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. 2008 Nov 5;27(21):2862-72.
doi: 10.1038/emboj.2008.199. Epub 2008 Oct 16.

Regulated association of misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3

Kseniya Petrova  1 , Seiichi Oyadomari, Linda M Hendershot, David Ron
Affiliations

Regulated association of misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3

Kseniya Petrova et al. EMBO J. .

Abstract

P58/DNAJc3 defends cells against endoplasmic reticulum (ER) stress. Most P58 molecules are translocated into the ER lumen, and here we report selective and stable binding to misfolded proteins by P58's TPR-containing N-terminal domain. In vitro, too, P58 binds selectively to a model misfolded protein and challenge of that complex with physiological concentrations of the ER lumenal Hsp70-type chaperone BiP encourages disassembly. BiP-induced dissociation of P58 from its substrate depends on the presence of ATP and on interactions with P58's J-domain, which are mediated by invariant residues BiP(R197) and P58(H422). A functional J-domain also accelerates dissociation of P58 from a model substrate, VSV-G(ts045), on the latter's re-folding in vivo. However, J-domain binding can be separated from the ability to promote substrate dissociation by the mutant BiP(E201G) and a wild-type J-domain fused ectopically to P58(H422Q) rescues the latter's inability to dissociate from substrate in response to BiP and ATP. These findings are consistent with a model whereby localized activation of the Hsp70-type partner is sufficient to promote substrate handover from the J-domain co-chaperone.

