Glycobiology Advance Access originally published online on June 22, 2007
Glycobiology 2007 17(9):913-921; doi:10.1093/glycob/cwm067
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Stable interaction of the cargo receptor VIP36 with molecular chaperone BiP
2 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan
3 Department of Anatomy, Yamanashi University School of Medicine, Tamaho-cho 1110, Yamanashi 409-3898, Japan
4 CREST, JST, Honcho 4-1-8, Kawaguchi 332-1102, Japan
1 To whom correspondence should be addressed: Tel: +81-4-7136-3614 Fax: +81-4-7136-3619; e-mail: yamamoto{at}k.u-tokyo.ac.jp
Received on February 26, 2007; revised on June 16, 2007; accepted on June 17, 2007
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Abstract |
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VIP36 is an intracellular lectin that cycles between the endoplasmic reticulum (ER) and the Golgi apparatus, and is thought to act as a cargo receptor in the transport and sorting of glycoproteins. Here we sought to identify the proteins that interact with VIP36 during the quality control of secretory proteins. VIP36 was crosslinked and immunoprecipitated from HEK293 cells that expressed Myc-tagged VIP36. An
80 kDa protein coprecipitated with VIP36 and LC/MS/MS analysis revealed it to be immunoglobulin-binding protein (BiP), a major protein of the Hsp70 chaperone family. A VIP36 mutant with defective lectin activity was also proficient for the coimmunoprecipitation of an equivalent amount of BiP, indicating that the interaction between VIP36 and BiP was carbohydrate-independent. Immunoelectron microscopy experiment demonstrated that the interaction between VIP36 and BiP occurred in the ER. However, the VIP36 coprecipitated with BiP was resistant to endo ß-N-acetylglucosaminidase H treatment. A pulse-chase experiment revealed that the amount of BiP interacting with VIP36 did not change over more than 2 h. These results suggest that the interaction of VIP36 and BiP is not due to chaperone–substrate complex. Surface plasmon resonance analysis using recombinant proteins confirmed these binding characteristics of VIP36 and BiP in vitro. The interaction between recombinant soluble VIP36 and BiP is dependent on divalent cations but not on ATP. This mode of interaction is also different from that observed between BiP and its chaperone substrates. These observations suggest a new role for VIP36 in the quality control of secretory proteins. Key words: BiP / cargo receptor / chaperone / interaction / VIP36
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Introduction |
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Secretory proteins are cotranslationally translocated into the lumen of the ER, where they interact with ER-resident chaperones such as the immunoglobulin-binding protein (BiP), calnexin, and/or calreticulin. Only secretory proteins that fold correctly are transported through the Golgi apparatus to their final destinations. Several proteins are known to be transported by specific receptors. Such receptors may include the membrane proteins ERGIC-53, the p24 family, and Erv29p, which cycle between the ER and the Golgi apparatus (Nichols et al. 1998
; Appenzeller et al. 1999; Muniz et al. 2000; Belden et al. 2001). ERGIC-53 bears homology to leguminous lectins and binds to mannose (Itin et al. 1996) and is therefore proposed to recognize the high mannose-type oligosaccharides attached to proteins and to transport these glycoproteins from the ER to the Golgi apparatus. Indeed, the lack of ERGIC-53 impairs the secretion of the procathepsin C and blood coagulation factors V (FV) and VIII (FVIII) glycoproteins (Nichols et al. 1998; Vollenweider et al. 1998). Chemical cross-linking studies have revealed that ERGIC-53 interacts with procathepsin Z in a mannose- and calcium-dependent manner (Appenzeller et al. 1999; Appenzeller-Herzog et al. 2005). However, ERGIC-53 and its mutant, which is unable to bind to mannose, both coimmunoprecipitate with FVIII, and treatment with tunicamycin does not reduce the interaction between ERGIC-53 and FVIII (Cunningham et al. 2003), which indicates that protein–protein interactions also contribute to this interaction. It is therefore possible that ERGIC-53 also acts as a molecular chaperone in addition to transporting glycoproteins.
