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J Biol Chem. 2013 Oct 25;288(43):31069-79. doi: 10.1074/jbc.M113.479345. Epub 2013 Aug 29.

Endoplasmic reticulum protein quality control is determined by cooperative interactions between Hsp/c70 protein and the CHIP E3 ligase.

Author information

1
From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 and.

Abstract

The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70.

KEYWORDS:

CFTR; CHIP; Co-chaperone; Cystic Fibrosis; E3 Ubiquitin Ligase; ERad; Heat Shock Protein; Hsp70; Reticulocyte Lysate; Ubiquitin Proteasome Pathway

PMID:
23990462
PMCID:
PMC3829420
DOI:
10.1074/jbc.M113.479345
[Indexed for MEDLINE]
Free PMC Article

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