Hsp70/CHIP E3 ligase complex triggers K149-linked ubiquitination and degradation of BEST1 mutants p.P233L and p.P346H, impairing chloride channel function and retinal integrity.

The mechanisms underlying ubiquitination-mediated degradation of bestrophinopathy-causing mutants and their in vivo effects on retinal pigment epithelium (RPE) localization and retinal structure remain poorly understood. Furthermore, the upstream signaling cascade that induces degradation of these mutants and the detailed ubiquitination mechanism are unknown. Here, we report a c.1037C > A (p.P346H) mutation and a c.698C > T (p.P233L) mutation that co-segregate with the phenotypes of pedigrees affected by RP50 and Best vitelliform macular dystrophy (BVMD), respectively. The BEST1 mutants p.P233L and p.P346H reduced chloride channel activity and induced mislocalization of bestrophin-1 in polarized MDCK II cells, significantly affecting the channel activity of wild-type bestrophin-1. Lys149 was identified as the site responsible for ubiquitination of p.P346H- and p.P233L-bestrophin-1, mediated by Hsp70 and the C-terminal Hsp70-interacting protein (CHIP). Mutant bestrophin-1 proteins p.P346H and p.P233L undergo ubiquitination and degradation, preventing their localization to the cell membrane of MDCK II cells and the RPE of zebrafish, thereby reducing chloride channel activity. Mislocalization of mutant bestrophin-1 to the RPE impaired the multicellular layered structure of the retina. Our study reveals a ubiquitination signaling pathway mediated by Hsp70 and CHIP that depends on Lys149 of bestrophin-1. Aberrant activation of this pathway leads to loss of function in the p.P233L and p.P346H mutants and triggers retinopathy.