Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase

• Published in: The Journal of biological chemistry Vol. 288; no. 43; pp. 31069 - 31079
• Main Authors: Matsumura, Yoshihiro, Sakai, Juro, Skach, William R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.10.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Diphosphate - chemistry; Adenosine Diphosphate - genetics; Adenosine Diphosphate - metabolism; Amino Acid Motifs; CFTR; CHIP; Co-chaperone; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator - chemistry; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; E3 Ubiquitin Ligase; Endoplasmic Reticulum - chemistry; Endoplasmic Reticulum - genetics; Endoplasmic Reticulum - metabolism; ERad; Heat Shock Protein; HSC70 Heat-Shock Proteins - chemistry; HSC70 Heat-Shock Proteins - genetics; HSC70 Heat-Shock Proteins - metabolism; Hsp70; Humans; Mutation; Protein Folding; Protein Structure, Tertiary; Protein Synthesis and Degradation; Proteolysis; Reticulocyte Lysate; Ubiquitin Proteasome Pathway; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; ERad; E3 Ubiquitin Ligase; Reticulocyte Lysate; Hsp70; CHIP; Cystic Fibrosis; Co-chaperone; Ubiquitin Proteasome Pathway; CFTR; Heat Shock Protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.479345

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. Background: The CHIP E3 ligase regulates Hsp70 pro-degradation activities. Results: The P269A CHIP U-box mutation induces CHIP oligomerization and modulates nucleotide- and substrate-dependent interactions between the TPR domain and Hsp70 C terminus. Conclusion: The U-box domain plays a key role in CHIP recruitment to Hsp70-client complexes, possibly by controlling oligomerization. Significance: Hsp70-CHIP substrate triage is governed by complex allosteric interactions between multiple domains in both proteins.