We recently demonstrated that plasma membrane KCa3. 1 is rapidly endocytosed and targeted for lysosomal degradation via a Rab7-and ESCRT-dependent pathway. Herein, we assess the role of ubiquitylation in this process. Using a biotin ligase acceptor peptide (BLAP)-tagged KCa3. 1, in combination with tandem ubiquitin binding entities (TUBEs), we demonstrate that KCa3. 1 is polyubiquitylated following endocytosis. Hypertonic sucrose inhibited KCa3. 1 endocytosis and resulted in a significant decrease in channel ubiquitylation. Inhibition of the ubiquitin-activating enzyme (E1) with UBEI-41 resulted in reduced KCa3. 1 ubiquitylation and internalization. The general deubiquitylase (DUB) inhibitor, PR-619 attenuated KCa3. 1 degradation, indicative of deubiquitylation being required for lysosomal delivery. Using the DUB Chip, a protein microarray containing 35 DUBs, we demonstrate a time-dependent association between KCa3. 1 and USP8 following endocytosis, which was confirmed by coimmunoprecipitation. Further, overexpression of wild-type USP8 accelerates channel deubiquitylation, while either a catalytically inactive mutant USP8 or siRNA-mediated knockdown of USP8 enhanced accumulation of ubiquitylated KCa3. 1, thereby inhibiting channel degradation. In summary, by combining BLAP-tagged KCa3. 1 with TUBEs and DUB Chip methodologies, we demonstrate that polyubiquitylation mediates the targeting of membrane KCa3. 1 to the lysosomes and also that USP8 regulates the rate of KCa3. 1 degradation by deubiquitylating KCa3. 1 prior to lysosomal delivery.—Balut, CM, Loch, CM, Devor, DC Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3. 1.