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1 Department of Biological Sciences, Tokyo Institute of Technology, Yokohama 226-8501, Japan
2 Department of Immunology and Microbiology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Two structurally unrelated deubiquitinating enzymes (DUBs), AMSH (associated molecule with the SH3 domain of STAM) and UBPY (Ub-specific protease Y), interact with the Src homology 3 (SH3) domain of STAM via a non-canonical SH3 domain-binding motif (SBM) (Tanaka et al. 1999; Kato et al. 2000). AMSH is a Ub-specific isopeptidase (McCullough et al. 2004) that belongs to the JAMM (JAB1/MPN/Mov34) family of metalloproteases (Amerik & Hochstrasser 2004; Nijman et al. 2005). This family also includes Rpn11/POH1, a component of the 19S regulatory complex of the 26S proteasome (Verma et al. 2002; Yao & Cohen 2002). In vitro, purified recombinant AMSH cleaves lysine 63 (K63)-linked, but not lysine 48 (K48)-linked, poly Ub chains to Ub monomers (McCullough et al. 2004). Recombinant AMSH is also shown to deubiquitinate epidermal growth factor receptor (EGFR) in vitro (McCullough et al. 2004), although the activity is much lower than that of UBPY (Mizuno et al. 2005). Furthermore, depletion of endogenous AMSH by RNA interference (RNAi) results in an acceleration of ligand-induced EGFR degradation (McCullough et al. 2004). These observations suggest that AMSH negatively regulates the down-regulation of activated EGFR by deubiquitinating EGFR or other regulatory proteins on endosomes. The other DUB, UBPY, is also designated USP8 (Ub-specific protease 8) and belongs to the USP (Ub-specific protease) family of cysteine proteases (Naviglio et al. 1998; Amerik & Hochstrasser 2004; Nijman et al. 2005). We have recently shown that UBPY also negatively regulates the down-regulation of EGFR by directly deubiquitinating endocytosed EGFR on endosomes (Mizuno et al. 2005).
On the early endosomal membrane, there is a microdomain which is coated with a flat clathrin lattice (Raiborg et al. 2002; Sachse et al. 2002; Murk et al. 2003). The Hrs-STAM complex localizes on this microdomain (Raiborg et al. 2002; Sachse et al. 2002), where it interacts with clathrin heavy chain (CHC) through the clathrin box motif located at the C-terminus of Hrs (Raiborg et al. 2001a). Therefore, the localization to this microdomain of endosomes is likely to be a prerequisite for AMSH and UBPY to play a role in receptor down-regulation. We have shown that UBPY localizes on Hrs-positive early endosomes via an unknown mechanism that does not require the Hrs-STAM complex (Mizuno et al. 2005). AMSH, when exogenously expressed, also localizes on early endosomes where it overlaps with EEA1 (early endosome antigen 1) as well as endocytosed transferrin receptor and EGFR (McCullough et al. 2004). However, the mechanism for the endosomal localization of AMSH is unclear as well. In addition, AMSH has a close homolog, AMSH-like protein (AMSH-LP), which is 55% identical to AMSH over the entire amino acid sequence (Kikuchi et al. 2003; Kitajima et al. 2003; Ibarrola et al. 2004). A lysine residue in the SBM of AMSH, which is critical for its binding to the SH3 domain of STAM, is replaced by threonine in AMSH-LP. This results in the inability of AMSH-LP to interact with STAM (Kikuchi et al. 2003), suggesting that AMSH and AMSH-LP are not functionally redundant. However, neither the function nor the subcellular localization of AMSH-LP has been studied.
In this study, we show that AMSH-LP is also a DUB that localizes on early endosomes. A very recent study demonstrated that AMSH binds directly to CHC (McCullough et al. 2006). Here we further show that AMSH, as well as AMSH-LP, interact with CHC via a novel binding site conserved in these DUBs, and that the interaction with clathrin is required for their endosomal localization.
