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 Cdc48Npl4-Ufd1 complex leads to much greater 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; 288(47). DOI:10.1074/jbc.M113.521088 · 4.57 Impact Factor
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ABSTRACT: The ubiquitin-proteasome pathway, involved in genetic recombination and sex-chromosome silencing during meiosis, plays critical roles in the specification of germ-line stem cells and the differentiation of gametes from gonocytes. Zygote-specific proteasome assembly chaperone (ZPAC) is expressed in the early mouse embryo, where it is important for progression of the mouse maternal-to-zygotic transition. The role of ZPAC during spermatogenesis in the adult gonads, however, remains unknown. In this study, rapid amplification of cDNA ends was used to determine the Zpac cDNA sequence, a 1584-bp transcript that includes a putative 1122-bp open reading frame coding for a 373 amino acid protein. Western blot and immunohistochemistry revealed that ZPAC was specifically expressed in gonads. To further dissect the function of ZPAC during spermatogenesis, we employed PiggyBac-based RNA interference vectors for transgenesis combined with cell transplantation to deplete Zpac during spermatogenesis. This RNAi-mediate depletion in Zpac expression disrupted normal spermatogenesis from spermatogonial stem cells. Two independent yeast two-hybrid screens further revealed an interaction between ZPAC and SYCE1. Together, these data suggest that ZPAC is required for normal spermatogenesis in mice. Mol. Reprod. Dev. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.Molecular Reproduction and Development 07/2015; DOI:10.1002/mrd.22507 · 2.68 Impact Factor