The anaphase-promoting complex (APC), or cyclosome, is an E3 ubiquitin-protein ligase that collaborates with E2 ubiquitin-conjugating enzymes to assemble polyubiquitin chains on proteins important for cell-cycle progression. It remains unclear how the APC - or many other E3s - promotes the multiple distinct reactions necessary for chain assembly. We addressed this problem by analyzing APC interactions with different E2s. We screened all budding yeast E2s as APC coenzymes in vitro and found that two, Ubc4 and Ubc1, are the key E2 partners for the APC. These proteins display strikingly different but complementary enzymatic behaviors: Ubc4 supports the rapid monoubiquitination of multiple lysines on APC targets, while Ubc1 catalyzes K48-linked polyubiquitin chain assembly on preattached ubiquitins. Mitotic APC function is lost in yeast strains lacking both Ubc1 and Ubc4. E2-25K, a human homolog of Ubc1, also promotes APC-dependent chain extension on preattached ubiquitins. We propose that sequential E2 proteins catalyze K48-linked polyubiquitination and thus proteasomal destruction of APC targets.

Newly synthesized proteins are triaged between biosynthesis and degradation to maintain cellular homeostasis, but the decision-making mechanisms are unclear. Here, we reconstitute the core reactions for membrane targeting and ubiquitination of nascent tail-anchored membrane proteins to understand how their fate is determined. The central six-component triage system is divided into an uncommitted client-SGTA complex, a self-sufficient targeting module, and an embedded but self-sufficient quality control module. Client-SGTA engagement of the targeting module induces rapid, private, and committed client transfer to TRC40 for successful biosynthesis. Commitment to ubiquitination is dictated primarily by comparatively slower client dissociation from SGTA and non-private capture by the Bag6 subunit of the quality control module. This provides a paradigm for how priority and time are encoded within a multi-chaperone triage system.Protein biosynthesis and quality control pathways are precisely balanced to provide nascent proteins an initial opportunity to mature, while favoring degradation over time (1-3). Deviations from this balance lead to premature degradation of normal maturation intermediates or persistence of misfolded proteins, either of which can cause disease (2,4,5). Although accurate triage between biosynthesis and degradation is essential for maintaining protein homeostasis, a mechanistic understanding of protein triage for any pathway has been hampered by the lack of a fully reconstituted system that faithfully recapitulates both the biosynthetic and quality control outcomes of a nascent protein.To achieve this goal, we chose the pathway of tail-anchored (TA) membrane protein insertion as a model. TA proteins engage a conserved and well-defined pathway for targeting and insertion at the ER (6-8), which in mammals is monitored by an embedded quality control (QC) pathway (9, 10) to degrade products that fail targeting ( fig. S1). Studies on the yeast TA targeting pathway (7,(11)(12)(13) and the mammalian TA targeting (6, 14-16) and ubiquitination (9, 10) reactions suggest that six core factors constitute a minimal mammalian triage system amenable to complete reconstitution.Three of the factors (SGTA, Bag6, and TRC40) can recognize and shield the transmembrane domain (TMD) of a TA protein client (6,(16)(17)(18) Analyses with purified factors rigorously established the sufficiency of these modules. In these experiments, radiolabeled TA protein is synthesized with purified E. coli translation factors supplemented with the desired TMD-binding chaperone ( fig. S5). This orthogonal system produces homogeneous TA-chaperone complexes that can be used for downstream functional assays (24). Isolated TA-TRC40 complex was competent for ER targeting and insertion ( Fig. 2A), but not ubiquitination by RNF126 ( fig. S6). By contrast, the TA-Bag6 complex could not mediate insertion (data not shown), but allowed ubiquitination via RNF126 recruitment to Bag6's N-terminal UBL domain (Fig. 2B).The TA-SGTA complex in iso...

SummaryApproximately 30% of eukaryotic proteins contain hydrophobic signals for localization to the secretory pathway. These proteins can be mislocalized in the cytosol due to mutations in their targeting signals, certain stresses, or intrinsic inefficiencies in their translocation. Mislocalized proteins (MLPs) are protected from aggregation by the Bag6 complex and degraded by a poorly characterized proteasome-dependent pathway. Here, we identify the ubiquitin ligase RNF126 as a key component of the MLP degradation pathway. In vitro reconstitution and fractionation studies reveal that RNF126 is the primary Bag6-dependent ligase. RNF126 is recruited to the N-terminal Ubl domain of Bag6 and preferentially ubiquitinates juxtahydrophobic lysine residues on Bag6-associated clients. Interfering with RNF126 recruitment in vitro prevents ubiquitination, and RNF126 depletion in cells partially stabilizes a Bag6 client. Bag6-dependent ubiquitination can be recapitulated with purified components, paving the way for mechanistic analyses of downstream steps in this cytosolic quality control pathway.

Summary Protein ubiquitination is catalyzed by ubiquitin-conjugating enzymes (E2s) in collaboration with ubiquitin-protein ligases (E3s). This process depends on nucleophilic attack by a substrate lysine on a thioester bond linking the C-terminus of ubiquitin to a cysteine in the E2 active site. Different E2 family members display specificity for lysines in distinct contexts. We addressed the mechanistic basis for this lysine selectivity in Ubc1, an E2 that catalyzes the ubiquitination of lysine 48 (K48) in ubiquitin, leading to the formation of K48-linked polyubiquitin chains. We identified a cluster of polar residues near the Ubc1 active site, as well as a residue in ubiquitin itself, that are required for catalysis of K48-specific ubiquitin ligation but not for general activity toward other lysines. Our results suggest that the active site of Ubc1, as well as the surface of ubiquitin, contain specificity determinants that channel specific lysines to the central residues involved directly in catalysis.