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Results |
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The DUB activity of AMSH-LP has not been examined. We tested it with an in vitro assay using recombinant AMSH-LP proteins. FLAG-tagged AMSH-LP and its mutant with the aspartic acid residue at position 360 replaced with alanine (AMSH-LPD360A) were expressed in HeLa cells, immunoprecipitated with an anti-FLAG antibody, and eluted with the FLAG competing peptide. The aspartic acid residue 360 corresponds to residue 348 in AMSH which is located in the JAMM domain and is essential for the AMSH DUB activity (McCullough et al. 2004). Coomassie Brilliant Blue (CBB) staining of the eluted proteins after SDS-polyacrylamide gel electrophoresis (PAGE) showed that they were purified to near homogeneity (Fig. 1A, arrowhead). The yields of the proteins were roughly estimated based on the CBB staining using bovine serum albumin as a standard, and the proteins (
0.3 µM) were incubated with K48- and K63-linked poly Ub chains. With similar substrate specificity to AMSH (McCullough et al. 2004), AMSH-LP cleaved K63-linked, but not K48-linked, Ub chains to Ub monomers (Fig. 1B). Also, similarly to AMSH, in which a mutation in the aspartic acid residue at 348 resulted in a loss of the enzymatic activity, AMSH-LPD360A did not exhibit detectable Ub isopeptidase activity on K63-linked Ub chains (Fig. 1B). This result excluded the possibility that the DUB activity detected in the wild-type AMSH-LP fraction is due to associated or contaminating proteases.
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Together, these results indicate that AMSH-LP is a DUB that belongs to the JAMM family of metalloproteases and exhibits a similar substrate specificity and subcellular localization to AMSH.
AMSH and AMSH-LP bind to CHC
To understand the role of AMSH and AMSH-LP on endosomes, we tried to identify AMSH-binding proteins using a combination of co-immunoprecipitation and mass spectrometry. FLAG-tagged AMSH was expressed in HeLa cells and immunoprecipitated with anti-FLAG antibody. Proteins that were co-precipitated with AMSH were separated by SDS-PAGE and detected by silver staining. A protein band of 190 kDa was precipitated from AMSH-transfected, but not from mock-transfected, cells (Fig. 2A, open arrowhead). This band was excised from the gel, digested with trypsin, and analyzed by mass spectrometry using peptide mass finger printing. This led to the identification of the protein as CHC with a high probability (MASCOT score = 170). To confirm this, we immunoblotted the anti-FLAG immunoprecipitates from FLAG-AMSH-transfected, as well as FLAG-AMSH-LP-transfected, cells with anti-CHC antibody. CHC was detected in the precipitates from AMSH- and AMSH-LP-transfected, but not from mock-transfected, cells (Fig. 2B, top), indicating that AMSH and AMSH-LP bind to CHC within cells.
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To identify the CHC-binding site in AMSH, we constructed various FLAG-tagged AMSH truncation mutants (Fig. 3A) and expressed them in HeLa cells. AMSH1–327 lacks a clathrin box-related sequence, LITLG, at amino acids 328–332 located within the JAMM domain. AMSH1–230 further lacks the SBM located at amino acids 231–239. On immunoprecipitation with anti-FLAG antibody, however, these mutants co-precipitated CHC as efficiently as the wild-type protein (Fig. 3B). Further truncation of the C-terminal region showed that the CHC-binding ability is decreased to some extent in AMSH1–171, and completely abolished in AMSH1–166. Similarly, the CHC-binding ability of N-terminally truncated mutants was examined. Whereas AMSH139–424 retained considerable CHC-binding ability, AMSH144–424 did not co-precipitate CHC at a detectable level (Fig. 3B). Therefore, it was suggested that the region between amino acids 139 and 171 was responsible for the CHC binding. We next constructed a FLAG-tagged AMSH deletion mutant lacking amino acids 139–166. Co-immunoprecipitation experiments with anti-FLAG antibody showed that the mutant AMSH
139–166 lost the ability to bind CHC (Fig. 3C).
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154–181, did not co-precipitate CHC at a detectable level (Fig. 3C), indicating that AMSH and AMSH-LP bind to clathrin via the conserved regions.
Finally, to examine whether the regions between amino acids 139–171 and 154–186 in AMSH and AMSH-LP, respectively, are capable of CHC binding by themselves, these regions were fused to green fluorescence protein (GFP) and expressed in HeLa cells. Immunoprecipitation with anti-GFP antibody followed by immunoblotting with anti-CHC antibody showed that the GFP-fusion proteins (GFP-AMSH139–171 and GFP-LP154–181), but not GFP alone, co-precipitate endogenous CHC (Fig. 3E). Together, the results shown in Fig. 3 indicate that these regions of AMSH and AMSH-LP are the minimum regions required for CHC binding. Hereafter, we therefore refer to AMSH139–166 and AMSH-LP154–181 as AMSHCBS and AMSH-LPCBS (CBS: clathrin-binding site), respectively.
