Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

Yi-Jheng Peng,Chih-Yung Tang,Chia-Ying Wu,Tsung-Yu Chen,Shu-Ching Chen,Hsin-Ying Hsieh,Hao-Han Wu,Jing-Jia Huang

doi:10.1038/srep32444

Abstract

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation.

Highlights

Employed for immunoblotting analyses or subject to streptavidin pull-down prior to immunoblotting analyses
We demonstrate that CLC-1 channels are associated with the molecular chaperones heat shock cognate protein 70 (Hsc70) and heat shock protein 90β(Hsp90β), as well as their co-chaperones FK506-binding protein 8 (FKBP8; known as FKBP38), activator of Hsp[90] ATPase homolog 1 (Aha1), and Hsp70/Hsp[90] organizing protein (HOP)
Over-expression of FKBP8/Aha[1] substantially increases the protein level of CLC-1 WT and A531V mutant heterologously expressed in HEK293T cells (Fig. 1A)

Summary

Introduction

Employed for immunoblotting analyses (total) or subject to streptavidin pull-down prior to immunoblotting analyses (surface). Expressions of GAPDH are displayed as the loading control. (Bottom) Quantification of surface protein level (Surface) and membrane trafficking efficiency (Surface/Total). The surface protein density was standardized as the ratio of surface signal to cognate total GAPDH signal, followed by normalization to that of the corresponding vector control. The total protein density was standardized as the ratio of input signal to GAPDH signal. The membrane trafficking efficiency was expressed as surface protein density divided by the corresponding standardized total protein density. The mean ratios in the presence of FKBP8/Aha[1] were normalized to those of corresponding vector controls. The protein density was standardized as the ratio of Flag-CLC-1 signal to cognate total tubulin signal, followed by normalization to that of the corresponding GFP control. We present additional evidence suggesting that, in addition to promoting CLC-1 protein folding, FKBP8 and Hsp90βmay play a critical role in regulating CLC-1 degradation by the CUL4A/B-DDB1-CRBN complex

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