cells normally strive to ensure that proteins get correctly folded and that damaged , mutant , or misfolded proteins are eliminated . to maintain protein homeostasis under adverse conditions , cells have evolved powerful and sophisticated protein quality control systems that normally operate very efficiently ( kim et  al . , 2013 ) . however , increasing evidence suggests that protein quality control gradually fails with age , which has  devastating consequences for cells and organisms ( kim et  al . , 2013 ; indeed , accumulation of misfolded , aggregation - prone proteins is the hallmark of a broad range of human diseases ( cuanalo - contreras et  al . , 2013 ; kim et  al . , 2013 ; identifying strategies to improve the cells ability to handle misfolded proteins could not only reveal novel aspects of the cellular defense systems against misfolded proteins but also uncover novel strategies to correct the diverse disorders that arise when protein quality control fails . the proteasome is a key component of protein quality control systems that degrades a large number of cellular proteins and thereby controls virtually all cellular processes ( hershko and ciechanover , 1998 ; goldberg , 2007 ; finley , 2009 ; tanaka et  al . , 2012 ) . the proteasome is evolutionarily conserved , and inhibition of the proteasome is lethal in all species ( kisselev and goldberg , 2001 ; navon and ciechanover , 2009 ) . when the proteasome is inhibited , cells accumulate undegraded proteins ( kisselev and goldberg , 2001 ; navon and ciechanover , 2009 ) . in addition , proteasome inhibition causes a lethal amino acid imbalance , since the undegraded proteins immobilize a pool of amino acids that would otherwise be recycled ( suraweera et  al . , 2012 ) . failure of the ubiquitin - proteasome system has been associated with a broad range of pathological conditions , such as the devastating neurodegenerative diseases characterized by the age - dependent deposition of aggregation - prone proteins ( sherman and goldberg , 2001 ; schwartz and ciechanover , 2009 ) . since the maintenance of adequate levels of proteasomes is vital for cells and organisms , the abundance of functional proteasome must be tightly regulated to enable cells to adapt and survive changes in their environment , in particular those that overwhelm the proteasome . indeed , it has been previously established that cells increase expression of proteasome subunits in a concerted manner when the demand for proteasomes increases , for example during protein misfolding stress ( hanna and finley , 2007 ) . in yeast , this is controlled by rpn4 , a transcription factor that regulates expression levels of proteasome subunits via a homeostatic negative feedback loop ( xie and varshavsky , 2001 ) . rpn4 is an unstable protein that is normally rapidly degraded but accumulates when the proteasome is overwhelmed ( hanna and finley , 2007 ) . increasing the levels of proteasome subunits , while necessary , is not sufficient to increase proteasome levels . indeed , the levels of functional proteasome depend not only on the expression of its subunits but also on their precise assembly . recent studies have shed light on the pathways for the ordered assembly of the proteasome ( murata et  al . , 2009 ; tomko and hochstrasser , 2013 ) . proteasome assembly is an extremely complex and challenging process in the crowded cellular environment that requires the precise arrangements of the 33 subunits ( murata et  al . , 2009 ; tomko and hochstrasser , 2013 ) . an understanding of the assembly of the regulatory particle ( rp ) of the proteasome began recently with the discovery of assembly intermediates and chaperone assembly factors ( funakoshi et  al . , 2009 ; le tallec et  al . , 2009 ; roelofs et  al . , 2009 ; saeki et  al . , the six atpases of the rp , rpt1rpt6 , assemble in a  unique order upon formation of a trimer of specific pairs of atpases ( frster et  al . the yeast rp assembly chaperones nas2 , nas6 , rpn14 , and hsm3 regulate base assembly by binding to the carboxyl termini of rpts , thereby preventing binding of immature complexes to the core particle ( cp ) ( park et  al . , 2013 ) . while atpase modules composed of rpt pairs bound to their carboxy - terminal chaperones have been isolated , it is unknown how rpts find