contained the first N-terminal coiled-coil region, and HZW10 N4 contained the second and third coiled-coil regions. Pull-down experiments revealed that the N-terminal 170-aa fragment (HZW10 N2), but not the N-terminal 55-aa fragment (HZW10 N1), binds FLAG-dynamitin (Fig. 1C, lanes 7 and 8). GST-HZW10 N3 (aa 170–316) and N4 (aa 45–170) bound FLAG-dynamitin with low affinities compared with GST-HZW10 N2 (lanes 9 and 10 vs. lane 8). Similar results were obtained for the binding of endogenous RINT-1 (Fig. 1D). To confirm that the interactions of the N-terminal fragments of HZW10 with dynamitin or RINT-1 are direct, we performed yeast two-hybrid analysis. The results of β-galactosidase filter assay showed that the HZW10 N2, but not N3, interacts directly with both dynamitin and RINT-1 (Fig. 1E). In the two-hybrid assay, the C-terminal region of HZW10 (HZW10 C) exhibited a very weak interaction with dynamitin. Perhaps this level of interaction is too low for the detection of the interaction between GST-HZW10 C and dynamitin expressed in 293T cells.

N-terminal region of HZW10 can be transported to the centrosomal region by dynein-dynactin

To examine whether the N-terminal region of ZW10 interacts with dynamitin in vivo, we treated HZW10-expressing HeLa cells with nordihydroguaiaretic acid (NDGA). Our recent study demonstrated that NDGA facilitates the minus-end-directed movement of dynein-dynactin and its interacting proteins including ZW10, leading to their accumulation at the centrosomal region in interphase cells (Arasaki et al. 2007a). Upon NDGA treatment (Fig. 2, +NDGA), expressed GST-HZW10 full (top row) and N (second row) accumulated at the perinuclear region and were markedly co-localized with the dynactin subunit p150Glued (Schroer 2004), as observed for endogenous ZW10 (Arasaki et al. 2007a). In contrast, no marked change was observed for the distribution of GST-HZW10 C (bottom row). In the case of GST-HZW10 M, slight accumulation was observed at the perinuclear region (third row). This may reflect an interaction between GST-HZW10 M and dynamitin with an affinity below the threshold of detection of our binding assays (Fig. 1). It should be noted that, upon NDGA treatment, p150Glued, a subunit of dynactin (Schroer 2004), accumulated at the centrosomal region in cells expressing any ZW10 mutants, ruling out the possibility that expression of HZW10 M or C affected the NDGA-induced movement of dynein-dynactin. These results suggest that the N-terminal region of ZW10, like endogenous ZW10, can interact with dynein-dynactin within cells.


Figure 2
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  		Figure 2  N-terminal region of HZW10 can be transported to the centrosomal region by dynein-dynactin. HeLa cells were transfected with 1 µg of the plasmid encoding the indicated GST-tagged constructs. At 24 h after transfection, the cells were treated with dimethyl sulfoxide (Vehicle) or 30 µM NDGA (+NDGA) for 1 h, and then double-stained with antibodies against GST and p150Glued. Arrows and arrowheads indicate GST-tagged constructs and p150Glued accumulated at the centrosomal region, respectively. Bar, 10 µm.

 
To gain another line of evidence for the interaction of the N-terminal region of HZW10 with endogenous dynamitin, we constructed a green fluorescent protein (GFP) chimera (GFP-HZW10(N)-Cb5TMD), in which the N-terminal fragment of ZW10 was fused to the transmembrane domain (TMD) of the bona fide ER-membrane protein cytochrome b5 (Cb5). As expected, expressed GFP-HZW10(N)-Cb5TMD was exclusively associated with membranes (Fig. 3A) and, in many cells, exhibited a widely spread/reticular pattern, typical for ER staining (Fig. 3B, upper two rows). However, approximately 30% of the GFP-HZW10(N)-Cb5TMD-expressing cells displayed a centrosomal/perinuclear aggregated pattern of the GFP construct and an ER marker, calnexin (Fig. 3B, lower two rows). In contrast, GFP-HZW10(full-length)-Cb5TMD exhibited a uniform distribution within the ER, and was redistributed to the centrosomal region when cells were treated with NDGA (data not shown). When cells expressing GFP-HZW10(N)-Cb5TMD were incubated with nocodazole to depolymerize microtubules, centrosomal/perinuclear GFP aggregates were dispersed throughout the cytoplasm with retaining some punctate structures (Fig. 3C, arrows). These results support the view that the N-terminal region of ZW10 can move to the centrosomal region along microtubule tracks by interacting with dynein-dynactin. The observation that the full-length construct without NDGA treatment did not accumulate at the centrosomal area may suggest that the middle and C-terminal region of ZW10 may coordinate the interaction of the N-terminal region of ZW10 with dynamitin.


