A mechanism for protein monoubiquitination dependent on a trans-acting ubiquitin-binding domain.

The length of the ubiquitin chain on a substrate dictates various functional outcomes, yet little is known about its regulation in vivo. The yeast arrestin-related protein Rim8/Art9 is monoubiquitinated in vivo by the Rsp5 ubiquitin ligase. This also requires Vps23, a protein that displays an ubiquitin-E2 variant (UEV) domain. Here, we report that binding of the UEV domain to Rim8 interferes with ubiquitin chain elongation and directs Rim8 monoubiquitination. We propose that Vps23 UEV competes with Rsp5 HECT N-lobe for binding to the first conjugated ubiquitin, thereby preventing polyubiquitination. These findings reveal a novel mechanism to control ubiquitin chain length on substrates in vivo.