The vesicular integral membrane protein VIP36 was originally identified as a component of apical post-Golgi vesicles in polarized Madin-Darby canine kidney cells (Fiedler et al. 1994). VIP36 shares significant homology with leguminous lectins as well as with ERGIC-53. Its ability to recognize high-mannose type glycans (Hara-Kuge et al. 1999; Kamiya et al. 2005) and its broad localization from the ER to the cis-Golgi (Fullekrug et al. 1999; Shimada et al. 2003a,2003b) indicates that VIP36 also functions as a cargo receptor that facilitates the transport of various glycoproteins. In this study, we sought to identify the proteins that associate with VIP36. One of these was found to be BiP, an Hsp70 homologue, and the interaction between VIP36 and BiP occurred in the ER constitutively.
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Results |
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BiP coimmunoprecipitates with VIP36
To search for VIP36-associated proteins, HEK293 cells transfected with a vector expressing Myc-tagged VIP36 were metabolically labeled for 3 h with [35S]methionine/cysteine, followed by immunoprecipitation with an anti-Myc antibody. The immunoprecipitated proteins were separated by SDS-PAGE and detected by autoradiography. As shown in Figure 1A, Myc-tagged VIP36 was copurified with a cellular protein that had a relative molecular mass of
80 kDa. This protein was absent from control immunoprecipitation reactions carried out using mock-transfected HEK293 cells.
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The luminal region of VIP36 has a mannose-specific lectin domain (Hara-Kuge et al. 1999
; Kamiya et al. 2005) that is structurally homologous to plant leguminous lectin. The key amino acids involved in sugar-binding are also conserved in VIP36 and it has been shown that the substitution of one of these residues, Asp131, with another amino acid abrogates the sugar-binding activity of VIP36 (Hara-Kuge et al. 2002). VIP36D131N is an example of such a mutant VIP36 protein that cannot bind sugars. We found that this mutant interacted with the 80 kDa protein to the same extent as wild-type VIP36 (Figure 1A), which indicates that the interaction between the two proteins is carbohydrate-independent.
To prepare a large amount of the 80 kDa protein, we performed an immunoprecipitation experiment on a large scale using HEK293 cells that stably express Myc-tagged VIP36. The protein thus obtained was treated with trypsin and the fragments were separated by reverse-phase liquid chromatography and analyzed by tandem mass spectrometry (LC/MS/MS). This analysis identified that the protein was BiP. Notably, the 80 kDa molecular weight of the protein estimated by SDS-PAGE agrees well with predicted molecular weight of BiP (78 kDa).
To confirm that the protein coprecipitating with VIP36 was BiP, the precipitated proteins were subjected to Western blotting using an anti-BiP antibody. HEK293 cells that stably expressed Myc-tagged VIP36 were lysed after having been exposed to the cross-linker DSP, which can penetrate cell membranes. The lysates were then incubated with an anti-Myc antibody and protein-G beads. To release any denatured proteins that may interact with BiP due to the lysis of the cells, we further incubated the immunocomplexes with 1 mM ATP after washing the beads. The coprecipitated proteins were blotted and stained with the anti-BiP antibody. BiP coprecipitated with Myc-tagged VIP36, whereas the BiP band was not detected when HEK293 cells were stably transfected with the mock vector (Figure 1B). Next, we transiently expressed 3x FLAG-tagged BiP in HEK293 cells that stably expressed Myc-tagged VIP36. After treating the cells with DSP, we subjected the lysates to immunoprecipitation with an anti-FLAG antibody. The coprecipitated proteins were separated on a SDS-polyacrylamide gel, blotted onto a membrane, and detected with an anti-VIP36 antibody. As shown in Figure 1C, the 36 kDa Myc-tagged VIP36 protein was detected in the lysates of these cells (Figure 1C) but not in the lysates of cells that had been cotransfected with mock vector lacking the 3x FLAG-tagged BiP cDNA. To confirm that VIP36 interacts with BiP under the physiological condition, untransfected HEK293 cells were used for coimmunoprecipitation assay. After treatment with DSP, immunoprecipitation was performed with anti-VIP36, and the coprecipitated proteins were blotted and stained with the anti-BiP antibody. BiP was coprecipitated with VIP36 from untransfected cell lysates (Figure 1D), indicating that VIP36 interacts with BiP under physiological condition.
