Ubiquitin chains are formed as structurally distinct polymers via different linkages, and several chain types including K33-linkage remain uncharacterized. Here, we describe a role for K33-polyubiquitination in protein trafficking. We show that the Cullin 3 (Cul3) substrate adaptor KLHL20 is localized to the trans-Golgi network (TGN) and is important for post-Golgi trafficking by promoting the biogenesis of TGN-derived transport carriers. The Cul3-KLHL20 ubiquitin E3 ligase catalyzes a nondegradable, K33-linked polyubiquitination on coronin 7 (Crn7), which facilitates Crn7 targeting to TGN through a ubiquitin-dependent interaction with Eps15. Blockage of K33-chain formation, Crn7 ubiquitination, or disruption of Crn7-Eps15 interaction impairs TGN-pool F-actin assembly, a process essential for generating transport carriers. Enforced targeting of Crn7 to TGN bypasses the requirement of K33-ubiquitination for TGN-pool F-actin assembly and post-Golgi trafficking. Our study reveals a role of KLHL20-mediated K33-ubiquitination of Crn7 in post-Golgi transport and identifies a cellular recognition mechanism for this ubiquitin chain type.
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