An allosteric interaction network promotes conformation state‐dependent eviction of the Nas6 assembly chaperone from nascent 26S proteasomes

Abstract

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.

Sem1/DSS1 accelerates ATP-dependent substrate unfolding by the proteasome through a conformation-dependent intercomplex contact

“…Our group and others recently discovered a role for another conformationdependent lid-base contact, between Rpn5 and Rpt3. This Rpn5-Rpt3 contact helps to enact conformational changes that both induce the eviction of the base assembly chaperone Nas6 from nascent proteasomes during biogenesis, and activate the proteasome for substrate degradation (Greene, et al, 2019;Nemec, et al, 2019).…”

“…Rpn10 was expressed from pRT205 as an N-terminal 6His fusion in bacterial strain LOBSTR (DE3) co-transformed with pRARE2 as previously described (Nemec, et al, 2019). Transformants were grown in LB and the appropriate antibiotics at 37°C, 250 rpm shaking until OD600 ≈ 0.6, at which point the temperature was reduced to 30°C and IPTG was added to 0.5 mM.…”

“…Fully recombinant lid complex and sem1Δ lid were expressed from pRT2226 and either pRT2214 (WT lid), pRT2216 (sem1Δ lid), or pRT2504 (sem1Δ, rpn7mut lid) as previously described (Nemec, et al, 2019). Lid was expressed in LOBSTR (DE3) cotransformed with pRARE2.…”