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2 Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701
3 Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530 Japan
Address correspondence to Ralph Henry, Dept. of Biological Sciences, 601 Science Engineering Building, University of Arkansas, Fayetteville, AR 72701. Tel.: (479) 575-2529. Fax: (479) 575-4010. email: Ralph.Henry{at}uark.edu
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Abstract |
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Key Words: protein transport; signal recognition particle; receptors; membrane proteins; Arabidopsis proteins
Abbreviations used in this paper: chl, chlorophyll; cpSRP, chloroplast signal recognition particle; cpTAT, chloroplast twin-arginine translocation; GMP-PNP, 5'-guanylyl-imidodiphosphate trisodium salt; LHCP, light harvesting chl a/b-binding protein; OE, oxygen-evolving complex; SE, stromal extract; Sec, secretory; SPDP, N-succinimidyl 3-[2-pyridyldithio]-propionate.
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Introduction |
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One of these, the spontaneous pathway, which is responsible for the integration of membrane proteins such as Elip2 into the thylakoid membrane, appears to lack proteinaceous and energetic requirements (Kim et al., 1999). The chloroplast twin-arginine translocation (cpTAT) pathway depends on a trans-thylakoidal pH gradient to supply the energy needed to transport substrates, including the lumenal 17-kD subunit of the oxygen-evolving complex (OE17; Cline et al., 1992). Although no soluble protein components have been reported for this pathway, a membrane translocase containing Tha4, Hcf106, and cpTatC, is required (Mori and Cline, 2001). The chloroplast Sec (cpSec) pathway, homologous to the bacterial and ER secretory (Sec) pathways, utilizes cpSecA, cpSecY, and cpSecE to transport a subset of lumenal proteins including the 33-kD subunit of the oxygen-evolving complex (OE33) in an ATP-dependent reaction (Mori and Cline, 2001). Based on homology to the bacterial Sec system, it is expected that cpSecY and cpSecE form a proteinaceous pore through which proteins are translocated (Muller et al., 2001). Homology between the translocase components cpSecY/E, bacterial SecY/E and Sec61/
in the ER membrane provides strong support for a common evolutionary history of these three translocation systems.
A chloroplast signal recognition particle (cpSRP) also functions in protein localization to the thylakoid (for review see Eichacker and Henry, 2001). Homologous SRPs in the cytosol of pro- and eukaryotes function exclusively to cotranslationally target proteins to the cytoplasmic and ER membranes, respectively (Walter and Johnson, 1994; Rapoport et al., 1996). cpSRP is unique in that it functions posttranslationally (Li et al., 1995) to transport a family of light-harvesting chlorophyll (chl) a/b-binding integral membrane proteins, the LHCs. The most studied of these is light harvesting chl a/b-binding protein (LHCP), the lhcb1 gene product. During or after import into the chloroplast, LHCP is bound by cpSRP, a heterodimer composed of an evolutionarily conserved 54-kD subunit (cpSRP54) and a unique 43-kD subunit (cpSRP43; Schuenemann et al., 1998; Groves et al., 2001). LHCP integration also requires cpFtsY, a homologue of the bacterial SRP receptor, FtsY, and the SR subunit of the SRP receptor in the ER (Kogata et al., 1999; Tu et al., 1999). Like FtsY in Escherichia coli (Zelazny et al., 1997), it is anticipated that cpFtsY functions at the thylakoid membrane as a cpSRP receptor during LHCP targeting. Consistent with the fact that both cpSRP54 and cpFtsY are GTPases, GTP is required for LHCP integration into isolated thylakoids (Hoffman and Franklin, 1994). Recently, we have shown that the purified recombinant proteins, cpSRP and cpFtsY, along with GTP, are necessary and sufficient for in vitro integration of LHCP into isolated thylakoid membranes (Yuan et al., 2002).