AMSH and AMSH-LP bind to the N-terminal region of CHC
The globular terminal domain located at the N-terminus of CHC serves as the site of interaction with various proteins (Lafer 2002). We therefore examined by in vitro pull-down experiments whether AMSH and AMSH-LP also bind to the terminal domain of CHC. The N-terminal region of CHC (amino acids 1–444) that encompasses the terminal domain (amino acids 1–330) was fused to glutathione S-transferase (GST) and incubated with the lysate of HeLa cells expressing FLAG-tagged AMSH, AMSHCBS, AMSH-LP or AMSH-LPCBS. Bound AMSH and AMSH-LP proteins were then detected by anti-FLAG immunoblotting. AMSH and AMSH-LP bound to the fusion protein GST-TD but not to GST alone (Fig. 4A, top). The CHC-binding ability of AMSH-LP was consistently higher than that of AMSH. In contrast, AMSHCBS and AMSH-LPCBS lacking CHC-binding ability were not pulled down by GST-TD (Fig. 4A, top). These results suggested that AMSH and AMSH-LP bind to the terminal domain of CHC.
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Localization of AMSH and AMSH-LP on CHC-positive puncta depends on CHC
To examine whether AMSH co-localizes with clathrin within cells, we stained HeLa cells expressing exogenous FLAG-AMSH by double immunofluorescence with anti-FLAG and anti-CHC antibodies. As reported previously (McCullough et al. 2004), FLAG-AMSH localized in the nucleus and on cytoplasmic puncta (Fig. 5A). AMSH co-localized with CHC on the puncta (Fig. 5A',A''). In contrast, AMSHCBS which can not bind CHC did not co-localize with CHC, although the nuclear localization was unaffected (Fig. 5B–B''). These results suggest that the binding to CHC is required for AMSH to localize on the CHC-positive puncta. The CHC-positive puncta in cells over-expressing wild-type AMSH exhibited higher anti-CHC staining than those in untransfected cells (Fig. 5A–A'', asterisks). However, such an effect was not observed on over-expression of AMSHCBS (Fig. 5B–B'').
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We next examined the co-localization of AMSH-LP with CHC within cells. Similarly to AMSH, FLAG-tagged AMSH-LP (Fig. 6A–A'') but not AMSH-LPCBS (Fig. 6B–B'') co-localized with CHC on cytoplasmic puncta. The AMSH-LP-positive puncta exhibited higher anti-CHC staining than CHC-positive puncta in untransfected (Fig. 6A–A'',B–B', asterisks) and AMSH-LPCBS-transfected (Fig. 6B–B'') cells. In addition, the localization of FLAG-AMSH-LP was completely lost in CHC-depleted cells transfected with the CHC siRNA-1 (Fig. 6C–C'') or siRNA-2 (our unpublished observations). Interestingly, the localization of AMSH-LP on the CHC-positive puncta was more clearly observable than that of AMSH (compare Fig. 6A–A'' with Fig. 5A–A''). This might reflect the greater CHC-binding affinity of AMSH-LP than of AMSH (Fig. 4, top).
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Both clathrin (Raiborg et al. 2001a) and AMSH (McCullough et al. 2004) have been shown to localize on early endosomes, suggesting that the AMSH- and CHC-positive puncta shown in Fig. 5 are early endosomes. To confirm this, we double-stained FLAG-AMSH-transfected cells with anti-FLAG and anti-EEA1 antibodies. Wild-type AMSH co-localized with EEA1 on the cytoplasmic puncta (Fig. 7A–A''). As expected from the results in Fig. 5, AMSHCBS lacking CHC-binding ability did not co-localize with EEA1 (Fig. 7B–B''). In contrast, AMSHSBM, a mutant lacking the ability to bind the SH3 domain of STAM, co-localized normally with EEA1 (Fig. 7C–C''). These results suggested that the localization of AMSH on the endosomal membrane is not mediated by the Hrs-STAM complex but by binding to clathrin. Similarly, AMSH-LPCBS did not co-localize with EEA1 on cytoplasmic puncta (Fig. 7D–D'') whereas wild-type AMSH-LP did (Fig. 1C–C'').