BiP interacts with an endo H-resistant form of VIP36
VIP36 has a N-glycosylation site and is resistant to digestion with endo ß-N-actylglucosaminidase H (endo H) at steady state (Fiedler et al. 1996). A kinetic analysis of the matulation of the N-linked sugar chains showed that more than half of the newly synthesized VIP36 was already endo H-resistant after 60 min (Fullekrug et al. 1999). To know which form of VIP36 interacts with BiP, VIP36 coimmunoprecipitated with BiP by the same procedure as Figure 1C was treated with endo H. The same experiment was performed after HEK293 cells stably expressing Myc-tagged VIP36 was treated with deoxymannojirimycin (DMJ) inhibiting 
1,2-mannosidase activity in the Golgi complex (Fullekrug et al. 1999). Although Myc-tagged VIP36 prepared in the presence of DMJ was susceptible to endo H, VIP36 coimmunoprecipitated with BiP was resistant to endo H-treatment (Figure 2). The endo H-resistance was acquired with modification by medial Golgi enzymes; hence this result indicates that BiP interacts with VIP36 that had already reached the Golgi apparatus.
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Interaction between VIP36 and BiP is stable over time
BiP is a molecular chaperone that transiently interacts with unfolded stretches of nascent polypeptides for as long as these regions remain unfolded. If VIP36 is a chaperone substrate of BiP, BiP should gradually release newly synthesized VIP36 after its initial binding. To test if this is the case, we performed pulse-chase label experiments in the presence of cycloheximide, which prevents new protein synthesis, and then immunoprecipitated the VIP36–BiP complexes. The ratio of BiP to VIP36 did not change over more than 2 h (Figure 3), which suggests that BiP binds to VIP36 stably over long periods of time.
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Colocalization of VIP36 and BiP in the ER
We used electron microscopy to investigate the localization of VIP36 and BiP in 293 cells expressing Myc-tagged VIP36. The number of gold particles of VIP36 and BiP per µm2 is shown in Table I, and the representative electron micrograph is shown in Figure 4. These data reveal that the 10-nm gold particle-labeled VIP36 and the 15-nm particle-labeled BiP mainly colocalized in the ER. Although VIP36 was also detected in the Golgi apparatus, colocalization with BiP was not observed. This indicates that VIP36 localizes from the ER to the Golgi apparatus and interacts with BiP particularly in the ER.
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In vitro binding of BiP to sVIP36 requires divalent cations
We used surface plasmon resonance (SPR) to analyze the in vitro interaction between recombinant sVIP36 and BiP prepared in Escherichia coli cells. Recombinant biotinylated VIP36 was immobilized on an SPR sensor chip and recombinant BiP was passed over it. In the presence of 20 mM HEPES (pH 7.4), 50 mM KCl, and 2 mM Mg2+, BiP bound to the VIP36-coated surface with high affinity (Kd = 3.62 x 10–8 M, TableII) but did not bind to a control surface immobilized with Nkrp1c (Figure 5). Since recombinant proteins expressed in E. coli lack glycosylation, the interaction between recombinant sVIP36 and BiP supports our earlier observation that the interaction does not depend on the carbohydrate moieties of these proteins.
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We next examined the effect of divalent cations on the interaction between sVIP36 and BiP. As divalent cations have not been reported to be required for the binding of BiP and chaperone substrates, we chose the P15 peptide, which mimics a BiP chaperone substrate (Misselwitz et al. 1998
), as a negative control. Peptide P15 was immobilized on a sensor chip and recombinant BiP was passed over it. The binding of BiP to peptide P15 was unchanged in the presence or absence of 2 mM Mg2+ (Figure 6A). In contrast, the binding of sVIP36 to BiP was substantially reduced in the absence of divalent cations. Ca2+ or Mn2+ at 2 mM strengthened the binding to the same extent as Mg2+ (Figure 6B).
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ATP does not affect the interaction between VIP36 and BiP
It has been reported that BiP releases its chaperone substrate in the presence of ATP (Wei et al. 1995
; Bukau et al. 1998), and that the binding of BiP to peptide P15 is markedly reduced in the presence of 1 mM ATP (Misselwitz et al. 1998). This was confirmed by SPR analysis using recombinant BiP and peptide P15 (Figure 7A), as the Kd was increased by three orders of magnitude in the presence of ATP (Table II). Next, we examined the effect of ATP on the interaction between VIP36 and BiP. As shown in Figure 7B, the interaction between VIP36 and BiP is ATP-independent since the Kd values of the interaction between VIP36 and BiP were almost the same in the presence or absence of ATP (Table II). Moreover, while the on and off rates of the binding of BiP to both VIP36 and peptide P15 were higher in the presence of ATP, the binding affinity of BiP and VIP36 was not altered by ATP, whereas ATP reduced the affinity between BiP and peptide P15 (Table II).