Though the soluble protein requirements for LHCP integration are well established, a detailed understanding of the membrane components is lacking. In an earlier study, we showed that the integral thylakoid protein, ALB3, functions in the LHCP integration mechanism (Moore et al., 2000). Antibodies bound to ALB3 were able to prevent LHCP integration without affecting transport by the cpSec or cpTAT pathways. Conversely, antibodies bound to cpSec or cpTAT translocase components had no effect on LHCP integration, but inhibited transport by the cpSec and cpTAT pathways specifically. These results implicate ALB3 as a necessary component of the LHCP integration machinery and are supported by recent genetic studies in Chlamydomonas reinhardtii (Bellafiore et al., 2002). The results also imply that the translocase used by LHCP is distinct from those used by other pathways. However, these findings do not rule out the possibility that ALB3 functions with the cpSec translocase in cotranslational integration of chloroplast synthesized proteins, a scenario suggested by results of cotranslational integration studies in E. coli (for reviews see Luirink et al., 2001; Chen et al., 2002).
In bacteria, the ALB3 homologue YidC appears to act in two functionally separate pools (Stuart and Neupert, 2000). One pool is associated with the Sec translocase (Scotti et al., 2000) and functions during cotranslational integration to interact with transmembrane segments that have exited the SecYE pore (Beck et al., 2001). YidC's association with SecYEG appears to stem from its ability to interact with a SecDFYajC complex (Nouwen and Driessen, 2002). The second pool of YidC operates in the absence of a functional Sec translocase to integrate a distinct subset of membrane proteins (Samuelson et al., 2000). Although sequence homologues of SecDF and YajC are absent in Arabidopsis thaliana, the fact that cpSecY and ALB3 can be cross-linked in thylakoid membranes (Klostermann et al., 2002) suggests that ALB3, like YidC, may also have two distinct functions. ALB3 associated with cpSecYE may serve in cotranslational integration activities, whereas a second pool of ALB3, functionally independent of cpSecYE, mediates LHCP integration posttranslationally. In this context, it is unclear whether cpSRP or cpFtsY play a direct role in delivering LHCP to ALB3, or simply act to target LHCP to the membrane in a conformation suitable to promote LHCP interaction with ALB3. A direct role would be supported by the ability of cpSRP or cpFtsY to associate with ALB3.
Currently, nothing is known about how cpSRP and cpFtsY interact with each other and/or with membrane proteins that function in LHCP integration. Here, we have used recombinant cpSRP and cpFtsY to explore these interactions. Our results show that cpSRP and cpFtsY interact with and target to the ALB3 translocase. Using 5'-guanylyl-imidodiphosphate trisodium salt (GMP-PNP), we can stabilize the association between cpSRP and cpFtsY at the membrane and show that they form a complex containing ALB3 and cpSecY. The complex occupies functional ALB3 translocation sites, demonstrated by decreased LHCP integration into thylakoids where this complex was stabilized before integration assays, whereas cpSecY function is not interrupted. Furthermore, we show that treatments of thylakoid membranes with anti-ALB3 serum are able to inhibit the association of a cpSRPcpFtsY complex with ALB3, which correlates with the antibody treatment's inhibitory effect on LHCP integration. Antibody against cpSecY, together with antirabbit IgG, removes cpSecY from complexes containing cpSRP, cpFtsY, and ALB3 without inhibiting ALB3 activity, indicating that cpSecY is likely not part of the functional complex. Interestingly, neither cpSRP43, nor LHCP, is required to form a complex with ALB3, suggesting that cpSRP43 functions to link the substrate to the true targeting components, cpSRP54 and cpFtsY, which form the targeting/translocation interface with ALB3.
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Results |
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Further evidence that cpSRP, cpFtsY, and ALB3 exist in a single complex comes from cross-linking data. The heterobifunctional cross-linker N-succinimidyl 3-[2-pyridyldithio]-propionate (SPDP) reacts with amino and sulfhydryl groups and is cleavable with reducing agents. When thylakoids loaded with the cpSRPcpFtsY complex are treated with 0.1 mM SPDP, a large portion of ALB3, cpSRP54, cpSRP43, cpFtsY, and cpSecY appears in very large complexes on nonreducing Western blots (unpublished data). To determine if these proteins were all cross-linked in a single complex, cross-linked thylakoids were solubilized in SDS, diluted with buffered Triton X-100, and mixed with anti-FLAG IgG and protein G agarose to repurify cpSRP43-FLAG and any cross-linking adducts under denaturing conditions. Precipitated samples were then treated with ß-mercaptoethanol to cleave cross-links, thereby allowing proteins to migrate as monomers during SDS-PAGE. As shown in Fig. 4, ALB3, cpSRP54, and cpFtsY were all coprecipitated with cpSRP43-FLAG in the presence of cross-linker. Furthermore, all four components were found in a single stained band, which was excised from a 5% polyacrylamide gel and treated with reducing agent (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200307067/DC1). Individual components were then separated by SDS-PAGE and identified by Western blotting. Both cpSecY and Tha4 were remarkably absent from the cross-linked complex suggesting that cpSecY is either not properly placed to cross-link to other complex components or is only loosely associated with the other protein components. It is noteworthy that cpSRP54 also coprecipitates with cpSRP43-FLAG in the absence of cross-linker. This suggests that cpSRP43 and cpSRP54 may be linked via a disulfide bond when in a complex on the thylakoids. Alternatively, cpSRP54 and cpSRP43 may refold upon addition of Triton to SDS-solubilized membranes allowing them to reunite. We are currently investigating these possibilities.