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Discussion |
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In cells over-expressing AMSH or AMSH-LP, endosomes which were positive for these DUBs exhibited higher anti-CHC staining than those in normal cells (Figs 5 and 6). However, these effects were abolished by deleting the clathrin-binding sites from these proteins (Figs 5 and 6). The observations suggest that at least when over-expressed, AMSH and AMSH-LP in turn stabilize the architecture of the endosomal clathrin coat through binding to CHC.
A recent proteomics study, in which ubiquitinated proteins were immunopurified from HEK293T cells with the antibody FK2 and were analyzed by liquid chromatography coupled to tandem mass spectrometry, showed that CHC is possibly ubiquitinated in the cells (Matsumoto et al. 2005). This raises the possibility that CHC serves as a substrate for the DUB activity of AMSH and AMSH-LP. This possibility remains to be tested. However, we have so far failed to detect the ubiquitination of CHC in HeLa cells (our unpublished observation).
The clathrin-binding sites in AMSH and AMSH-LP (Fig. 3) are located in predicted coiled-coil regions that roughly span amino acids 100–180 and 120–180 of AMSH and AMSH-LP, respectively (COILS; available at <http://www.ch.embnet.org/software/COILS_form.html>). This is consistent with the function of coiled-coil regions as protein–protein interaction domains (Burkhard et al. 2001). On the other hand, AMSH and AMSH-LP bound to the N-terminal region of CHC (Fig. 4). The N-terminal fragment that we used for the GST pull-down experiments encompasses amino acid residues 1–444 of CHC. This region contains a ß-propeller globular terminal domain (amino acids 1–330) as well as a part of the 
-zigzag helix region (amino acids 331–444) that links the terminal domain to the distal leg region (ter Haar et al. 1998). The globular terminal domain binds to several regulatory proteins including adaptor complexes such as AP1 and AP2, amphiphysin, and ß-arrestin (Lafer 2002). Hrs and STAM1 also bind to this domain (Raiborg et al. 2001a; McCullough et al. 2006). Therefore, it is likely that AMSH and AMSH-LP also bind to the globular terminal domain. Whether the Hrs-STAM complex and AMSH bind simultaneously to a single terminal domain of CHC or they compete for CHC binding is unknown. The latter might not be the case because the efficiency of CHC co-immunoprecipitation by AMSH was not affected by over-expressing Hrs or STAM1 (Fig. 4). In the prototype clathrin coat, terminal domains from different clathrin triskelions are closely positioned to one another (Smith et al. 1998; Musacchio et al. 1999; Kirchhausen 2000). Therefore in any case, binding to CHC probably allows AMSH to be located in close proximity to the Hrs-STAM complex on the endosomal membrane.
Deletion of the SBM that interacts with STAM did not affect the localization of AMSH on early endosomes (Fig. 7). In addition, AMSH-LP, which has a replacement in the critical lysine residue in the SBM and thus does not bind to STAM (Kikuchi et al. 2003), also localized on early endosomes (Fig. 1). These results suggest that the localization of AMSH and AMSH-LP is not regulated by interaction with the Hrs-STAM complex. This is analogous to the mechanism of UBPY localization to endosomes. Although how UBPY localizes on endosomes remains unclear, experiments using a UBPY mutant lacking the SBMs and RNAi-mediated depletion of the Hrs-STAM complex suggest that the localization does not depend on the Hrs-STAM complex (Mizuno et al. 2005). Upon ligand stimulation, endocytosed EGFR binds to UBPY on endosomes. This interaction is inhibited when the endogenous Hrs-STAM complex is depleted using RNAi for Hrs. In addition, a UBPY mutant lacking the SBMs does not bind to endocytosed EGFR. These observations suggest that the interaction between the Hrs-STAM complex and UBPY is required for the transfer of ubiquitinated EGFR from the sorting complex to UBPY (Mizuno et al. 2005). Therefore, the interaction of AMSH with the Hrs-STAM complex could also be important for receiving sorted cargo proteins from the Hrs-STAM complex. This is supported by a recent observation that the in vitro DUB activity of AMSH is increased by the addition of STAM to the reaction (McCullough et al. 2006).
