The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum

Abstract

The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain-binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding-induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain-binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates.

Figure 1.

ATP hydrolysis rates of rBiP derivatives. Inorganic phosphate production was measured spectroscopically to determine rates of steady-state ATP hydrolysis by purified rBiP variants (WT, R197H, T229G) or Kar2p. Measurements were conducted under basal conditions (lanes 1, 7, 13, and 17) or in the presence of purified ERdj4 J domain (fourfold molar excess, lanes 2, 8, and 14), pepCL9C (200-fold molar excess, lanes 3 and 9), or both (lanes 4 and 10), as indicated. Control reactions contained the ATPase mutant rBiP T229G (lane 17) or rBiP WT in the absence of ATP (lane 18). To determine the effect of reconstitution conditions on BiP ATPase activity, rBiP (and ERdj4, where indicated) was exposed to high pH (lanes 5, 6, 11, 12, 15, and 16) before being returned to neutral pH. The mean values from 3–5 independent experiments are shown ± SD.

Figure 2.

NBD-labeled translocation intermediates engaged with KRMs, XRMs, or RRMs. (a) Short nascent chains (e.g., pPL₆₄) are sealed off from the lumen by the BiP-mediated gate (hatched oval), whereas longer nascent chains (e.g., pPL-sK₇₈) are exposed to the lumen. Probe positions are indicated by open circles and the nascent chain within the ribosome tunnel is depicted as a dashed line. (b–d) Mean K_SV values (n = 3–12 ± SD) are shown for NBD-pPL₆₄ (b) or NBD-pPL-sK₇₈ (c and d) translocation intermediates functionally engaged with KRMs, XRMs, or RRMs as indicated. For RRMs, reconstitutions were performed either in the presence of ATP (b and c) or ADP (d). Measurements were made in the absence (•, I⁻ on cytosolic side of sealed membranes only) or presence (○, I⁻ both on cytosolic side and in lumen) of melittin.

Figure 3.

Dependence of translocon gating on the SBD of BiP. (a) Mean fluorescence anisotropy values of NBD-pepCL9C (n = 3–5 ± SD) preincubated with increasing concentrations of rBiP (WT or 44K) with ATP, ADP, and unlabeled pepCL9C (fourfold molar excess) as indicated. (b) Mean K_SV values (n = 3 ± SD) for NBD-pPL-sK₇₈ associated with RRMs reconstituted with rBiP 44K or full-length rBiP (WT or T37G) measured in the absence (•) or presence (○) of melittin. RRMs contained pepCL9C (20-fold molar excess over rBiP) and ATP or ADP as indicated. In the final lane, KRMs were incubated with pepCL9C before fluorescence measurements (n/a, not applicable).

Figure 4.

Dependence of translocon gating on the J domain–binding region of BiP. (a) GST-J recombinant protein was immobilized on glutathione agarose and incubated with purified rBiP (WT or R197H) in the presence of ADP (left) or ATP (right). Unbound protein was removed by extensive washing, and the bound protein was released from the glutathione agarose by SDS sample buffer, subjected to SDS-PAGE, and visualized by Coomassie blue staining. (b) Mean K_SV values (n = 3 ± SD) for NBD-pPL-sK₇₈ RNCs bound to RRMs reconstituted with rBiP R197H and ATP or ADP measured in the absence (•) or presence (○) of melittin.

Figure 5.

Co-reconstitution of rBiP WT and sealing-incompetent rBiP. Mean K_SV values (n = 3 ± SD) for NBD-pPL-sK₇₈ RNCs bound to RRMs reconstituted with ATP and equimolar concentrations of rBiP WT and either rBiP T229G, rBiP R197H, or Kar2p measured in the absence (•) or presence (○) of melittin.

Figure 6.

Proposed mechanism by which BiP gates the ER translocon pore. Step 1: ADP-bound BiP seals the lumenal end of the translocon via an interaction both between the SBD and a translocon-associated component (represented here as a lumenal loop) and also between BiP and a J protein (J). Step 2: after SRP-dependent targeting of a RNC, the BiP-mediated gate continues to seal the translocon from the lumen for short nascent chains. Step 3: opening of the BiP-mediated gate after the nascent chain reaches a threshold length of ∼70 residues requires that BiP assumes the ATP-bound, open-binding pocket conformation. Step 4: after translation of substrate, ribosome-free translocons are resealed by the BiP-mediated gate. Steps blocked by BiP mutations are indicated.