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Antibody inhibition of LHCP integration correlates with the loss of cpSRP and cpFtsY binding to ALB3
In an earlier report, we demonstrated that antibodies bound to ALB3 inhibited the subsequent in vitro integration of LHCP (Moore et al., 2000). To better understand these results in the context of the proteinprotein interaction data presented above, we examined the influence of ALB3 and cpSecY antibodies on the formation of complexes between cpSRP, cpFtsY, ALB3, and cpSecY (Fig. 7). Salt-washed membranes were first treated with various rabbit-produced antibodies, washed, incubated again in the presence or absence of antirabbit IgG, and then split into two aliquots. One was used to verify the effect of antibody treatment on integration of the ALB3 substrate, LHCP, or the cpSec substrate, OE33. As shown previously, only immune antibody against ALB3 was able to inhibit LHCP integration, whereas anti-cpSecY serum inhibited only OE33 transport (Fig. 7 A). Also, the presence of antirabbit antibody does not appear to inhibit the function of ALB3 or cpSecY, demonstrated by transport of both LHCP and OE33 into thylakoids treated with preimmune antibody followed by antirabbit IgG. The second aliquot of antibody-treated thylakoids was used to perform complex formation assays by mixing with GMP-PNP, cpSRP, and cpFtsY. After removal of unbound recombinant proteins, thylakoids were solubilized and cpFtsY was repurified along with associated proteins using S-protein agarose. Although immune antibodies had no influence on the amount of precipitated targeting components, thylakoid treatment with anti-ALB3 severely inhibited cpFtsY association with ALB3 (Fig. 7 B, lanes 2 and 5), further establishing the specificity of cpFtsY-ALB3 association. In contrast, formation of a cpFtsYcpSRPcpSecY complex at the membrane was unaffected by anti-cpSecY alone. However, by adding antirabbit IgG to bind the anti-cpSecY on the thylakoids, cpSecY was prevented from interacting with the cpSRPcpFtsYALB3 complex without affecting LHCP integration. These data indicate that whereas cpSecY may be present in a complex with ALB3, its presence is not needed for posttranslational ALB3 function. In no case was the association between cpFtsY and cpSRP affected, signifying that these proteins do not require ALB3 or cpSecY to associate. Together, with data shown above, these results suggest that the direct association of a cpSRPcpFtsY complex with ALB3, or with an unidentified ALB3-associated protein, is a required event for LHCP integration.
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Discussion |
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An association between ALB3 and cpSecY was recently reported by the finding that the two proteins form a cross-linking adduct (Klostermann et al., 2002). The mitochondrial homologue of ALB3, Oxa1p, does not have an available SecY homologue to associate with and is thought to form homooligomeric complexes in the inner membrane (Nargang et al., 2002). However, the ALB3 homologue in E. coli, YidC, can be found in SecY-associated and nonassociated pools (Scotti et al., 2000; Nouwen and Driessen, 2002); and YidC functions in both a Sec-dependent and -independent manner (Samuelson et al., 2000). So far, only ALB3 is known to be necessary for posttranslational translocation of LHCs. Co-translationally integrated thylakoid membrane proteins that appear to use both cpSRP54 and cpSecY, e.g., D1 of photosystem II (Eichacker and Henry, 2001; Nilsson and van Wijk, 2002; Zhang and Aro, 2002), may also require ALB3, but this has yet to be demonstrated.