Figure 8 illustrates the mode of interaction between the endosomal clathrin coat, AMSH, and the Hrs-STAM complex, based on the assumption that the endosomal clathrin coat is structurally similar to the prototype coat. In clathrin-coated vesicles, the terminal domains of CHC project toward the vesicular membrane from the outer polyhedral cage (Smith et al. 1998; Kirchhausen 2000). While the Hrs-STAM complex is anchored on the early endosomal membrane via the interaction of the Hrs FYVE domain with phosphatidylinositol(3)-phosphate (Komada & Soriano 1999; Raiborg et al. 2001b), it also interacts with the CHC terminal domain via the clathrin box in Hrs (Raiborg et al. 2001a). The binding of AMSH to the terminal domain of CHC thus suggests that one of the roles of the endosomal clathrin coat is to serve as a scaffold to increase the efficiency of AMSH-mediated deubiquitination of cargo proteins which are sorted by the Hrs-STAM complex. In this way, AMSH may be able to regulate the rate of lysosomal trafficking of cargo proteins negatively by removing their sorting signals (i.e. Ub moieties), as proposed for ligand-activated EGFR (McCullough et al. 2004). Both Hrs and STAM undergo ubiquitination (Katz et al. 2002; Polo et al. 2002). Moreover, over-expression of a catalytically inactive AMSH mutant causes the accumulation of the ubiquitinated form of STAM (McCullough et al. 2004). Therefore, it is also possible that AMSH deubiquitinates regulatory proteins in the MVB sorting machinery, thereby regulating their functions in MVB sorting.
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In conclusion, we demonstrated that AMSH and AMSH-LP bind to CHC via a novel conserved clathrin-binding site and that these DUBs localize on early endosomes via interaction with clathrin. To understand the functions of AMSH and AMSH-LP on endosomes, further efforts should be made toward the identification of their substrate proteins.
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Experimental procedures |
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The expression vectors for FLAG-tagged human AMSH and its mutant AMSHSBM were constructed as previously described (Kato et al. 2000). Construction of those for HA-tagged Hrs and STAM1 was also previously described (Komada et al. 1997; Mizuno et al. 2003). The expression vector for FLAG-tagged human AMSH-LP was constructed by inserting its cDNA (Kikuchi et al. 2003) into pME-FLAG (Kato et al. 2000). AMSH truncation mutant cDNAs (AMSH1–166, 1–171, 1–230, 1–327, 139–424, and 144–424) were amplified by polymerase chain reaction using its full-length cDNA as a template, and cloned into pME-FLAG. Point and deletion mutant cDNAs (AMSHCBS, AMSH-LPD360A, and AMSH-LPCBS) were obtained by in vitro mutagenesis using the QuikChange site-directed mutagenesis system (Stratagene, La Jolla, CA, USA). To construct GFP-AMSH139–171 and GFP-LP154–186 expression vectors, the regions between amino acids 139–171 and 154–186 for AMSH and AMSH-LP, respectively, were amplified by polymerase chain reaction and inserted into pEGFP-C1 (Clontech, Mountain View, CA, USA). The sequences of the mutant cDNAs were verified by DNA sequencing. The expression vectors were transfected into HeLa cells for two days using the FuGENE 6 Transfection Reagent (Roche Diagnostics, Indianapolis, IN, USA).
Immunoprecipitation and immunoblotting
Cell lysates were prepared by solubilizing cells with lysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, 1 µg/mL leupeptin, and 1 µg/mL pepstatin A) for 30 min on ice and collecting the supernatants after centrifugation at 12 000 g for 15 min at 4 °C. The lysates were used directly for immunoblotting or immunoprecipitated with anti-FLAG (4 µg, Sigma-Aldrich, St. Louis, MO, USA) or anti-GFP serum (0.5 µL, Molecular Probes, Eugene, OR, USA) antibodies. Immunoblot analysis was performed by standard procedures. Primary antibodies used were mouse anti-FLAG (4 µg/mL, Sigma-Aldrich), rabbit anti-FLAG (0.8 µg/mL, Sigma-Aldrich), anti-Ub (5 µg/mL, Covance, Princeton, NJ, USA), anti-CHC (TD.1, 2 µg/mL, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GFP (2 µg/mL, Nacalai Tesque, Kyoto, Japan), anti-HA (0.4 µg/mL, Roche Diagnostics), and anti--tubulin (1 µg/mL, Sigma-Aldrich) antibodies. Secondary antibodies were peroxidase-conjugated anti-mouse, anti-rabbit, and anti-rat IgG antibodies (GE Healthcare, Piscataway, NJ, USA). Blots were detected using the ECL reagent (GE Healthcare).
