Abstract
SummaryNewly synthesized membrane proteins are queried by ubiquitin ligase complexes and triaged between degradative and nondegradative fates. The mechanisms that convert modest differences in substrate-ligase interactions into decisive outcomes of ubiquitination are not well understood. Here, we reconstitute membrane protein recognition and ubiquitination in liposomes using purified components from a viral-mediated degradation pathway. We find that substrate-ligase interactions in the membrane directly influence processivity of ubiquitin attachment to modulate polyubiquitination. Unexpectedly, differential processivity alone could not explain the differential fates in cultured cells of degraded and nondegraded clients. Both computational and experimental analyses identified continuous deubiquitination as a prerequisite for maximal substrate discrimination. Deubiquitinases reduce polyubiquitin dwell times preferentially on clients that dissociate more rapidly from the ligase. This explains how small differences in substrate-ligase interaction can be amplified into larger differences in net degradation. These results provide a conceptual framework for substrate discrimination during membrane protein quality control.
Highlights
Eukaryotic integral membrane proteins insert, assemble, and mature at the endoplasmic reticulum (ER) (Skach, 2009)
We find that substrate-ligase interactions in the membrane directly influence processivity of ubiquitin attachment to modulate polyubiquitination
We discovered that modest differences in client-ligase interaction are converted into clear differences in polyubiquitination by a combination of processivity differences and deubiquitinases (DUBs) that impact the dwell time of a degradation mark on potential clients
Summary
Synthesized membrane proteins are queried by ubiquitin ligase complexes and triaged between degradative and nondegradative fates. Differential processivity alone could not explain the differential fates in cultured cells of degraded and nondegraded clients. Both computational and experimental analyses identified continuous deubiquitination as a prerequisite for maximal substrate discrimination. Deubiquitinases reduce polyubiquitin dwell times preferentially on clients that dissociate more rapidly from the ligase. This explains how small differences in substrate-ligase interaction can be amplified into larger differences in net degradation. These results provide a conceptual framework for substrate discrimination during membrane protein quality control
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