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Discussion |
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The ER has a quality control system for proofreading newly synthesized proteins in eukaryotic cells. If a protein does not attain a correctly folded conformation, it is not transported to its final destination and is instead degraded. The molecular chaperones and folding sensors that are involved in these processes often have the dual functions of assisting the folding process and dispatching improperly folded proteins for destruction in a process called ER-associated degradation (McCracken et al. 1996
). Calnexin, calreticulin, UDP-glucose glycoprotein glucosyltransferase (UGGT), protein disulfide isomerase (PDI), ERp57, BiP, GRP94, and ER-degradation-enhancing 1,2-mannosidase-like protein (EDEM) all participate in this quality control process and sometimes function together in complexes (Kleizen et al. 2004). For example, calnexin, calreticulin, and ERp57 form a complex (Corbett et al. 1999; Oliver et al. 1999), as do calnexin and EDEM (Molinari et al. 2003; Oda et al. 2003). These proteins have common inducible characteristics under several ER-stress conditions.
Cargo receptors participate in secondary quality control, which refers to various selective mechanisms that regulate the export of individual protein species or protein families in the secretory pathway (Ellgaard et al. 2003). These cargo receptors are considered to participate not only in the export of folded proteins from the ER but also in the retrieval of misfolded proteins from the Golgi to the ER (Ellgaard et al. 1999; Arvan et al. 2002). ERGIC-53 and VIP36 are induced by ER-stress conditions (Nyfeler et al. 2003), and ERGIC-53 forms a complex with MCFD2, which is also induced by ER-stress conditions (Spatuzza et al. 2004), but VIP36 has not been found to be a complex so far. To investigate the possibility that VIP36 also forms a complex with other molecules, we searched for proteins that interact with the cargo receptor VIP36.
To identify the proteins that associate with VIP36, cell lysates were prepared from HEK293 cells expressing Myc-tagged VIP36 and an anti-Myc antibody was used for immunoprecipitation. LC/MS/MS analysis of the trypsin-digested fragments of the coprecipitated 80 kDa protein revealed that the molecular chaperone BiP is a VIP36-interacting protein. Coprecipitaition of VIP36 with BiP was also confirmed from untransfected cell lysates, indicating that VIP36 interacts with BiP under the physiological condition. BiP is a major protein of the Hsp70 family. It mainly localizes in the ER (Haas 1994), where it binds to various nascent and newly synthesized proteins and assists their folding as a chaperone. BiP consists of an ATPase domain, a peptide-binding pocket, and a lid domain covering the peptide-binding pocket (Bukau et al. 1998). ATP/ADP bound to the ATPase domain regulates reversible peptide binding, and hydrolysis of the ATP bound to BiP enhances the binding of peptide chains in its immediate vicinity. There was the possibility that BiP associated with VIP36 still in the process of folding. However, coimmunoprecipitated VIP36 with BiP was resistant to endo H, strongly indicating that the interaction is not the chaperone–substrate association. Several kinds of proteins, including thyroglobulin (Kim et al. 1992), human immunodeficiency virus type 1 envelope glycoprotein gp160 (Knarr et al. 1999), and blood coagulation factor VIII (Dorner et al. 1987) have been reported to be BiP chaperone substrates. These proteins associate with BiP in a time-limited manner with association half-lives of about 10, 30, and 60 min, respectively. To test whether newly synthesized VIP36 also binds transiently to BiP, we performed a time-course pulse-chased experiment. In contrast to what is seen with BiP chaperone substrates, the amount of BiP that coprecipitated with Myc-tagged VIP36 did not change over 120 min, which indicates that VIP36 associates with BiP in a stable manner. It is reported that J proteins also associate with BiP constitutively and function as cochaperones to enhance the ATPase activity of BiP (Bukau et al. 1998). However, the ATPase activity of BiP did not change in the presence or absence of VIP36 (data not shown), which indicates that VIP36 does not function as a cochaperone.