their dedicated partners . proteasome assembly is challenging under normal conditions . how cells manage to assemble proteasomes under stress conditions associated with an accumulation of undesirable proteins this represents a problem of vital importance , since cell and organismal survival depends on adequate levels of functional proteasomes . through an unbiased screen in yeast designed to identify suppressors of proteasome defects , we discovered a previously uncharacterized stress - induced protein , adc17 , which facilitates early steps in rp assembly . the discovery of this stress - inducible chaperone reveals that proteasome assembly is an adaptive process . aiming at understanding how cells adapt to proteasome deficiencies , we performed an unbiased genome - wide multicopy suppressor screen in the yeast rpt6 thermosensitive proteasome mutant cim3 - 1 ( ghislain et  al . , 1993 ; suraweera et  al . , in addition to wild - type rpt6 , we isolated two potent suppressors of the cim3 - 1 growth defects ( figure  1a ) . one encoded the previously reported suppressor , rpn14 ( funakoshi et  al . , 2009 ) , an assembly factor that binds to the carboxyl terminus of rpt6 . the second potent suppressor of the cim3 - 1 defects was tma17 , encoding a protein of unknown function , previously found in association with ribosomes ( fleischer et  al . , 2006 ) . we renamed the protein adc17 for atpase dedicated chaperone of 17  kda , following its functional characterization , as will be described below . high - copy adc17 rescued growth of cim3 - 1 at nonpermissive temperature ( figure  1a ) , but not of the rpt1 ts mutant cim5 - 1 ( ghislain et  al . , 1993 ) or the cp ts mutant pre1 - 1 ( heinemeyer et  al . , 1991 ) this suggests a genetic interaction between rpt6 and adc17 . increased accumulation of polyubiquitin conjugates and sensitivity to canavanine , an arginine analog , are hallmarks of proteasome mutants . high - copy adc17 or rpn14 markedly reduced accumulation of polyubiquitin conjugates in the cim3 - 1 cells at 30c and 35c ( figure  1b ) and abrogated the canavanine sensitivity of the cim3 - 1 ( figure  1c ) . these results suggest that the function of adc17 , a previously uncharacterized protein , is related to the proteasome . to further investigate whether the function of adc17 was related to the proteasome , we next deleted adc17 . the deletion of adc17 decreased cell fitness ( figure  2a ) , and this defect was specific as it was rescued upon overexpression of adc17 ( figure  2b ) . in the cim3 - 1 cells , deletion of adc17 caused severe growth defects ( figure  2c ) , and this was associated with a proteasome defect , as manifested by an overload of polyubiquitinated conjugates in the cim3 - 1 adc17 cells ( figure  2d ) . these results reveal that adc17 is important for cell fitness and that its  function is related to the proteasome . to characterize the function of adc17 , we next analyzed proteasome integrity by in - gel peptidase assays on whole - cell lysates . active 26s proteasomes exist as a mixture of cp bound to one  or  two rp , but the specific functional properties of rpcp and rp2cp are currently unknown ( finley , 2009 ) . as expected , both rpcp and rp2cp were detected in wild - type cells , and these two complexes appeared equally abundant ( figure  3a ) . the cim3 - 1 cells at 30c also contained similar amounts of  rpcp and rp2cp ( figure  3a ) . while equal amounts of total proteins were loaded in each condition ( see experimental procedures for details ) , the abundance of 26s proteasome ( rpcp and rp2cp ) appeared consistently higher than in the wild - type cells ( figure  3a ) . this attests of a proteasome defect in the cim3 - 1 cells , as already noticed with the accumulation of polyubiquitinated conjugates in this mutant ( suraweera et  al . , cells attempt to compensate for proteasome defects by increasing proteasome levels ( xie and varshavsky , 2001 ) . at 37c , the proteasome profiles of the cim3 - 1 cells were markedly abnormal , with a nearly complete loss of rpcp complexes ( figure  3a ) . in the cim3 - 1 cells , the 26s proteasome complexes had reduced mobility relative to their wild - type counterparts , further highlighting proteasome abnormalities in the mutant cells . free cp , revealed with low concentration of