The data presented here also demonstrate that the GMP-PNPbound complex of cpSRPcpFtsY occupies functional ALB3 integration sites as evidenced by the ability of the complex to inhibit LHCP integration. The inhibition was pathway specific, not inhibiting any of the other three posttranslational protein targeting pathways. The fact that posttranslational cpSecY function was not abolished by cpSRP and cpFtsY binding provides additional support for the hypothesis that ALB3 and cpSecY function independently during posttranslational transport. Further experiments showed that a complex lacking cpSRP43, but containing cpSRP54 and cpFtsY, was similarly able to associate with ALB3 and inhibit LHCP integration. Therefore, we hypothesize that cpSRP43 is not required for targeting to ALB3, but functions as a bridge between the substrate molecule and the actual targeting components, cpSRP54 and cpFtsY.
It has been proposed that cpSRP acts as a chaperone during posttranslational targeting to maintain hydrophobic substrates in an integration competent form, and once at the membrane, the substrate is recognized by the translocase and consequently released for integration (Eichacker and Henry, 2001). The observation that the substrate LHCP was unnecessary for complex formation between cpSRP, cpFtsY and ALB3 argues that cpSRP-bound full-length (posttranslational) substrate molecules will be directed to ALB3, or an associated protein, because of the affinity of cpSRP and cpFtsY for the ALB3 translocase. We hypothesize that the formation of a complex between cpSRPcpFtsY and ALB3 is a necessary step in the integration mechanism. Further support for this hypothesis stems from our finding that anti-ALB3 antibodies, which inhibit LHCP integration, also prevent formation of a complex containing cpSRP, cpFtsY, and ALB3. Using antibody against cpSecY, coupled with antirabbit IgG, we were able to eliminate cpSecY from the cpSRPcpFtsYALB3 complex. Under the same conditions, ALB3 was still active and LHCP was properly integrated, strongly suggesting once more that cpSecY is not involved in ALB3-dependent LHCP integration. However, we cannot completely rule out the possibility that treatment with antirabbit IgG inhibits cpSecY coprecipitation yet still allows cpSecY to function with ALB3. Based on data presented in this report and homology with bacterial translocation pathways, we propose that ALB3 and cpSecY act separately during posttranslational transport activities, but complexes between the two may form in the thylakoid membrane for cotranslational translocation purposes as found in E. coli. Therefore, cpSecY may be found in cpSRPcpFtsYALB3 complexes due to its interactions with both cpSRP54 and ALB3 during cotranslational transport activities.
Interestingly, cpSRP and cpFtsY form a complex at the membrane, even when ALB3 is made unavailable by bound antibody. This leads us to hypothesize that the formation of a cpSRPcpFtsY complex at the membrane is a step in the targeting mechanism that precedes interaction with ALB3. We anticipate that the ordered assembly of this complex must take place for efficient integration of substrates.
In view of the data shown here, we propose the following model for cpSRP-based targeting to the ALB3 translocase in thylakoids (Fig. 8). Chloroplast FtsY is found both in the stroma and at the thylakoid membrane. Chloroplast SRP arrives at the membrane loaded with substrate and in a guanine nucleotide-free form (Yuan et al., 2002). cpFtsY interacts with cpSRP to promote GTP binding by both cpSRP54 and cpFtsY, which stabilizes the subunits together on the membrane. In the absence of accessible ALB3, the complex containing cpSRP, cpFtsY, and LHCP remains associated with the membrane until the ALB3 translocase is available. There, the substrate is released to ALB3 and/or possible unknown translocase components for integration, and GTP hydrolysis liberates the cpSRP and cpFtsY for a successive round of targeting.
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Materials and methods |
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Antibody production against cpSRP54 and cpSRP43
Metal affinity purified mature cpSRP54 from A. thaliana was expressed with a His6-tag at the COOH terminus and used as antigen for antibody production. Similarly purified mature cpSRP43 from A. thaliana was expressed with an NH2-terminal His6-tag and used as antigen. Both antibodies were prepared in rabbits (Cocalico Biologicals).