Figure 3
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  		Figure 3  N-terminal fragment of HZW10 anchored to the ER membrane accumulates at the centrosomal region. (A) HeLa cells were transfected with 1 µg of the plasmid for GFP-HZW10(N)-Cb5TMD. After 24 h, the post-nuclear supernatant of cell extracts was separated into the cytosol and membrane fractions. Portions of the fractions (10 µg each) were analyzed by immunoblotting with the indicated antibodies. (B) HeLa cells were transfected with the plasmid for GFP-HZW10(N)-Cb5TMD. At 24 h after transfection, the cells were fixed and stained with an antibody against calnexin. A widely spread/reticular pattern (upper two rows) and a centrosomal/perinuclear aggregation pattern (lower two rows) are shown. Bar, 5 µm. The quantitative results of the analysis of 160 cells expressing the GFP construct are shown on the bottom. Error bars represent the SEM for three experiments. (C) GFP-HZW10(N)-Cb5TMD-expressing cells were treated with 10 µg/mL nocodazole for 120 min, fixed, and stained with an antibody against calnexin. A reticular distribution (top row) and a punctate pattern (middle and bottom rows) are shown. Arrows indicate large puncta positive for GFP and calnexin. Bar, 5 µm. The quantitative results of the analysis of 150 cells expressing the GFP construct are shown on the bottom. Error bars represent the SEM for three experiments.

 
Dynamitin and RINT-1 bind to ZW10 in a mutually exclusive manner

Because dynamitin and RINT-1 occupy the overlapping region on HZW10, we next examined whether their binding to ZW10 occurs in a mutually exclusive manner. We first performed pull-down experiments using GST-HZW10 N2 under the conditions where the expression of FLAG-dynamitin increased gradually. As shown in Fig. 4A, the amount of pulled down RINT-1, as well as a RINT-1-interacting protein, BNIP1 (Nakajima et al. 2004), decreased as the expression of FLAG-dynamitin increased (lanes 4–6).


Figure 4
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  		Figure 4  Binding of dynamitin and RINT-1 to ZW10 is mutually exclusive. (A) 293T cells were transfected with 1 µg of the plasmid encoding GST-HZW10 N2 and FLAG-dynamitin (0, 0.5 or 3.0 µg). After 24 h, cell lysates were prepared, and GST-tagged proteins were pulled down. The pulled down proteins were analyzed by immunoblotting with the indicated antibodies (lanes 4–6). Lanes 1–3 show the amounts of FLAG-dynamitin, RINT-1, BNIP1 and GST-HZW10 N2 in 5.5% of the lysates. The quantitative results are shown on the right. Error bars represent the SEM for three experiments. (B) Coomassie staining of purified GST-HZW10 N2 and His6-RINT-1 (aa 1–264) (arrowheads). Stars denote putative degradation products. The positions of molecular weight markers are indicated on the left. (C) His6-RINT-1 (aa 1–264) (0.4 µM) was incubated with GST (0.45 or 0.15 µM) or GST constructs (0.15 µM), and then pull-down was performed. Because the GST-HZW10 N2 preparation contained large amounts of putative degradation products, the concentration of authentic GST-HZW10 N2 was determined by comparing its Coomassie staining intensity with that of bovine serum albumin. 5% of the total mixture (lanes 1–5) and the pulled down proteins (lanes 6–10) were analyzed by immunoblotting with antibodies against penta His and GST. (D) 293T cells were transfected with 1 µg of the plasmid encoding FLAG-dynamitin. After 24 h, cell lysates were prepared, and incubated with GST (0.45 or 0.15 µM) or GST constructs (0.15 µM). The pulled down proteins were analyzed by immunoblotting with antibodies against FLAG and GST. 0.5% of the lysates was also analyzed (lanes 1–5). (E) 293T cells were transfected with 1 µg of the plasmid encoding the FLAG-dynamitin. After 24 h, cell lysates were prepared, and incubated with GST-HZW10 N2 (0.24 µM) without or with His6-RINT-1 (aa 1–264) (0.9 or 2.7 µM). The pulled-down proteins were analyzed by immunoblotting with the indicated antibodies. The quantitative results are shown on the bottom. Error bars represent the SEM for three experiments.