sds ( kisselev and goldberg , 2005 ) , hardly detectable in the wild - type strain at either 30c or 37c , increased in cim3 - 1 cells ( figure  3a ) , confirming that the proteasome is defective in the mutant cells . the suppressors adc17 or rpn14 noticeably increased the levels of rpcp in the cim3 - 1 at 37c , although free cp remained ( figure  3b ) . these results reveal that the proteasome is defective in cim3 - 1 cells and overexpression of adc17 or rpn14 partly rescued 26s proteasome defects . deletion of adc17 caused a modest but reproducible increase in the levels of both rpcp and rp2cp ( figure  3c ) , similar to what was observed for the cim3 - 1 mutant at 30c ( figure  3a ) . this suggests that deletion of adc17 causes proteasome defects to which cells adapt by increasing proteasome levels . furthermore , the deletion of adc17 impaired the proteasome of the cim3 - 1 cells , as cim3 - 1 adc17 cells completely lacked rpcp already at 30c ( figure  3c ) . these findings explain the proteolytic defects observed in cim3 - 1 , their rescue upon rpn14 and adc17 overexpression , and the aggravated proteasome defects of the cim3 - 1 adc17 ( figures 1 and 2 ) . to understand the molecular basis of the observed proteasome defects , we next examined the levels of different proteasome subunits in different cells . we found that the levels of rpt6 were dramatically decreased in the cim3 - 1 cells ( rpt6 thermosensitive mutant ) at 30c and even more at 37c ( figure  3d ) . in contrast to the protein levels , the levels of mrna encoding rpt6 were significantly higher in cim3 - 1 ( figure  s2 ) . this implies that rpt6 is destabilized by the ts mutation in cim3 - 1 , and cells attempt to compensate for this defect by increasing rpt6 mrna . in contrast , the protein levels of rpt5 or the cp subunit 7 rather increased in cim3 - 1 ( figure  3d ) . the expression of the  suppressors rpn14 and adc17 specifically increased the abundance of rpt6 in cim3 - 1 cells at both at 30c and at 37c ( figure  3e ) . since rpt6 is essential ( rubin et  al . , 1998 ) , the absence of rpt6 at 37c in cim3 - 1 explains the growth defects , and restoring viable levels of rpt6 upon adc17 or rpn14 overexpression in cim3 - 1 provides the basis for the rescued growth ( figure  1a ) and rescued proteasome function ( figure  3b ) . interestingly , the levels of adc17 , revealed with a specific adc17 antibody ( figure  s3 ) , were also reduced in cim3 - 1 ( figure  3d ) , and overexpression of rpt6 rescued this defect without noticeably altering the levels of rpt5 or 7 ( figure  3e ) . as previously noticed ( figure  3d ) , the levels of rpt6 were reduced in cim3 - 1 ( figure  3f ) . deletion of adc17 in the cim3 - 1 cells further reduced the levels of rpt6 ( figure  3f ) . thus , adc17 is important to maintain adequate levels of rpt6 required for proteasome integrity ( figures 3a3c ) and to sustain cell viability ( figure  2 ) . our results described above revealed that overexpression of adc17 rescues the proteasome defects while deletion of adc17 compromises proteasome integrity . to verify this , we next analyzed the mobility of adc17 on native - page followed by immunoblots . endogenous adc17 , from wild - type cell lysates , migrated as a predominant species , which was absent from adc17 cells or cim3 - 1 mutant cells and clearly distinct from mature proteasomes ( figures 4a and 4b ) . the mobility of the endogenous adc17 species ( adc17 ) was much slower than recombinant adc17 ( adc17 , figure  4a ) . to determine whether adc17 was engaged in a complex in cells , adc17-flag was immunoprecipitated and  the immunoprecipitated proteins were analyzed by mass spectrometry . rpt6 was the only protein absent from the control immunoprecipitations that specifically coprecipitated with adc17-flag ( figure  4c ) . note that adc17-flag was functional ( figure  s4 ) and that tagged protein exhibited the same mobility as the untagged adc17 on native - page ( figure  4a ) . these results reveal that in wild - type cells , adc17 is engaged in a complex with rpt6 . this complex is distinct from mature proteasome complexes and is likely to be a proteasome assembly intermediate . the results obtained so far suggested that adc17 might play a  role in rp assembly . the known rp assembly chaperones nas2 , nas6 , rpn14 , and hsm3 have overlapping functions ( funakoshi et  al . we thus examined possible genetic interactions between adc17 and some known proteasome rp assembly chaperones , using dosage suppression as a robust experimental paradigm . the single deletion of  either rpn14 , nas2 , nas6 , or hsm3 does not cause growth defects , in contrast to combined deletions ( funakoshi et  al . , 2009 ; le tallec et  al . , 2009 ; roelofs et  al . 