Construction, expression, and purification of affinity-tagged cpSRP43
The nucleotide sequence coding for the mature region of cpSRP43, beginning AAVQRN, was subcloned from pGEX-6P-2 (Yuan et al., 2002) into pGEX-4T-2 (Amersham Biosciences) using BamHI and SmaI sites to create pGEX-4T-m43. This clone contains two residues, which differ from the published sequence (GenBank/EMBL/DDBJ accession no. AAD01509): K140R and R192L. The mature cpSRP43 was then PCR amplified from pGEX-4T-m43 using a forward sequencing primer, which amplified a BamHI site at the 5' end. The reverse primer added all but the last residue of the FLAG antigenic sequence to the 3' end of the cpSRP43 coding sequence. The PCR product was inserted into pGEX-4T-2 using the BamHI and SmaI restriction sites to make pGEX-4T-m43-FLAG. Sequencing of the plasmid indicated that the amino acid sequence DYKDDDDGSTRAAAS was added to the COOH terminus of the translated protein.
BL21 Star (Invitrogen) cells harboring pGEX-4T-m43-FLAG were grown to mid-log phase and induced with 0.3 mM IPTG for 2 h for expression of GST-m43-FLAG. Glutathione SepharoseTM fast flow (Amersham Biosciences) was used for initial purification. After overnight treatment with thrombin and desalting, cleaved GST was removed by a second pass over Glutathione SepharoseTM. To complete the process, anion exchange was used and proteins were eluted with a linear KCl gradient in 10 mM Hepes-KOH, pH 8.0, and 10 mM MgCl2.
Chloroplast SRP used for assays was made by combining equimolar amounts of isolated cpSRP54-his and m43-FLAG and incubating overnight at 4°C. Further purification by gel filtration using a HiLoad 26/60 Superdex 75 (Amersham Biosciences) with 10 mM Hepes-KOH, pH 8.0, and 10 mM MgCl2 buffer yielded cpSRP-FLAG.
Complex formation and precipitation assays
Complexes between thylakoid membrane proteins and cpSRP-FLAG and Trx-cpFtsY were formed by incubating indicated components (i.e., salt-washed thylakoids, nucleotide, and purified proteins) at 25°C for 30 min. Membranes were recovered by centrifugation and washed with 50 mM Hepes-KOH, pH 8.0, and 0.33 M sorbitol (IB) plus 10 mM MgCl2 (IBM). Thylakoids equal to 25 µg chl were removed and resuspended in 250 µl SDS solubilization buffer for subsequent examination of bound recombinant proteins. For precipitation assays, membranes equal to 50 µg chl were solubilized in 50 µl IB containing 1% n-dodecyl ß-D-maltoside and 1.5% BSA for 10 min followed by centrifugation at 70,000 g for 12 min to pellet insoluble material. The soluble portion was added to 50 µl S-protein agarose (Novagen) as a 50% slurry in IB or 10 µl anti-FLAG M2 (Sigma-Aldrich) and 50 µl protein G agarose (Sigma-Aldrich) as a 50% slurry in IB and incubated for 30 min at RT with gentle mixing. Afterward, the agarose beads were washed three times with 0.1% n-dodecyl ß-D-maltoside in IB, resuspended in IB and transferred to new tubes. Coprecipitating proteins were eluted with 100 µl SDS solubilization buffer.
Cross-linking and denatured precipitation assays
Salt-washed thylakoids containing 350 µg chl and 0.5 mM GMP-PNP were incubated with IBM or 7 µg cpSRP-FLAG and 3.5 µg Trx-cpFtsY as indicated in the Fig. 4 legend to form complexes on the membrane. Afterward, membranes were washed with IBM, a total bound protein sample was removed (50 µg chl) and the remaining thylakoids were aliquoted for cross-linking. Pre-treated thylakoids containing 150 µg chl were incubated in 333 µl IBM containing DMSO or 0.1 mM SPDP (Pierce Chemical Co.) in DMSO for 30 min at 25°C. The cross-linker was quenched by the addition of Tris, pH 8.0 to a final concentration of 3 mM. Membranes were washed with IB containing 3% BSA and subsequently resuspended in 2% SDS at 1 mg/ml chl. After 30 min at 25°C, insoluble material was pelleted. The soluble fraction was then mixed with 10 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, and 1% BSA so that the final SDS concentration was 0.03%. 5 µl anti-FLAG IgG and protein G agarose were added and the solution was mixed overnight at 4°C. Pelleted agarose was washed twice in 10 mM Tris, pH 8, 150 mM NaCl, 0.2% Triton X-100, and once with buffer lacking detergent. Precipitated proteins were eluted with 100 µl SDS solubilization buffer containing ß-mercaptoethanol and heated at 70°C for 12 min.