 
To corroborate this observation, we performed in vitro competition assay using recombinant proteins. To this end, we purified bacterially expressed His6-RINT-1 fragment containing the N-terminal 264 aa, which is sufficient for the binding to ZW10 (Arasaki et al. 2006), and GST-HZW10 N2 (Fig. 4B). As shown in Fig. 4C, His6-RINT-1 (aa 1–264) was pulled down with GST-HZW10 N2 (lane 10), whereas little binding was observed for GST and other GST-constructs (lanes 6–9). Because we were unable to express and isolate functional recombinant dynamitin from Escherichia coli, we used FLAG-dynamitin expressed in 293T cells and performed pull-down experiments using recombinant GST-HZW10 N2. As shown in Fig. 4D, FLAG-dynamitin was pulled down with GST-HZW10 N2 (lane 10), whereas little co-precipitation was observed for GST and other GST-constructs (lanes 6–9). Using recombinant GST-HZW10 N2 and FLAG-dynamitin expressed in 293T cells, together with recombinant His6-RINT-1 (aa 1–264), we performed competition experiments. As shown in Fig. 4E, the amount of FLAG-dynamitin pulled down with GST-ZW10 N2 markedly decreased as that of His6-RINT-1 (aa 1–264) increased. These results unequivocally demonstrated that dynamitin and RINT-1 bind to the N-terminal region of ZW10 in a mutually exclusive manner.

Over-expression of RINT-1 blocks NDGA-induced redistribution of ZW10 to the centrosomal region

To verify that RINT-1 and dynein-dynactin bind to ZW10 in a competitive manner in vivo, we examined the effect of over-expression of RINT-1 on NDGA-induced redistribution of ZW10 to the centrosomal region in HeLa cells. Our previous study showed that RINT-1 over-expression does not affect the distribution of ZW10, whereas the N-terminal RINT-1 fragment causes redistribution of ZW10 (Arasaki et al. 2006). If RINT-1 competes with dynamitin for binding to ZW10 in vivo, RINT-1 over-expression would prevent ZW10 to move to the centrosomal region mediated by dynein-dynactin. In control cells, HZW10, p150Glued and a recycling endosome maker, transferrin receptor (TfR), displayed reticular, filamentous and cytoplasmic dot-like patterns, respectively (Fig. 5, left column). Upon NDGA treatment, these proteins were redistributed to the centrosomal region (right column), as reported previously (Arasaki et al. 2007a), whereas the distribution of RINT-1 was not changed significantly (data not shown). When RINT-1 was over-expressed, HZW10 was not redistributed to the centrosomal region upon NDGA treatment (top and second panels in the middle column). However, redistribution of p150Glued and TfR was not blocked by over-expression of RINT-1 (bottom four panels), suggesting that the effect of RINT-1 over-expression is specific for ZW10.


Figure 5
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  		Figure 5  Over-expression of RINT-1 suppresses the NDGA-induced redistribution of HZW10 to the centrosomal region. HeLa cells were transfected without (right column) or with 1 µg of the plasmid encoding FLAG-RINT-1 (middle column). After 24 h, the cells were treated with 30 µM NDGA (+NDGA) for 1 h, and then double-stained with antibodies against FLAG (data not shown) and HZW10 (top two rows), p150Glued (third and fourth rows) or TfR (bottom two rows). Cells treated with dimethyl sulfoxide (Vehicle) are shown on the left. Arrows indicate HZW10, p150Glued and TfR accumulated at the centrosomal region. Bar, 5 µm. The quantitative results are shown on the bottom. Error bars represent the SEM for three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Although a previous yeast two-hybrid study showed that ZW10 interacts with dynamitin through its C-terminal region (Starr et al. 1998), our present analyses using cultured cells, a yeast two-hybrid system, and recombinant proteins unequivocally verified that the N-terminal region of ZW10 is the major binding site for dynamitin, as well as RINT-1. Moreover, using NDGA, a reagent that stimulates dynein-dynactin-mediated processes (Arasaki et al. 2007a), and a GFP-ZW10 construct with an ER-anchor domain, we showed that the N-terminal domain interacts with endogenous dynein-dynactin. Similar to the present case, inconsistent results have been obtained in the analysis of the interaction between HZW10 and its interacting protein, Zwint-1. Although Starr et al. (2000) showed that Zwint-1 binds to the C-terminal region of HZW10 (aa 468–779) in a yeast two-hybrid system, another group reported that Zwint-1 directly binds to the N-terminal region of ZW10 (Wang et al. 2004). Using extensive mutant library of ZW10, Famulski et al. (2008) also recently showed that the N-terminal region of ZW10 is responsible for the binding to Zwint-1.