We prepared recombinant BiP and soluble VIP36 proteins in E. coli cells and examined their direct interaction by SPR analysis. The binding of VIP36 to BiP differs significantly from the binding between BiP and its unfolded ligands with regard to the effect of ADP/ATP and the requirement for divalent cations. With respect to the effect of ADP/ATP, our SPR binding analysis showed that unfolded ligands bind to BiP faster and are also released faster in the presence of ATP than in the presence of ADP: Kd values between BiP and p15 peptide in the presence of ATP and ADP were 4.41 x 10–5 and 6.6 x 10–8 M, respectively (Table II). This is in good agreement with previous reports that suggest the binding of ATP to BiP opens up its binding site for unfolded ligands so that they can rapidly enter and exit the binding pocket, resulting in a high Kd (Palleros et al. 1993; Schmid et al. 1994). When ADP is present, this peptide exchange is much slower and the affinity is higher. In contrast, the Kd for the interaction between VIP36 and BiP did not change in the presence of ATP and ADP (1.13 x 10–8 and 3.62 x 10–8 M, respectively), which indicates that BiP interacts with VIP36 in a stable manner and probably not via a peptide-binding pocket. With regard to the requirement for divalent cations, we showed that the binding between BiP and VIP36 is dependent on the presence of Ca2+, Mg2+, or Mn2+ (Figure 6B). In contrast, BiP binds to its chaperone substrates without the need for divalent cations (Figure 6A) (Palleros et al. 1991). These two findings strongly indicate that VIP36 does not interact with BiP in the same way as it binds to unfolded ligands.
VIP36 has sugar-binding activity that is specific for N-linked high mannose-type sugar chains, especially higher molecular weight chains (Hara-Kuge et al. 1999; Kamiya et al. 2005). So some target glycoproteins may interact with VIP36 in carbohydrate-binding manner in the cells. However, major bands considered to be target glycoproteins were not detected (Figure 1A). If many kinds of target glycoproteins are coprecipitated with VIP36, the amount of each glycoprotein may be much smaller than BiP. This may be explained also by the fact that the Ka values of VIP36 are three or four orders of magnitude lower for sugar ligands (104 M–1, Kamiya et al. 2005) than BiP.
VIP36 cycles between the ER and the Golgi apparatus (Fullekrug et al. 1999) and has been suggested to be involved in the retrieval of misfolded proteins (Hauri et al. 2000). In our experiment, VIP36 coimmunoprecipitated with BiP was found to have endo H-resistant sugar chains. This indicates that VIP36 associated with BiP had already transported to the medial Golgi, in which sugar moieties on VIP36 were processed from endo H-sensitive high mannose type to endo H-resistant complex-type glycans. Immunoelectron microscopy experiment using gold-labeled anti-VIP36 and anti-BiP demonstrated that colocalization of VIP36 and BiP occurred especially in the ER but not in the Golgi (Figure 4). These observations may indicate that VIP36 interacted with BiP in the ER is a cargo receptor that has been retrotransported with misfolded glycoproteins to effectively deliver such proteins to ER-resident molecular chaperone BiP.
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Recombinant DNAs
To express VIP36 in eukaryotic cells, a human VIP36 cDNA lacking a signal sequence was cloned into pRC/CMV (Invitrogen, Carlsbad, CA), which was modified to include an N-terminal Myc-tag sequence after the CD8 signal sequence. Site-directed mutagenesis was performed by using a QuickChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. To express recombinant VIP36 (sVIP36) in E. coli cells, a cDNA encoding the luminal domain of VIP36 (amino acids 45–322) was cloned into a pET3c vector (Studier et al. 1990
) that was modified to include a C-terminal enzymatic biotinylation signal (Schatz 1993). To express BiP in eukaryotic cells, a human BiP cDNA lacking a signal sequence was cloned into p3xFLAG-CMV-8 (Sigma, St. Louis, MO). For E. coli expression of recombinant BiP, a cDNA encoding BiP without its signal sequence was cloned into pET21b (Studier et al. 1990), which contains a hexahistidine-tag.
Protein purification
sVIP36 was expressed in the BL21(DE3)pLysS strain of E. coli and obtained as inclusion bodies. It was refolded by dilution as described (Matsumoto et al. 2001) and then purified by sequential anion exchange and gel filtration chromatography steps and biotinylated with BirA biotin ligase (Avidity, Denver, CO). BiP was expressed in the BL21(DE3)pLysS strain of E. coli and then purified by sequential Ni2+-affinity and anion exchange chromatography steps.