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Figures

Figure 1
Figure 1
P58 selectively associates with misfolded VSV-G in vivo. (A) Immunoblot of GFP-tagged VSV-Gts045 recovered in complex with FLAG–P58 from lysates of FLAG–P58-expressing CHO cells that had been maintained at the permissive temperature (32°C) or the non-permissive temperature (41°C) for 12 h before harvest or switched back to the permissive temperature for the last hour before harvest (41–>32°C). Where indicated, the cells were exposed to the proteasome inhibitor MG132 for 3 h before harvest. The middle panel reports on the amount of VSV-Gts045–GFP in the cell lysates (1.5% of the input) and the lower panel on the recovery of FLAG–P58 in the immunoprecipitate. (B) Autoradiograph of immunopurified VSV-Gts045–GFP from transfected CHO cells metabolically labelled for 30 min at the permissive (32°C) or non-permissive temperature (41°C) in the absence or presence of brefeldin A (BFA). Where indicated, the immunopurified proteins were digested by an endoglycosidase that does not react with Golgi-modified N-linked saccharides (Endo H). The slower migrating Endo H-resistant (Endo HR) and faster migrating Endo H-sensitive (Endo HS) forms of VSV-G–GFP are indicated. (C) Autoradiograph of proteins immunopurified from CHO cells co-expressing FLAG–P58 and VSV-Gts045–GFP. The cells were briefly radiolabelled (30 min) in the presence of BFA at the permissive (32°C) or non-permissive temperature (41°C) and where indicated, chased with unlabelled media for 30 min in the continued presence of BFA, before lysis and immunoprecipitation with anti-FLAG antibody or anti-GFP serum. (D) Same as (C) except that cells were pulse labelled in the presence of BFA at the non-permissive temperature (41°C) and a cold chase was performed for 1 h at the permissive (32°C, lane 4) or non-permissive temperature (41°C, lane 5) in the continued presence of BFA. The lysate of cells lacking FLAG–P58 (CHO parental, lanes 1 and 6) serves as a specificity control for the recovery of VSV-Gts045–GFP in complex with FLAG–P58.
Figure 2
Figure 2
The complex between P58 and misfolded VSV-G is independent of BiP. (A) Autoradiograph of proteins immunopurified from CHO cells expressing VSV-Gts045–GFP and FLAG–P58 (lanes 1–4) or control, parental CHO cells (lanes 5 and 6), metabolically labelled at the non-permissive temperature. Where indicated, the immunopurified FLAG–P58-containing complex was challenged by incubation at room temperature (25°C) in the presence or absence of ATP (to dissociate bound BiP). The VSV-Gts045–GFP, BiP and FLAG–P58 that remained associated with the immune complex are revealed by autoradiography. (B) BiP and FLAG immunoblot of material prepared as in (A), except that lane 1 contains 1% of the input of the lysate used in lanes 2–4.
Figure 3
Figure 3
Direct binding of P58 to misfolded RNase A in vitro. (A) Immunoblots of full-length FLAG–P58 (P58WT) and C-terminally truncated FLAG–P58 (P58ΔJ) from lysates of transfected 293T cells associated with an affinity resin consisting of native (N) or denatured (D) RNase A coupled covalently to sepharose beads. The resin was reacted with cell lysates, washed, followed by incubation in the presence of ATP where indicated, further washed and the remaining bound material was eluted and revealed by SDS–PAGE. Lanes 1 and 2 report on the input of the binding reactions. The lower panel (a BiP immunoblot) reports on the association of endogenous BiP with the resin. (B) As in (A), except that the crosslinker DSP was added to the assembled complex followed by sequential elution (indicated by the arrow), first with SDS (to recover non-covalently bound proteins) and then with DTT (to reverse the crosslink and recover covalently bound proteins). Where indicated, the pre-formed complex was reacted with ATP and washed extensively before adding cross linker. (C) Coomassie-stained gel of wild-type FLAG–P58 purified from stably expressing CHO cells by FLAG immuno-affinity chromatography and elution by FLAG peptide competition. The crude lysate (input), the column flow-through (FT) and the first and second elution by FLAG peptide competition elution are shown (E1 and E2). (D) Immunoblot of the purified FLAG–P58 (shown in (C)) that associated with the native (N) and denatured (D) RNase A affinity resin. Where indicated, the complex was challenged with ATP. Lane 1 shows 5% of the input used in the other lanes.
Figure 4
Figure 4
Addition of BiP and ATP dissociates P58 from misfolded RNase A. (A) Immunoblot of purified FLAG–P58 (shown in Figure 3C and D) associated with denatured RNase A. Where indicated, the pre-formed complex was challenged with 9 μM bacterially expressed wild-type BiP (WT) or mutant BiPR197E in the presence of ADP or ATP. The ratio of P58 retained on the resin was measured by the LI-COR IR imaging system and is indicated for each experimental pair (under the curved arrow). The bound BiP was revealed by staining the blot with Ponceau S (lower panel). (B) As in (A) but using wild-type FLAG–P58 (WT) and a C-terminal truncation mutant FLAG–P58 lacking the J-domain (ΔJ) from mammalian cell lysates associated with denatured RNase A. Where indicated, the pre-formed complex was challenged with 9 μM wild-type BiP (WT), BiP lacking its C-terminal substrate-binding domain (44K) and mutant BiPR197E. The BiP that remained bound to the resin is revealed in this experiment as a residual fluorescent signal on the P58 immunoblot. (C) Same as (B), but using mammalian cell lysates expressing mutant FLAG–P58H422Q.
