Unassembled CD147 is an endogenous endoplasmic reticulum-associated degradation substrate

Abstract

Degradation of folding- or assembly-defective proteins by the endoplasmic reticulum-associated degradation (ERAD) ubiquitin ligase, Hrd1, is facilitated by a process that involves recognition of demannosylated N-glycans by the lectin OS-9/XTP3-B via the adaptor protein SEL1L. Most of our knowledge of the machinery that commits proteins to this fate in metazoans comes from studies of overexpressed mutant proteins in heterologous cells. In this study, we used mass spectrometry to identify core-glycoslyated CD147 (CD147(CG)) as an endogenous substrate of the ERAD system that accumulates in a complex with OS-9 following SEL1L depletion. CD147 is an obligatory assembly factor for monocarboxylate transporters. The majority of newly synthesized endogenous CD147(CG) was degraded by the proteasome in a Hrd1-dependent manner. CD147(CG) turnover was blocked by kifunensine, and interaction of OS-9 and XTP3-B with CD147(CG) was inhibited by mutations to conserved residues in their lectin domains. These data establish unassembled CD147(CG) as an endogenous, constitutive ERAD substrate of the OS-9/SEL1L/Hrd1 pathway.

FIGURE 1:

Identification of CD147 as a putative ERAD substrate. (A) Diagram depicting the human CD147 protein and important features (SS, signal peptide; Ig, immunoglobulin domain; TM, transmembrane domain). Peptides identified in LC-MS/MS analysis of S-OS-9.2 affinity purifications are shown. (B) Profile of CD147 expression in Triton X-100–solubilized whole-cell HEK293 lysates treated with or without Endo H (Mat., mature; CG, core-glycosylated; DeG, deglycosylated). (C) HEK293 cells expressing S-OS-9.2 and infected with lentivirus expressing an shRNA targeting SEL1L (+) or an empty virus as a control (−) were solubilized in 1% digitonin, and S-OS-9.2 complexes were affinity-purified from cell lysates and probed for the indicated proteins. (D) CD147 turnover was assessed by immunoblotting Triton X-100 cell extracts for CD147 after treating cells with cycloheximide for the indicated times. (E) Autoradiogram of a CD147 pulse–chase analysis in HEK293 cells treated with either dimethyl sulfoxide (DMSO; control) or 10 μM MG132 during the chase period. CD147 was isolated from cell lysates with anti-human CD147 8G6, and immunoprecipitates were separated by SDS–PAGE and analyzed with a phosphorimager. (F and G) Quantification of the pulse–chase data experiment from (E), in which degradation of CG and formation of Mat. glycoforms of CD147 are calculated as a percent of starting CG material. CTL, control; IB, immunoblot; SAP, S-peptide affinity purification; S-protein, S-protein-HRP.

FIGURE 2:

Degradation of CD147(CG) is mediated by the Hrd1-SEL1L E3 ligase complex. (A) HEK293 cells expressing either gp78-S or Hrd1-S were lysed in 1% digitonin, and S-protein affinity-purified complexes were immunoblotted for the indicated proteins. (B) Cells infected with either control (−) or SEL1L (+) shRNA-expressing retrovirus were lysed in 1% digitonin, and anti-CD147 immunoprecipitates were probed for the indicated proteins. IB, immunoblot; SAP, S-peptide affinity purification; IP, immunoprecipitation. (C) Pulse–chase analysis of CD147 in HEK293 cells infected with retroviruses harboring shRNAs that target firefly luciferase (FF, control), Hrd1, SEL1L, or gp78. Anti-CD147 immunoprecipitates were separated by SDS–PAGE and analyzed on a phosphorimager. KD, knockdown. (D and E) Quantification of the data in (C) for CD147(CG) degradation (D) or formation of CD147 (Mat.). (E) Each time point represents the average of three independent experiments. The error bars represent the SEM.

FIGURE 3:

The role of N-glycans and the ER lectins OS-9 and XTP3-B in the degradation of CD147(CG). (A) Pulse–chase analysis of CD147 in cells stably transduced with retroviruses expressing shRNAs targeting firefly luciferase (FF, control), in the presence of vehicle control (DMSO) or 5 μg/ml kifunensine during the chase. Cells stably expressing shRNAs targeting Hrd1 were also analyzed as a positive control. Anti-CD147 immunoprecipitates were separated by SDS–PAGE and analyzed on a phosphorimager. (B) Quantification of the data in (A) for CD147(CG) turnover. (C) Pulse–chase analysis of CD147 in HEK293 cells infected with retroviruses expressing shRNAs that target firefly luciferase (FF, control) XTP3-B or OS-9. Anti-human CD147 immunoprecipitates were separated by SDS–PAGE and analyzed on a phosphorimager. (D) Quantification of the data in (C) for CD147(CG) turnover, with each time point representing the average of at least three independent experiments. Error bars represent SEM. (E) HEK293 cells were cotransfected with vectors expressing S-OS-9.2 and shRNAs targeting SEL1L (+) or firefly luciferase (−). S-OS-9.2 complexes were affinity-purified from 1% digitonin-solubilized cell lysates and analyzed by immunoblotting for the indicated proteins. (F) HEK293 cells stably expressing XTP3-B-S were transduced with lentiviruses harboring shRNAs targeting SEL1L (+) or firefly luciferase (−). S-protein-affinity purifications from 1% digitonin-solubilized lysates were processed as in (E). (G) S-OS-9.2 or XTP3-B-S affinity-purified complexes were isolated from cells expressing firefly luciferase (−) or SEL1L (+) shRNAs and lysed in 1% Triton X-100. * indicates nonspecific immunoreactive bands in (E) and (G). IB, immunoblot. SAP, S-peptide affinity purification; KD, knockdown.

FIGURE 4:

Glycan dependence of the interaction of OS-9 and XTP3-B with CD147(CG). (A) HEK293 cells were cotransfected with wild-type S-OS-9.2 or the indicated S-OS-9.2 MRH domain mutant together with shRNAs targeting firefly luciferase (−) or SEL1L (+). S-OS-9.2 complexes were affinity-purified from 1% digitonin-solubilized cell lysates and analyzed by immunoblotting for the indicated proteins. (B) HEK293 cells were transfected with wild-type XTP3-B-S or the indicated XTP3-B-S MRH domain mutants, and affinity-purified complexes from 1% digitonin-solubilized cell lysates were analyzed by immunoblotting for the indicated proteins. (C) S-OS-9.2 affinity-purified complexes from 1% Triton X-100–solubilized cell lysates were isolated from cells expressing shRNAs targeting SEL1L and treated with either DMSO (−) or 5 μg/ml kifunensine (+), and probed for the indicated proteins. IB, immunoblot. SAP, S-peptide affinity purification. S-protein, S-protein-HRP.