The present results raise the possibility that the localization of ZW10 in interphase cells is regulated by two proteins; RINT-1, which serves as an ER anchor for ZW10, and dynamitin, which links ZW10 to dynein function. The competitive relationship of dynamitin and RINT-1 in the binding to ZW10 predicts a model in which dynamitin is recruited to ER membranes and displaces ZW10 from RINT-1 (Fig. 6). Given that ZW10 is the orthologue of yeast Dsl1p, which interacts with several COPI coat proteins (Andag et al. 2001; Reilly et al. 2001; Andag & Schmitt 2003), it is tempting to speculate that ZW10 acts as a receptor for dynein-dynactin on COPI-coated vesicles. Indeed, our preliminary results showed the interaction between ZW10 and some COPI subunits (K. Arasaki and M. Tagaya, unpublished data). Although COPI-coated vesicles are known to be involved in retrograde transport from the Golgi to the ER in yeast, they likely participate in anterograde transport from the ER to the Golgi, as well as retrograde transport, in mammalian cells (Watson & Stephens 2005). If ZW10 functions as a dynein-dynactin receptor on ER-to-Golgi transport vesicles, one may speculate that ZW10 is localized in the Golgi apparatus as a consequence of dynein-dynactin-mediated transport. Indeed, Varma et al. (2006) and we (Arasaki et al. 2007b) found that ZW10 is localized in the Golgi apparatus in certain cells. Golgi localization of ZW10 appears to correlate with the tightness of the interaction between ZW10 and dynactin (Arasaki et al. 2007b).


Figure 6
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  		Figure 6  Model for ZW10 to switch partners. ZW10 is attached to the ER-associated SNAP receptors (p31, BNIP1 and syntaxin 18) through RINT-1. Dynein-dynactin may be recruited to the ER membrane via the interaction with ZW10. This may lead to the switching of ZW10 partner from RINT-1 to dynamitin. A similar mechanism may work for the interaction of ZW10 with Zwint-1 and dynamitin.

 
The mode of interaction between ZW10 and its interphasic partner, RINT-1, may be similar to that between ZW10 and its mitotic partner, Zwint-1. ZW10 interacts with Zwint-1 through its N-terminal region (Wang et al. 2004), as in the case of RINT-1, and the ZW10–ROD–Zwilch complex is linked to the inner kinetochore via Zwint-1 (Cheeseman et al. 2004; Obuse et al. 2004; Wang et al. 2004; Kops et al. 2005). Although dynein was found to be associated with the ZW10–ROD–Zwilch complex, dynactin including dynamitin appeared not to be present in immunoaffinity-purified ZW10–ROD–Zwilch complexes from Drosophila cells (Williams et al. 2003). Therefore, dynactin may be recruited to kinetochores through the interaction between dynamitin and ZW10, leading to the dissociation of ZW10 from Zwint-1. In support of this idea, Zwint-1 remains at kinetochores until late in anaphase (Starr et al. 2000), whereas the ZW10–ROD–Zwilch complex is transported away by dynein-dynactin from kinetochores to the spindle poles (Basto et al. 2004).


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Antibodies

Polyclonal antibodies against RINT-1, ZW10, p31, BNIP1 and syntaxin 18 were prepared as described (Hirose et al. 2004; Nakajima et al. 2004). A monoclonal antibody against TfR was a generous gift from Dr T. Yoshimori (Osaka University, Suita, Japan). Monoclonal antibodies against calnexin and p150Glued were obtained from BD Transduction Laboratories. A monoclonal antibody against penta His and a polyclonal antibody against GST were purchased from Qiagen, Venlo, the Netherlands and Santa Cruz Biotechnology, Santa Cruz, CA, respectively. Monoclonal and polyclonal antibodies against FLAG were obtained from Sigma-Aldrich, Seelze, Germany.

Cell culture

HeLa cells were cultured in Eagle's minimum essential medium supplemented with 50 IU/mL penicillin, 50 µg/mL streptomycin and 10% fetal calf serum. 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with the same materials.