2009 ) . among the double - deletion strains , rpn14 nas6 showed the most severe phenotype at elevated temperatures ( funakoshi et  al . , 2009 ; 2009 ) , probably because both rpn14 and nas6 are components of the same assembly module ( rpt3-rpt6 ) . having found that adc17 forms a complex with rpt6 and selectively suppresses the defects of the cim3 - 1 mutant , we wondered whether adc17 might be involved in the formation of the rpt6-rpt3 module . to assess this possibility , we examined whether adc17 could suppress the defects of the yeast mutant lacking the two assembly chaperones of the rpt3-rpt6 module , rpn14 and nas6 . overexpression of adc17 suppressed the severe growth defects of rpn14 nas6 at 34c and 35c ( figure  4d ) . the suppression of the rpn14 nas6 defects by adc17 was robust , albeit slightly less effective than the rescue with rpn14 ( figure  4d ) . interestingly , adc17 overexpression failed to rescue the hsm3nas6 defects while nas6 did ( figure  4e ) . thus , adc17 is a selective suppressor of defects in the nas6-rpt3-rpt6-rpn14 module , whether the defects are caused by a ts mutation in rpt6 or due to the lack of its assembly chaperones . in addition , we found that the growth defects of adc17 cells were rescued by adc17 but not rpn14 or nas6 ( figure  4f ) . this reveals that the function of adc17 is functionally distinct , albeit related to rpn14 . we then next examined this function in greater detail . to further characterize the function of adc17 in proteasome assembly , we next examined the interaction between adc17 and rpt6 in several independent experimental systems . yeast two - hybrid analyses confirmed that adc17 specifically interacted with rpt6 ( figure  5a ) . surprisingly , unlike all the previously known rpt assembly factors that bind to the carboxy - terminal region of their cognate atpase , thereby preventing binding of intermediates to the cp ( funakoshi et  al . , 2009 ; le tallec et  al . , 2009 ; park et  al . , 2013 ; roelofs et  al . , 2009 ; saeki et  al . , 2009 ) , adc17 selectively interacted with the amino - terminal domain of rpt6 ( rpt6-nt ) ( figure  5b ) . when coexpressed in e.  coli , gst - rpt6-nt and his - adc17 formed a complex , demonstrating that adc17 directly interacted with rpt6 ( figure  5c ) . in contrast , adc17 did not form a complex with gst - rpt3-nt under the same conditions ( figure  5d ) . sequence analysis of adc17 revealed the presence of a putative coiled - coil domain ( figure  5e ) , and analysis by circular dichroism showed that adc17 was -helical ( figure  s5 ) . to determine whether the coiled - coil domain in adc17 was important for adc17 s function , we replaced leucine 93 with an aspartate . this substitution abrogated the ability of adc17 to rescue the cim3 - 1 defects ( figure  5f ) and to interact with gst - rpt6-nt ( figure  5 g ) , establishing the importance of this interaction for adc17 function . the six atpases , rpt1rpt 6 , of the rp assemble in a unique order ( frster et  al . 2010 ) upon formation of a trimer of specific pairs of atpases ( tomko and hochstrasser , 2013 ) . how the highly related rpt subunits assemble with their cognate partner in the complex cellular environment since adc17 binds to the amino - terminal region of rpt6 , and since structural analyses of the proteasome have revealed that rpt dimers are held together by the amino - terminal regions of each rpt ( beck et  al . , 2012 ; da fonseca et  al . , 2012 ; lander et  al . , 2012 ) , we wondered whether adc17 played a role in the rpt6-rpt3 heterodimer formation . rpt3 and rpt6 amino - terminal regions were tagged at their carboxyl termini , to keep their amino termini free , and expressed in e.  coli in the presence or absence of adc17 . following coexpression of rpt6-nt - mbp and rpt3-nt - gst from a polycistronic vector , we only recovered rpt6-nt - mbp on amylose