The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control.

Protein quality control (PQC) is a critical process wherein misfolded or damaged proteins are cleared from the cell to maintain protein homeostasis. In eukaryotic cells, the removal of misfolded proteins is primarily accomplished by the ubiquitin-proteasome system. In the ubiquitin-proteasome system, ubiquitin-conjugating enzymes and ubiquitin ligases append polyubiquitin chains onto misfolded protein substrates signaling for their degradation. The kinetics of protein ubiquitylation are paramount as a balance must be achieved between the rapid removal of misfolded proteins versus providing sufficient time for protein chaperones to attempt refolding. To uncover the molecular basis for how PQC substrate ubiquitylation rates are controlled, the reaction catalyzed by nuclear ubiquitin ligase San1 was reconstituted in vitro Our results demonstrate that San1 can function with two ubiquitin-conjugating enzymes, Cdc34 and Ubc1. Although Cdc34 and Ubc1 are both sufficient for promoting San1 activity, San1 functions preferentially with Ubc1, including when both Ubc1 and Cdc34 are present. Notably, a homogeneous peptide that mimics a misfolded PQC substrate was developed and enabled quantification of the kinetics of San1-catalyzed ubiquitylation reactions. We discuss how these results may have broad implications for the regulation of PQC-mediated protein degradation.

Ubiquitin-conjugating enzyme Cdc34 and ubiquitin ligase Skp1-cullin-F-box ligase (SCF) interact through multiple conformations.

In the ubiquitin-proteasome system, protein substrates are degraded via covalent modification by a polyubiquitin chain. The polyubiquitin chain must be assembled rapidly in cells, because a chain of at least four ubiquitins is required to signal for degradation, and chain-editing enzymes in the cell may cleave premature polyubiquitin chains before achieving this critical length. The ubiquitin-conjugating enzyme Cdc34 and ubiquitin ligase SCF are capable of building polyubiquitin chains onto protein substrates both rapidly and processively; this may be explained at least in part by the atypically fast rate of Cdc34 and SCF association. This rapid association has been attributed to electrostatic interactions between the acidic C-terminal tail of Cdc34 and a feature on SCF called the basic canyon. However, the structural aspects of the Cdc34-SCF interaction and how they permit rapid complex formation remain elusive. Here, we use protein cross-linking to demonstrate that the Cdc34-SCF interaction occurs in multiple conformations, where several residues from the Cdc34 acidic tail are capable of contacting a broad region of the SCF basic canyon. Similar patterns of cross-linking are also observed between Cdc34 and the Cul1 paralog Cul2, implicating the same mechanism for the Cdc34-SCF interaction in other members of the cullin-RING ubiquitin ligases. We discuss how these results can explain the rapid association of Cdc34 and SCF.

Multimodal mechanism of action for the Cdc34 acidic loop: a case study for why ubiquitin-conjugating enzymes have loops and tails.

Together with ubiquitin ligases (E3), ubiquitin-conjugating enzymes (E2) are charged with the essential task of synthesizing ubiquitin chains onto protein substrates. Some 75% of the known E2s in the human proteome contain unique insertions in their primary sequences, yet it is largely unclear what effect these insertions impart on the ubiquitination reaction. Cdc34 is an important E2 with prominent roles in cell cycle regulation and signal transduction. The amino acid sequence of Cdc34 contains an insertion distal to the active site that is absent in most other E2s, yet this acidic loop (named for its four invariably conserved acidic residues) is critical for Cdc34 function both in vitro and in vivo. Here we have investigated how the acidic loop in human Cdc34 promotes ubiquitination, identifying two key molecular events during which the acidic loop exerts its influence. First, the acidic loop promotes the interaction between Cdc34 and its ubiquitin ligase partner, SCF. Second, two glutamic acid residues located on the distal side of the loop collaborate with an invariably conserved histidine on the proximal side of the loop to suppress the pKa of an ionizing species on ubiquitin or Cdc34 which greatly contributes to Cdc34 catalysis. These results demonstrate that insertions can guide E2s to their physiologically relevant ubiquitin ligases as well as provide essential modalities that promote catalysis.