Cell culture and transfection
Human embryonic kidney cell line HEK293 cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100 µg/mL penicillin, and 100 U/mL streptomycin. The cells were transfected by using Lipofectamine (Invitrogen) according to the manufacturer's instructions.
Metabolic labeling, crosslinking of proteins, and immunoprecipitation
Prior to the metabolic labeling of the newly synthesized proteins, the cells were washed with labeling medium (methionine/cysteine-free RPMI-1640 medium supplemented with 10% heat-inactivated and dialyzed FCS, 2 mM glutamine, 100 µg/mL penicillin, and 100 U/mL streptomycin) (Sigma). Twenty-four hours after transfection, the cells were metabolically labeled with 100 µCi of [35S]methionine/cysteine (Amersham Biosciences Corp., Piscataway, NJ) for 3 h in labeling medium. The labeled cells were washed with cold PBS and then incubated at 4°C for 30 min with 2 mM dithiobis(succinimidyl propionate) (DSP, Pierce) in PBS. Cells were subsequently washed twice with PBS containing 0.04% (w/v) EDTA before further processing. For immunoprecipitation, 35S-labeled cells were lysed by incubation on ice for 30 min in lysis buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, and 1 µg/mL leupeptin). The lysates were centrifuged at 12,000 x g for 30 min at 4°C to remove insoluble materials and then incubated with an anti-Myc antibody (9E10) (American Type Culture Collection, Manassas, VA). Immunocomplexes were precipitated with protein G-Sepharose (Amersham Pharmacia Biotech), washed three times with wash buffer (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.1% Triton X-100, and 1 mM PMSF), and resolved by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The labeled proteins were then visualized with a BAS 5000 system (Fuji Film, Kanagawa, Japan).
Pulse-chase analysis
Cells were metabolically labeled as described above. The pulse was ended by adding prewarmed RPMI-1640 supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 µg/mL penicillin, 100 U/mL streptomycin, and 100 µg/mL cycloheximide. Cycloheximide is an inhibitor of protein translation. After various chase periods of various lengths, the cells were transferred to ice and washed with cold PBS, followed by immunoprecipitation with an anti-Myc antibody. The immunoprecipitated proteins were separated by SDS-PAGE and the gels were exposed to an imaging plate. Quantification of the autoradiograms was carried out with the BAS 5000 system.
Coimmunoprecipitation assay
Twenty-four hours after transfection, HEK293 cells were harvested, lysed in lysis buffer, and clarified by centrifugation at 12,000 x g for 30 min at 4°C. After crosslinking of proteins by 2 mM DSP, the soluble supernatant was incubated with either an anti-Myc antibody or a mouse monoclonal anti-FLAG antibody (M2) (Sigma) for 2 h at 4°C. When using untransfected HEK293 cells, the immunoprecipitation was performed with anti-VIP36. The immunocomplexes were then precipitated with protein G-Sepharose and then washed three times with wash buffer containing 1 mM ATP to remove unexpected denatured proteins bound to BiP. The products were resolved with 12.5% SDS-PAGE under reducing conditions and detected by Western blotting with either a rabbit polyclonal anti-BiP antibody (ET-21) (Sigma) or an anti-VIP36 antibody. The anti-VIP36 antibody was raised against synthetic peptides conjugated with keyhole limpet haemocyanin (KLH) using the stalk portion corresponding to 299–316 residues of VIP36 (NFLKSPKDNVDDPTGNFR) by m-maleimidobenzoyl-N-hydroxysuccimide ester (MBS). Rabbits were immunized with 0.2 mg of the KLH-conjugated peptide in Freund's incomplete adjuvant three times at 2-week intervals. The IgG was purified from the antisera by using a Melon Gel IgG Spin Purification Kit (Pierce), according to the manufacturer's instructions and then affinity-purified on a column conjugated with the synthetic peptides.
Endo H treatment
Immunoprecipitated samples with antibodies and protein G-sepharose were eluted by boiling for 5 min in PBS containing 1% SDS. SDS was quenched with NP-40 and the samples were incubated with 7.5 mU endo-N-acetylglucosaminidase H (endo H) (New England Biochem, Beverly, MA) for 18 h at 37°C.