Transport inhibition assays
Complexes of proteins on the thylakoid membranes were formed using salt-washed thylakoids, 1 mM GMP-PNP, and protein components as indicated in Figs. 5 A and 6 B legends. Afterward, membranes were washed with IBM, a total bound protein sample was removed and the remaining thylakoids were aliquoted for integration assays. Pre-treated thylakoids containing 25 µg chl were incubated with 1 mM ATP, 0.1 mM GTP, SE (equal to 100 µg chl), and 12.5 µl radiolabeled translation product for 15 min at 25°C. Thylakoids were recovered by centrifugation and treated with thermolysin.
Antibody inhibition assays
Salt-washed thylakoids contiaining 250 µg chl were incubated with 75 µl of the indicated antisera as described previously (Mori et al., 1999; Moore et al., 2000) for 1 h on ice. Pelleted membranes were washed with IBM, divided in half, and resuspended in 125 µl 3% BSA in IB. 50 µl of 3 mg/ml antirabbit IgG (Sigma-Aldrich) was added to one of each pair and both brought to a final volume of 750 µl with 10 mM Hepes-KOH, pH 8. After 30 min at 4°C with light mixing, membranes were again pelleted, washed with IBM, and aliquoted for individual assay procedures. Treated thylakoids containing 25 µg chl were used for transport assays as described previously (Mori et al., 1999; Moore et al., 2000). The remaining 75 µg chl from sera-treated thylakoids were used for complex formation and precipitation assays. These membranes were incubated with 0.5 mM GMP-PNP, 20 µg cpSRP-FLAG, and 10 µg Trx-FtsY in a final volume of 450 µl. Bound and precipitated samples were obtained as described earlier (Complex formation and presentation assays).
Analysis of samples
After integration assays, pelleted thylakoids were resuspended in 10 µl 20 mM EDTA and 15 µl 2x SDS solubilization buffer. After heating, proteins from each sample (10 µl) were separated by SDS-PAGE and analyzed by phosphorimaging using a Typhoon 8600 and IQ Solutions software (Molecular Dynamics). Thylakoid membranes with bound recombinant proteins and precipitation samples (10 µl) were separated on 12.5% SDSpolyacrylamide gels, blotted, and probed according to standard Western blotting methods. Secondary antibodies conjugated to horseradish peroxidase were used and detected by ECL. Images were recorded using a Fujifilm LAS-1000 Plus and individual spot intensities were quantified with Science Lab 98 for Windows software (Fujifilm).
Online supplemental material
Salt-washed thylakoids containing 15 mg chl were mixed with 0.1 mM GMP-PNP, 300 µg cpSRP-FLAG, and 150 µg Trx-cpFtsY in a final volume of 45 ml. After incubation at 25°C for 30 min, membranes were washed with IBM and resuspended in 30 ml IBM. SPDP was added to a final concentration of 0.1 mM and the solution was incubated for 30 min at RT. The cross-linker was quenched by the addition of 1 M Tris, pH 8.0 to a final concentration of 50 mM. After a wash with IBM, membranes were treated as above (Cross-linking and denatured precipitation assays) for solubilization in SDS and dilution with buffered Triton X-100. 500 µl of a 50% slurry of anti-FLAG M2 Affinity Gel (Sigma-Aldrich) was added to the solution and incubated overnight before reisolation by centrifugation. The resin was washed as described earlier (Cross-linking and denatured precipitation assays) and coprecipitating proteins were eluted by incubation for 1 h with SDS solubilization buffer lacking ß-mercaptoethanol, but containing 8 M urea. A sample of the eluted proteins was electrophoresed on a 5% SDSpolyacrylamide gel and stained; a sample was also run on an identical unstained gel. A gel piece corresponding to the stained band was excised from the unstained gel and treated with DTT. Proteins from segments of the treated gel slice were analyzed on a 12.5% polyacrylamide gel and Western blotted. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200307067/DC1.
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Acknowledgments |
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This work was supported by grants from the Department of Energy (DE-FG02-01ER15161 to R. Henry) and the National Institutes of Health through the Center for Protein Structure and Function at the University of Arkansas (NIH NCRR COBRE grant 1 P20 RR15569-02).
Submitted: 10 July 2003
Accepted: 6 August 2003
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References |
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