p97 functions as an auxiliary factor to facilitate TM domain extraction during CFTR ER-associated degradation

Abstract

The AAA-ATPase (ATPase associated with various cellular activities) p97 has been implicated in the degradation of misfolded and unassembled proteins in the endoplasmic reticulum (ERAD). To better understand its role in this process, we used a reconstituted cell-free system to define the precise contribution of p97 in degrading immature forms of the polytopic, multi-domain protein CFTR (cystic fibrosis transmembrane conductance regulator). Although p97 augmented both the rate and the extent of CFTR degradation, it was not obligatorily required for ERAD. Only a 50% decrease in degradation was observed in the complete absence of p97. Moreover, p97 specifically stimulated the degradation of CFTR transmembrane (TM) domains but had no effect on isolated cytosolic domains. Consistent with this, p97-mediated extraction of intact TM domains was independent of proteolytic cleavage and influenced by TM segment hydrophobicity, indicating that the relative contribution of p97 is partially determined by substrate stability. Thus, we propose that p97 functions in ERAD as a nonessential but important ancillary component to the proteasome where it facilitates substrate presentation and increases the degradation rate and efficiency of stable (TM) domains.

Figure 1

CFTR is degraded into TCA-soluble peptides. (A) Schematic representation of CFTR showing relative location of methionine residues and topology of TM domains (TMDs), NBDs and regulatory (R) domain. Residues V358, S589 and D835 are also indicated. (B) CFTR translated in the presence of CRMs (lane 2) yields the expected full-length ∼150–160 kDa glycosylated protein. (C) Carbonate extraction of CFTR (lanes 1–4) and control TM and secretory (sec) proteins (lanes 5–8) (Skach and Lingappa, 1993). (D) In vitro CFTR degradation showing the percent of [³⁵S]methionine-labeled protein that is converted into radiolabeled TCA-soluble fragments at each time point. The inset shows brief delay and then linear degradation during the first 30 min. (E) RRL was preincubated at 37^oC for 30, 60, 120 and 240 min before addition of substrate. Degradation activity remains intact for 2 h but is severely decreased after 4 h. (F) Degradation was carried out as in panel D except that at 240 min, membranes containing residual CFTR protein were isolated and added to a fresh degradation assay and reactions were continued for an additional 2 h. All values are reported as mean of three or more experiments ±s.e.m.

Figure 2

RRL depletion of p97 and p97 complexes. (A) Coomassie-stained gel of recombinant His-tagged proteins used as bait for RRL affinity depletion. (B) Immunoblots for p97 (top) and ufd1 (bottom) of intact RRL (lane 1), RRL following a single adsorption with Ni-NTA beads (lane 2), uu5-coated beads (lane 4) or p47-coated beads (lane 6), and two sequential adsorptions with Ni-NTA beads (lane 3) or uu5 followed by p47 (lane 5). (C) Immunoblots for 19S RC subunit Rpt5 (top) and 20S α3 subunit (bottom) using intact, mock- and p47-adsorbed RRL. (D) Immunoblot of membrane-associated p97. Data show p97 recovered in pellet (P) and supernatant (S) after pelleting microsomes (lanes 2 and 3) and the amount of p97 from the same starting material that remained associated with membranes after dilution indicated (lanes 4–7). (E) CRMs (lanes 1 and 5) and p97-depleted membranes (100-fold dilution) were reincubated with RRL (lane 4) or recombinant p97 (lane 7), pelleted and analyzed by immunoblotting.

Figure 3

p97 effect on CFTR degradation. (A) Degradation was carried out as in Figure 1, using mock-depleted RRL (Ni RRL), RRL depleted of p97 using p47 (depl. RRL) or p97-depleted RRL plus recombinant p97 (depl.+p97). (B) Initial degradation rates were calculated from panel A based on the percent of intact [³⁵S]methionine-labeled protein converted into radiolabeled TCA-soluble fragments (% CFTR converted/min) during the first 30 min. (C) CFTR degradation in sequential mock-depleted RRL (Ni-Ni), ufd1 and p97-depleted RRL (depl. RRL) and depleted RRL with the addition of recombinant p97 (depl.+p97). (D) Degradation rates calculated from panel C. The slight decrease in the baseline degradation was caused by dilution of RRL with beads used during affinity adsorption. (E) RRL was serially depleted (× 2) with p47 Ni-NTA beads and degradation reactions were supplemented with the indicated amounts of undepleted RRL (inset) to quantitate the % inhibition of CFTR degradation at very low p97 concentrations. Values represent mean of three or more experiments ±s.e.m.

Figure 4

Degradation of CFTR cytosolic domains is unaffected by p97. Purified NBD1 (A) or NBD1-R (C) was added directly to degradation reactions containing mock-depleted RRL or p97-depleted RRL with or without added recombinant p97 as indicated. Also shown are degradation rates in ATP-depleted RRL (−ATP) and in the presence of MG132. (B, D) Initial degradation rates were calculated from panels A and C. Location of domain in the CFTR polypeptide is indicated in Figure 1A.

Figure 5

Degradation of CFTR TMD1. (A) TMD1 topology showing location of methionines and length of cytosolic and lumenal loops. Carbonate extraction of in vitro-expressed TMD1. (B) TMD1 degradation was assayed in the presence and absence of p97 as in Figure 4. Also shown are effects of MG132 and ATP depletion. (C) Initial rates of TMD1 degradation in mock RRL, p97-depleted RRL and after p97 supplementation.

Figure 6

p97 effects are influenced by TM segment composition. (A) Topology and methionine distribution of TM1–2 constructs. Location of residues Glu92 and Lys95 is shown within the TM1 sequence. (B) Carbonate extraction of wild-type and E92A/K95A polypeptides. (C, E) Degradation assays performed in mock-depleted RRL and p97-depleted RRL with or without added recombinant p97. Effects of MG132 and ATP depletion are also shown. (D, F) Initial degradation rates calculated from panels C and E.

Figure 7

p97 facilitates membrane extraction independently of cleavage. TMD1 degradation reactions were carried out in (A) mock- or (B) p97-depleted RRL, and cytosolic supernatants were analyzed for CFTR fragments before (total released) or after (TCA sol) TCA precipitation. Vertical arrow indicates TCA-insoluble peptide fragments that accumulate in the supernatant. (C) Autoradiograms showing dislocated full-length TMD1 degradation products generated in the presence of MG132 (lanes 1–10), in mock-depleted (lanes 1–5) or p97-depleted (–p97, lanes 6–10) RRL. No fragments were visualized by SDS–PAGE in the absence of either ATP (lanes 11–16) or MG132 (lanes 16–20). The extent (D) and initial rate (E) of total TMD1 released in the presence of MG132 in mock-depleted (Ni), p97-depleted (depl.) or depleted RRL supplemented with recombinant p97 (depl.+p97).

Figure 8

P97 effect is inversely related to the rate of degradation. The stimulatory effect (% increase) of p97 on the degradation rate was plotted as a function of the initial degradation rate in the absence of p97.