Decoding polyubiquitin regulation of K7. 1 (KCNQ1) surface expression with engineered linkage-selective deubiquitinases.

Polyubiquitin chain diversity generates a 'ubiquitin code' that universally regulates protein abundance, localization, and function. Functions of polyubiquitin diversity are mostly unknown, with lack of progress due to an inability to selectively tune protein polyubiquitin linkages in live cells. We develop linkage-selective engineered deubiquitinases (enDUBs) by fusing linkage-selective DUB catalytic domains to GFP-targeted nanobody and use them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. YFP-KCNQ1 in HEK293 cells has polyubiquitin chains with K48/K63 linkages dominant. EnDUBs yield unique effects on channel surface abundance with a pattern indicating: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling; and K48 is necessary for forward trafficking. EnDUB effects differ in cardiomyocytes and on KCNQ1 disease mutants, emphasizing ubiquitin code mutability. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 abundance and subcellular localization and introduce linkage-selective enDUBs as potent tools to demystify the polyubiquitin code.