Proteasomal degradation of preemptive quality control (pQC) substrates is mediated by an AIRAPL-p97 complex

Abstract

The initial folding of secreted proteins occurs in the ER lumen, which contains specific chaperones and where posttranslational modifications may occur. Therefore lack of translocation, regardless of entry route or protein identity, is a highly toxic event, as the newly synthesized polypeptide is misfolded and can promiscuously interact with cytosolic factors. Mislocalized proteins bearing a signal sequence that did not successfully translocate through the translocon complex are subjected to a preemptive quality control (pQC) pathway and are degraded by the ubiquitin-proteasome system (UPS). In contrast to UPS-mediated, ER-associated degradation, few components involved in pQC have been identified. Here we demonstrate that on specific translocation inhibition, a p97-AIRAPL complex directly binds and regulates the efficient processing of polyubiquitinated pQC substrates by the UPS. We also demonstrate p97's role in pQC processing of preproinsulin in cases of naturally occurring mutations within the signal sequence of insulin.

FIGURE 1:

(A) VCAM1-HA was transfected into cells, and 48 h posttransfection, VCAM1 content was evaluated by HA immunoblots. Cell lysates were analyzed directly or after in vitro PNGase or Usp2 treatment. Where indicated, cells were treated overnight with Velcade (100 nM) and/or CAM741 (250 nM) as indicated. Asterisk marks an additional LMW band of VCAM-1. Glycosylated VCAM-1 is labeled Glc-Nac-VCAM-1. (B) Cells expressing VCAM1 were treated with Velcade in the presence or absence of CAM741 as indicated. Cell lysates were evaluated directly (input) or subjected to a HA IP to evaluate Bag6 and p97 interaction. (C) VCAM-1 IP in the presence of CAM741 and Velcade as in B was subjected to an in vitro deubiquitination by Usp2 as indicated. (D) Recombinant P97 was layered under a discontinuous sucrose gradient in the absence of membranes (–M) or presence of ribosome-stripped PK-RMs or purified reconstituted trimeric Sec61-PLs. After centrifugation, fractions were collected from top to bottom and immunoblotted against p97. The membrane binding of p97 is evident by its flotation together with the membranes to the top of the gradient.

FIGURE 2:

(A) Illustration of VCAM-1 containing the Flag signal sequence (F-SS), the glycosylation sites, transmembrane domain (TM), and C-terminus HA tag. (B) Cells expressing the Flag-SS VCAM-1 and the indicated p97 were treated with Velcade and CAM741 as indicated, and VCAM-1 content was evaluated by Flag and HA IB. Cellular expression levels of p97 are indicated, as well as Rpt6 levels serving as a loading control. Glycosylated VCAM-1 is labeled Glc-Nac-VCAM-1, signal peptide–bearing VCAM-1 is labeled SP-VCAM-1, and signal peptide–bearing glycosylated VCAM-1 is labeled Glc-Nac-SP-VCAM-1. VCAM-1 that does not contain a signal peptide is labeled as VCAM-1.

FIGURE 3:

(A) Left, cellular lysates of CAM741- and Velcade-treated cells expressing VCAM-1 and AIRAPL WT or UIM mutant were subjected to an AIRAPL IP. Copurification of VCAM-1 is observed to be UIM dependent, whereas p97 copurification is not UIM dependent. Right, AIRAPL IP was performed from cellular lysates of cells treated with Velcade or usp2 as indicated. Bag6 and ubiquitin copurification was evaluated by the indicated immunoblots. (B) Cells expressing AIRAPL and the indicated Bag6 were treated with CAM741 with or without Velcade (as indicated), and cellular lysates were subjected to an AIRAPL IP. Copurification of Bag6 and VCAM-1 was evaluated by immunoblots, and increased levels of VCAM-1 upon Bag6 88–387 expression are noted (input). Note the reactive band at the size of the Bag6 mutant that appears even in the absence of Bag6 mutant expression, indicating a nonspecific or partial Bag6 product expressed at this molecular weight. Nevertheless, the reduced amount of Bag6 mutant copurified upon AIRAPL IP (compare to WT) implies a role for additional Bag6-mediated processes in AIRAPL binding. Long exposures of cellular input content reveal the reduced levels of HMW VCAM-1 upon Bag6 mutant expression. (C) Cells treated with CAM741 and Velcade expressing the indicated AIRAPL isoform were subjected to an AIRAPL IP, and VCAM-1, Bag6, ubiquitin, and p97 interaction was evaluated. Longer exposure of VCAM-1 content in the AIRAPL IP reveals the presence of a HMW VCAM-1 isoform in the WT and VIM mutants of AIRAPL (bottom, long exp.).

FIGURE 4:

(A) Cells expressing VCAM-1 and the indicated p97 isoform were treated with CAM741 alone or with Velcade as indicated. Cellular lysate content was evaluated directly (input) or subjected to a Rpt6 IP to evaluate proteasomal association (Rpn11) of VCAM-1 and p97. The asterisk represents a nonspecific band in the Rpt6 IP. (B) VCAM-1 and p97 expressing cells were treated with CAM741 or Velcade as indicated. Cellular lysates were fractionated into supernatant and pellet and VCAM-1, and proteasomal (PSMA1) content distribution was evaluated by IB. Sec61 immunoblot serves as a positive marker for ER membrane distribution.

FIGURE 5:

(A) Cells expressing p97 and insulin isoforms (as indicated) were treated with Velcade or left untreated as indicated. Preproinsulin and proinsulin migration is indicated, and GFP expression was evaluated to demonstrate equal transfection and loading of the gel. (B) N-glycosylation–bearing insulin was expressed in cells, and cellular content was resolved to discriminate between the indicated insulin isoforms. Velcade treatment and p97 expression were performed as in A. PNGaseF treatment was performed on cellular lysate, and PSMA1 content served as a loading control for cell lysates.

FIGURE 6:

Based on the presented data, a plausible scenario can be drawn in which a mislocalized protein (MLP) that has not translocated into the ER is bound to the ER membrane by virtue of hydrophobic segments of its transmembrane (TM) domain and/or hydrophobic segments within its signal sequence (SS). Bag6 interaction with hydrophobic segments within the MLP would then enable recruitment of the p97 adaptor Ubxd8 (via its UBA domain). Polyubiquitination of the MLP (by virtue of the ribosome quality control ligase LTN1, RNF126, or another E3 ligase) would enable recruitment of AIRAPL, an additional p97 adaptor to the polyubiquitinated MLP.