Mammalian expression plasmid construction and transfection

The plasmid (pEBG) encoding GST was a kind gift from Dr B. Mayer (Harvard Medical School, Boston, MA). The cDNAs encoding HZW10 N (aa 1–316), HZW10 M (aa 257–537), HZW10 C (aa 468–779), HZW10 N1 (aa 1–55), HZW10 N2 (aa 1–170), HZW10 N3 (aa 170–316) and HZW10 N4 (aa 45–170) were amplified by PCR and inserted into the BamHI/SmaI site of pEBG vector. The plasmids encoding FLAG-dynamitin and FLAG-RINT-1 were constructed as described (Hirose et al. 2004; Arasaki et al. 2006). To construct the plasmid encoding GFP-HZW10 with the C-terminal Cb5TMD (pEGFP-C3-HZW10-Cb5TMD), the TMD sequence of Cb5 was amplified by PCR using forward and reverse primers containing XhoI and BamHI sites, respectively. The amplified fragment was inserted into pEGFP-C3-HZW10. pGFP-HZW10(N)-Cb5TMD was constructed in a similar way. Transfection was performed using LipofectAMINE PLUS reagent (Invitrogen, Carlsbad, CA) according to the manufacture's protocol.

Yeast two-hybrid assay

Yeast two-hybrid assays were performed essentially according to the manufacturer's protocol (Clontech. Mountain View, CA). pGBT9 and pACT2 vectors were used for the construction of bait and prey plasmids, respectively. The two types of plasmids were transformed into yeast strain SFY526. Transformants were plated on SD-Trp-Leu medium, and colonies that grew on this medium were filter assayed for β-galactosidase activity.

Immunofluorescence analysis

Immunofluorescence microscopy was performed as described (Tagaya et al. 1996). Cells were fixed with methanol at –20 °C for 5 min. Confocal microscopy was performed with an Olympus Fluoview 300 laser scanning microscope.

Immunoprecipitation and GST pull-down assay

Cells were lysed in lysis buffer (20 mM Hepes, pH 7.2, 150 mM KCl, 2 mM EDTA, 0.5 µg/mL leupeptin, 2 µM pepstatin, 2 µg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol and 1% Triton X-100). The lysates were centrifuged in microcentrifuge at 15 000 rpm for 10 min. Immunoprecipitation and GST pull-down assay were carried out as described previously (Hatsuzawa et al. 2000; Hirose et al. 2004).

GST pull-down using recombinant proteins

The cDNAs encoding GST-HZW10 N2 (aa 1–170) and GST-Rab6 were inserted into the BamHI/SmaI site of pGEX4T-1. The cDNA for His6-RINT-1 (aa 1–264) was inserted into the BamHI/SmaI site of pQE30 vector. The plasmid encoding GST-Sec22b (aa 1–195) lacking the transmembrane domain was constructed previously (Aoki et al. 2008). Recombinant proteins were purified according to the standard protocol. Recombinant GST or GST-HZW10 N2 (aa 1–170) was mixed with His6-RINT-1 (aa 1–264) in incubation buffer (180 µL) comprising 10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl2, and 1% Triton X-100, and incubated at 4 °C for 2 h with gentle rotation. After incubation, the mixtures were mixed with 5–10 µL glutathione-Sepharose 4B beads, and then rotated at 4 °C for 1 h. The beads were washed with incubation buffer twice, and the bound proteins were eluted by 30 mM glutathione and mixed with SDS sample buffer.

To detect the binding between dynamitin and HZW10, extracts from 293T cells expressing FLAG-dynamitin were prepared and incubated with recombinant GST or GST-HZW10 N2 (aa 1–170) in the presence or absence of His6-RINT-1 (aa 1–264). Pull-down experiments were conducted as described above.


   Acknowledgements
 
We thank Drs T. Yoshimori and B. Mayer for gifts of reagents. We are grateful to Dr K. Tani for her helpful comments and Mr T. Kamimura for his excellent technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research (#18370081, #18050036 and #18657044) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. K.A. was a recipient of a Japan Society for the Promotion of Science research fellowship.


   Footnotes
 
Communicated by: Yoshinori Ohsumi

aPresent address: Section of Microbial Pathogenesis, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, USA. Back

{dagger}These authors contributed equally to this work. Back

* Correspondence: tagaya{at}ls.toyaku.ac.jp


   References
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 Abstract
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 Results
 Discussion
 Experimental procedures
 References
 
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Received: 1 June 2007
Accepted: 26 May 2008




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