Figure 5
Figure 5
A J-domain mutation that compromises interaction with BiP stabilizes P58's binding to VSV-Gts045–GFP in cells. (A) Autoradiograph of 35S-radiolabelled proteins recovered in an anti-FLAG–M1 immunoprecipitate from lysates of CHO cells stably expressing wild-type P58 or the H422Q J-domain mutant. The cells were transfected with a VSV-Gts045–GFP expression plasmid, pulsed at the non-permissive temperature (41°C) and chased for the indicated time at the permissive temperatures, before isolation of the immune complexes. The location of the radiolabelled P58 and VSV-G is indicated. (B) Autoradiograph of 35S-radiolabelled VSV-G–GFP recovered by immunoprecipitation with anti-GFP serum from the flow through of the reactions shown in (A). (C) Immunoblot comparing the steady-state levels of wild-type and H422Q mutant FLAG–P58 in the CHO cells. (D) Plot of the signal of labelled VSV-G bound to P58 from (A). CHO cells stably expressing FLAG–P58WT (-▴-) and CHO cells stably expressing FLAG–P58H422Q (-▪-).
Figure 6
Figure 6
Addition of purified P58 J-domain compromises BiP's ability to promote dissociation of P58 from denatured RNase A. (A) Immunoblot of FLAG–P58WT and a J-domain mutant FLAG–P58H422Q from mammalian cell lysates associated with denatured RNase A. Where indicated, the pre-formed complex was challenged with bacterially expressed BiP (9 μM) in the presence of ADP or ATP and the presence or absence of equimolar wild-type or mutant versions of P58's isolated J-domain (residues 384–470) expressed as a fusion protein with GST (GST–JWT and GST–JH422Q). The amount of P58 retained on the resin following each manipulation was measured by the LI-COR IR imaging system and is indicated (as a percentage of the signal in the unmanipulated sample). BiP's association with the resin is revealed by Ponceau S staining of the blot (lower panel). (B) Coomassie-stained SDS–PAGE of BiP retained on glutathione sepharose beads to which GST, GST–JWT or GST–JH422Q had been pre-bound. Where indicated, the binding buffer was supplemented with ADP or ATP. Lanes 1–4 report on the input proteins used in the experimental lanes 5–10. (C) ATPase activity of BiP alone or in the presence of GST–JWT or GST–JH422Q, measured by the conversion of ATP to ADP. The mean±s.e.m. of the fluorescence polarization signal of AlexaFluor 633-labelled ADP tracer, which is dissociated from an anti-ADP antibody in the presence of ADP generated in the reaction from BiP's ATPase activity, is shown (n=3). The fluorescent polarization signal is inversely proportional to the ATPase activity.
Figure 7
Figure 7
The BiPE201G mutation separates J-domain binding from dissociation of P58 from denatured RNase A. (A) Coomassie-stained SDS–PAGE of BiP or BiPE201G mutant retained on glutathione sepharose beads to which GST, GST–JWT or GST–JH422Q had been pre-bound. Where indicated, the binding buffer was supplemented with ADP or ATP. Lanes 1–5 report on the input proteins used in the experimental lanes 6–13. (B) Immunoblot of bacterially expressed and purified P58 associated with denatured RNase A. Where indicated, the pre-formed complex was challenged with bacterially expressed BiPWT or BiPE201G (9 μM) in the presence of ADP or ATP. (C) ATPase activity of wild-type BiP and the E201G mutant alone or in the presence of GST–JWT or GST–JH422Q, measured by the conversion of ATP to ADP. The mean±s.e.m. of the fluorescence polarization signal is shown (as in Figure 6C).
Figure 8
Figure 8
Ectopic fusion of the J-domain to the N terminus of P58H422Q rescues the protein's inability to dissociate from misfolded RNase A in response to BiP/ATP. (A) Coomassie-stained gel of bacterially expressed proteins associated with the native (N) and denatured (D) RNase A affinity resin. The resin was reacted with purified bacterially expressed P58, or fusion proteins consisting of P58's J-domain (either wild-type or H422Q) fused ectopically to P58H422Q's N terminus, washed and the remaining proteins were eluted in SDS. Lanes 1–3 contain 10% of the input used in the respective binding reactions. The migration of P58, J–P58 fusion proteins and their N-terminal fragments are indicated. (B) Immunoblot of material as in (A). Where indicated, the pre-formed complex with denatured RNase A was challenged with bacterially expressed BiPWT (9 μM) in the presence of ADP or ATP. The amount of P58 retained on the resin following each manipulation was measured by immunoblot with a LI-COR IR imaging system and is indicated (as a percentage of the signal in the unmanipulated sample). Note that the N-terminal fragments of JWT–P58H422Q (but not those of the JH422Q–P58H422Q control) were also dissociated by BiP/ATP.
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