Disulfide engineering to map subunit interactions in the proteasome and other macromolecular complexes.
ABSTRACT In studies of protein complexes for which high-resolution structural data are unavailable, it is often still possible to determine both nearest-neighbor relationships between subunits and atomic-resolution details of these interactions. The eukaryotic 26S proteasome, a ∼2.5 MDa protein complex with at least 33 different subunits, is a prime example. Important information about quaternary organization and assembly of proteasomes has been gained using a combination of sequence alignments with related proteins of known tertiary structure, molecular modeling, and disulfide engineering to allow oxidative cross-linking between predicted polypeptide neighbors. Here, we provide detailed protocols for engineered cysteine cross-linking of yeast proteasome subunits in whole-cell extracts, in active 26S proteasome complexes first isolated by native polyacrylamide gel electrophoresis, and in subcomplexes that function as potential assembly intermediates.
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ABSTRACT: Regulated protein degradation mediated by the ubiquitin-proteasome system (UPS) is critical to eukaryotic protein homeostasis. Often vital to degradation of protein substrates is their disassembly, unfolding, or extraction from membranes. These processes are catalyzed by the conserved AAA-ATPase Cdc48 (also known as p97). Here we characterize the Cuz1 protein (Cdc48-associated UBL/zinc-finger protein-1), encoded by a previously uncharacterized arsenite-inducible gene in budding yeast. Cuz1, like its human ortholog ZFAND1, has both an AN1-type zinc finger (Zf_AN1) and a divergent ubiquitin-like domain (UBL). We show that Cuz1 modulates Cdc48 function in the UPS. The two proteins directly interact, and the Cuz1 UBL, but not Zf_AN1, is necessary for binding to the Cdc48 N-terminal domain. Cuz1 also associates, albeit more weakly, with the proteasome, and the UBL is dispensable for this interaction. Cuz1-proteasome interaction is strongly enhanced by exposure of cells to the environmental toxin arsenite, and in a proteasome mutant, loss of Cuz1 enhances arsenite sensitivity. Whereas loss of Cuz1 alone causes only minor UPS degradation defects, its combination with mutations in the Cdc48(Npl4-Ufd1) complex leads to substantial further impairment. Cuz1 helps limit the accumulation of ubiquitin conjugates on both the proteasome and Cdc48, suggesting a possible role in the transfer of ubiquitylated substrates from Cdc48 to the proteasome or in their release from these complexes.Journal of Biological Chemistry 10/2013; · 4.65 Impact Factor