Deoxymannojirimycin treatment
HEK293 cells were incubated with 1 mM deoxymannojirimycin (DMJ) (Sigma) for 24 h at 37°C. Lysis, immunoprecipitation, and endo H treatment were carried out as described above.
Mass spectrometry and protein identification
HEK293 cells stably expressing Myc-tagged VIP36 (5.0 x 108 cells) were crosslinked with 2 mM DSP and lysed. VIP36 was immunoprecipitated from the cell lysate with an anti-Myc antibody and the precipitates were separated by SDS-PAGE under reducing conditions. The band corresponding to 80 kDa was excised and subjected to in-gel trypsin digestion. LC/MS/MS analysis of trypsin-digested fragments and protein identification were performed as described previously (Nagaike et al. 2001; Suzuki et al. 2001a,2001b).
Surface plasmon resonance (SPR) experiments
The interaction between sVIP36 and BiP was measured with a Biacore biosensor system (Biacore International AB, Uppsala, Sweden) using CM5 research grade chips. Streptavidin was covalently coupled to the CM5 sensor chip by using an amine coupling kit (Biacore International AB), and biotinylated sVIP36 was immobilized on the chip through its interaction with streptavidin. Biotinylated Nkrp1c, an NK receptor, was immobilized on the chip via steptavidin as a control. Peptide P15 (ALLLSAPRRGAGKKC) was immobilized by using a thiol coupling kit (Biacore International AB) as described previously (Misselwitz et al. 1998). The experiments were performed at 25°C at a flow rate of 5–10 µL/mL of a running buffer containing 20 mM HEPES (pH 7.4), 50 mM KCl, and the indicated divalent cation (2 mM) or 1 mM ATP. To test the effect of pH on the interaction between sVIP36 and BiP, we used 20 mM MES (pH 7.0, 6.5, or 6.0) containing 50 mM KCl and 2 mM CaCl2 as the running buffer (Grabe et al. 2001). The sensorgrams were analyzed using BIAevaluation 3.0 software (Biacore International AB). Kinetic constants were obtained by fitting curves to a single-site binding model.
Immunoelectron microscopy
Cells on a cover glass were fixed by 5% acrolein in 1/15 M phosphate buffer (PB), pH 7.4 for 15 min, washed well with PB for 30 min three times, and then postfixed in 1% osmium tetroxide for 2 h at 4°C and 7% sucrose in PB for 2 h at 4°C. The cells were dehydrated with a graded ethanol series at 0°C, embedded in Lowicryl K4M (Polysciences Inc., Niles, IL) and cured under ultraviolet light for 3 days at –35°C. For quenching any remaining aldehyde, the ultrathin sections were treated with 3% hydrogen peroxide for 10 min at 15°C, washed three times with water and then treated with 0.1% ammonium chloride for 10 min at 15°C. The ultrathin sections were washed thoroughly with PBS, treated with 10% normal goat serum (NGS) (Sigma) in PBS for 1 h at room temperature and then incubated with rabbit anti-VIP36 antibody (0.8 µg/mL) in PBS containing 1% bovine serum albumin (BSA) for 24 h at 4°C. The sections were rinsed thoroughly with PBS, incubated for 1 h at 20°C with 10-nm colloidal gold-labeled anti-rabbit IgG (1:200) (British Biocell International Ltd., London, UK), and then rinsed with PBS followed by water. For double-staining immunoelectron microscopy, the reverse side of the thin section was treated with hydrogen peroxide and ammonium chloride as described above, and then treated with NGS under the same conditions. We did not stain on the same side with a cocktail of antibodies in order to interferences of antibodies. The section was incubated with mouse anti-BiP/GRP78 antibody (1:500) (BD Biosciences, San Jose, CA) in PBS containing 1% BSA for 24 h at 4°C, washed, and then incubated with 15-nm gold-labeled anti-mouse IgG (British Biocell International Ltd.) for 1 h at 20°C. The sections were stained with uranyl acetate for 5 min and stained with lead citrate for 1 min, then observed by an electron microscope (H7500, Hitachi, Tokyo, Japan).
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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CREST of the Japan Science and Technology Agency and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (16390019).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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None declared.
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Abbreviations |
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BiP, immunoglobulin-binding protein; SPR, surface plasmon resonance; VIP36, 36-kDa vesicular integral membrane protein
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References |
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