The 26S proteasome is the central ATP‐dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated in part by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation‐specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP‐binding by the ATPase subunit Rpt3 then promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly‐coupled allosteric signals promote chaperone eviction, and provide a framework for understanding the eviction of other chaperones from this biomedically important molecular machine. Support or Funding Information 1R01GM118600 to R.J.T. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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The 26S proteasome is the central ATP‐dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated in part by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation‐specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP‐binding by the ATPase subunit Rpt3 then promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly‐coupled allosteric signals promote chaperone eviction, and provide a framework for understanding the eviction of other chaperones from this biomedically important molecular machine.Support or Funding Information1R01GM118600 to R.J.T.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The 26S proteasome is a chambered protease that uses ATP‐powered movements of pore loops within a ring of AAA+ ATPase subunits to drive unfolding and translocation of substrates into the peptidase core for proteolysis. Whereas most AAA+ family proteases contain a homohexameric ATPase ring in which each ATPase pore loop is thought to function identically and cooperatively, the proteasome contains six highly similar but non‐identical ATPase subunits. Some limited evidence suggests their contributions to proteolysis are non‐equivalent. We hypothesized that inactivation of each pore loop may distinctly impact substrate selection and/or processing by the proteasome. Using a collection of congenic pore loop mutant cells, we discovered many unique impacts on cell health, stress response, and ubiquitin homeostasis. Mutating the pore loop of one particular ATPase, Rpt3, led to a significant decrease in overall proteolytic activity. Genetic and biochemical analyses in yeast indicate that this pore loop transmits a signal to the peptidase component of the proteasome to trigger translocation of the substrate into the peptidase core for proteolysis. Together, our findings suggest an allosteric mechanism that couples pore loop movements with access to the peptidase centers for substrate processing.