In vitro DUB assay
HeLa cells were transfected with FLAG-tagged AMSH, AMSH-LP or AMSH-LPD360A and then lyzed with lysis buffer without EDTA. The lysates were immunoprecipitated with anti-FLAG antibody conjugated to agarose beads (anti-FLAG M2 affinity gel, Sigma-Aldrich). Precipitated proteins were eluted by incubation with 100 µL of phosphate-buffered saline (PBS), pH 7.0, containing 300 µg/mL of FLAG peptide (Sigma-Aldrich). The purity and concentration of recombinant proteins in the eluates were assessed by CBB staining after SDS-PAGE using purified bovine serum albumin as a standard. K48- and K63-linked poly Ub chains (Ub2–7) were purchased from Affiniti Research Products (Exeter, UK) and Boston Biochem (Cambridge, MA, USA), respectively. The FLAG peptide-eluted recombinant proteins (0.3 µM) were incubated with the Ub chains (0.25 µg) in 50 µL of PBS, pH 7.0, containing 5 mM MgCl2 and 2 mM dithiothreitol at 37 °C for 16 h. Reaction products were separated by SDS-PAGE and detected by immunoblotting with anti-Ub antibody.
Mass spectrometry
FLAG-AMSH expressed in HeLa cells was immunoprecipitated with anti-FLAG antibody-conjugated agarose beads (anti-FLAG M2 affinity gel, Sigma-Aldrich). Immunoprecitated FLAG-AMSH was eluted by incubation with 100 µL of PBS, pH 7.0, containing 300 µg/mL of FLAG peptide (Sigma-Aldrich). The eluted proteins were separated by SDS-PAGE, detected by silver staining (Silver Stain MS kit, Wako, Osaka, Japan), and excised from the gel. In-gel digestion with trypsin followed by an analysis using UltraFlex MALDI-TOF/TOF-MS (Bruker Daltonics, Billerica, MA, USA) was performed according to the manufacturer's instructions. Data were analyzed using the MASCOT search program (Matrix Science, Boston, MA, USA).
GST pull-down
A cDNA encoding an N-terminal region of rat CHC (amino acids 1–444) was excised from pET28a/rat CHC 1–444 (a gift from Dr K. Nakayama, Kyoto University, Kyoto, Japan), and cloned into pGEX4T-3 (GE Healthcare) to generate a GST fusion construct. The GST fusion protein (GST-TD) was purified from Escherichia coli using glutathione-Sepharose affinity beads (GE Healthcare). GST-TD as well as GST alone (3 µg) was coupled to 10 µL of glutathione beads and incubated with lysates of HeLa cells transfected with FLAG-tagged AMSH or AMSH-LP constructs. After the beads were washed with lysis buffer three times, bound AMSH and AMSH-LP proteins were detected by immunoblotting with anti-FLAG antibody.
Immunofluorescence staining
Cells were fixed with 4% paraformaldehyde in PBS for 10 min on ice, permeabilized with 0.2% Triton X-100 in PBS, and stained with rabbit polyclonal anti-FLAG (0.4 µg/mL, Sigma-Aldrich), mouse monoclonal FK2 (0.7 µg/mL, MBL, Nagoya, Japan), goat polyclonal anti-CHC (C20, 10 µg/mL, Santa Cruz Biotechnology), and mouse monoclonal anti-EEA1 (1 µg/mL, BD Transduction Laboratories, Lexington, KY, USA) antibodies using standard procedures. Secondary antibodies were Alexa488-, Alexa555- and Alexa594-conjugated anti-mouse, rabbit and goat IgG antibodies (Molecular Probes). Fluorescence images were captured with a confocal microscope (Axiovert 200M, Carl Zeiss, Oberkochen, Germany) using the LSM5 PASCAL system (Carl Zeiss).
RNAi
Using the siRNA expression vector pSilencer 1.0-U6 (Ambion, Austin, TX, USA), two vectors that allow the production of siRNAs for human CHC were constructed. As target sequences, 5'-GAAAGAATCTGTAGAGAAA-3' and 5'-GCAATGAGCTGTTTGAAGA-3' were chosen according to Huang et al. (2004). These vectors or an empty mock vector were transfected into HeLa cells twice at 36 h intervals. To express other constructs in these cells, the expression vectors were co-transfected with the siRNA vectors in the second round of transfection.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: makomada{at}bio.titech.ac.jp
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