Dynamic Ubiquitination/Deubiquitination Regulates Plasma Membrane KCa3.1 Endocytosis and Targeting to the Lysosomes

Abstract

We recently demonstrated that plasma membrane KCa3.1 is rapidly endocytosed and targeted for lysosomal degradation via the ESCRT family of proteins. In this study, we assess the role of ubiquitination in this process. Using a biotin-ligase acceptor peptide tagged KCa3.1, which allows rapid biotinylation and streptavidin labeling of the channel at the cell surface, in conjunction with tandem ubiquitin-binding entities (TUBEs), which enable isolation of ubiquitinated proteins, we demonstrate that KCa3.1 is heavily poly-ubiquitinated following endocytosis. Further, by inhibiting channel endocytosis using hypertonic sucrose, we demonstrate that KCa3.1 undergoes ubiquitination at the plasma membrane. The role of ubiquitination in channel endocytosis was confirmed by treating the cells with the ubiquitin-activating enzyme (E1) inhibitor UBEI-41, which dramatically reduced channel ubiquitination and inhibited its internalization, as assessed by both confocal microscopy and pull-down assays. To determine if KCa3.1 undergoes deubiquitination before delivery to the lysosomes, we used the general deubiquitinating enzyme inhibitor PR619. This induced a dramatic delay in channel degradation and an increased level of channel ubiquitination. Using the DUB-CHIP, a protein microarray impregnated with thirty-five deubiquitylase enzymes, we identified Usp8 as the specific DUB involved in deubiquitination of the endocytosed KCa3.1. This result was confirmed in HEK cells, by measuring membrane KCa3.1 ubiquitination and degradation rate in the presence of the wild type or catalytically inactive mutant of Usp8. Thus, overexpression of wild type Usp8 accelerates channel deubiquitination, while the mutant Usp8 strongly enhanced accumulation of ubiquitinated KCa3.1. Interestingly, in both cases the rate of channel degradation was delayed. In conclusion, we demonstrate that poly-ubiquitination mediates the targeting of membrane KCa3.1 to the lysosomes and also that Usp8 regulates the rate of KCa3.1 degradation by deubiquitinating KCa3.1 before delivery to lysosomes.