Quality Control of Protein Complex Assembly by a Transmembrane Recognition Factor.

The inner nuclear membrane (INM) is continuous with the endoplasmic reticulum (ER) but harbors a distinctive proteome essential for nuclear functions. In yeast, the Asi1/Asi2/Asi3 ubiquitin ligase complex safeguards the INM proteome through the clearance of mislocalized ER membrane proteins. How the Asi complex selectively targets mislocalized proteins and coordinates its activity with other ER functions, such as protein biogenesis, is unclear. Here, we uncover a link between INM proteome identity and membrane protein complex assembly in the remaining ER. We show that lone proteins and complex subunits failing to assemble in the ER access the INM for Asi-mediated degradation. Substrates are recognized by direct binding of Asi2 to their transmembrane domains for subsequent ubiquitination by Asi1/Asi3 and membrane extraction. Our data suggest a model in which spatial segregation of membrane protein complex assembly and quality control improves assembly efficiency and reduces the levels of orphan subunits.

Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36.

Ubiquitin and ubiquitin-like proteins typically use distinct machineries to facilitate diverse functions. The immunosuppressive ubiquitin-like protein Fubi is synthesized as an N-terminal fusion to a ribosomal protein (Fubi-S30). Its proteolytic maturation by the nucleolar deubiquitinase USP36 is strictly required for translationally competent ribosomes. What endows USP36 with this activity, how Fubi is recognized and whether other Fubi proteases exist are unclear. Here, we report a chemical tool kit that facilitated the discovery of dual ubiquitin/Fubi cleavage activity in USP16 in addition to USP36 by chemoproteomics. Crystal structures of USP36 complexed with Fubi and ubiquitin uncover its substrate recognition mechanism and explain how other deubiquitinases are restricted from Fubi. Furthermore, we introduce Fubi C-terminal hydrolase measurements and reveal a synergistic role of USP16 in Fubi-S30 maturation. Our data highlight how ubiquitin/Fubi specificity is achieved in a subset of human deubiquitinases and open the door to a systematic investigation of the Fubi system.

Native Semisynthesis of Isopeptide-Linked Substrates for Specificity Analysis of Deubiquitinases and Ubl Proteases.

Post-translational modifications with ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are regulated by isopeptidases termed deubiquitinases (DUBs) and Ubl proteases. Here, we describe a mild chemical method for the preparation of fluorescence polarization substrates for these enzymes that is based on the activation of C-terminal Ub/Ubl hydrazides to acyl azides and their subsequent functionalization to isopeptides. The procedure is complemented by native purification routes and thus circumvents the previous need for desulfurization and refolding. Its broad applicability was demonstrated by the generation of fully cleavable substrates for Ub, SUMO1, SUMO2, NEDD8, ISG15, and Fubi. We employed these reagents for the investigation of substrate specificities of human UCHL3, USPL1, USP2, USP7, USP16, USP18, and USP36. Pronounced selectivity of USPL1 for SUMO2/3 over SUMO1 was observed, which we rationalize with crystal structures and biochemical assays, revealing a SUMO paralogue specificity mechanism distinct from SENP family deSUMOylases. Moreover, we investigated the recently identified Fubi proteases USP16 and USP36 and found both to act as bona fide deFubiylases, harboring catalytic activity against isopeptide-linked Fubi. Surprisingly, we also noticed the activity of both enzymes toward ISG15, previously not identified in chemoproteomics, which makes USP16 and USP36 the first human DUBs with specific isopeptidase activity toward three distinct modifiers. The methods described here for the preparation of isopeptide-linked, fully folded substrates will aid in the characterization of further DUBs/Ubl proteases. More broadly, our findings highlight possible limitations associated with fluorogenic substrates and Ubl activity-based probes and stress the importance of isopeptide-containing reagents for validating isopeptidase activities and quantifying substrate specificities.