resins ( figure  s5 ) . thus , under these conditions rpt6-nt - mbp and rpt3-nt - gst did not form a complex ( figure  s5 ) . in contrast , coexpression of adc17 with rpt6-nt - mbp and rpt3-nt - gst led to the formation of two abundant complexes : a trimeric complex containing adc17 with the rpt6 , rpt3 derivatives and a dimeric complex containing rpt6-nt bound to adc17 ( figure  5h ) . this reveals that adc17 is required for rpt6 and rpt3 amino termini to interact . since adc17 did not interact with rpt3 ( figures 5a and 5d ) and because in the absence of adc17 , rpt3-nt - gst was not recovered ( figure  s5 ) , these results imply that without a productive interaction with its  dedicated partner , rpt3-nt was unstable ( figure  s5 ) . together , these results reveal that the binding of adc17 to rpt6-nt renders rpt6-nt competent to engage a productive interaction with rpt3-nt . the assays described above establish that adc17 assists pairing of rpt6-rpt3 amino termini and explains why decreased levels of adc17 are detrimental to cim3 - 1 . having identified the function of adc17 , the physiological importance of adc17 remained to be elucidated . the results obtained so far revealed that the lack of adc17 reduced cell fitness ( figure  2a ) , compromised proteasome integrity , and aggravated the defects of the cim3 - 1 mutant ( figure  2c ) . however , a proteasome regulatory complex was recently obtained by coexpression of nine integral base subunits and the four known proteasome assembly chaperones nas2 , nas6 , rpn14 , and hsm3 in e.  coli ( beckwith et  al . , 2013 ) . while adc17 was dispensable in this study having found that deletion of adc17 aggravated the cim3 - 1 defects ( figures 2c and 2d ) , we wondered whether the requirement of adc17 might be exacerbated when the proteasome is overwhelmed . to test this possibility , we deleted rpn4 , encoding the homeostatic transcription regulator of proteasome abundance ( xie and varshavsky , 2001 ) . as expected ( xie and varshavsky , 2001 ) , the levels of the proteasome subunits rpt5 , rpt6 , and 7 were markedly reduced in cells lacking rpn4 ( figure  6a ) . in contrast , the levels of adc17 dramatically increased in absence of rpn4 ( figure  6a ) . this suggests that cells adapt to reduced levels of proteasome resulting from the deletion of rpn4 by increasing adc17 . this result prompted us to examine whether this increase was only occurring following the deletion of rpn4 or whether the increased abundance of adc17 represents a generic adaptive response to overwhelming demands on the proteasome . thus , we next tested whether adc17 levels increased upon different stresses that impose a high burden on the proteasome . indeed , we found that treatment of yeast cells with tunicamycin , which causes accumulation of misfolded proteins in the endoplasmic reticulum , dramatically increased adc17 levels ( figure  6b ) . although robust , this increase was less dramatic than that caused by tunicamycin ( figure  6b ) . the different stressors tested are also expected to increase proteasome levels ( xie and varshavsky , 2001 ) , and this was manifested here by an increase in rpt6 levels ( figure  6b ) . the increased levels of adc17 upon stress together with the increased requirement of adc17 for the fitness of cim3 - 1 ( figure  2c ) suggest that the importance of adc17 is exacerbated when the proteasome is overwhelmed . having found that the levels of adc17 dramatically increased upon tunicamycin treatment , we tested whether adc17 had an increased sensitivity to tunicamycin . note that the experimental condition used here ensured that the cells were in exponential growth phase before tunicamycin treatment ( figure  6c ) . we found that cells lacking adc17 had an increased sensitivity to tunicamycin ( figure  6d ) . together , our results suggest that adc17 has evolved to facilitate proteasome assembly when the need increases , as is the case under stress conditions . to test this possibility , proteasomes were examined by in - gel peptidase assays on whole - cell lysates collected from unstressed or stressed cells , either wild - type or lacking adc17 . as expected , tunicamycin treatment increased the levels of rp2cp detected by in - gel peptidase assays ( figures 6e and 6f ) . western blot analysis of native gels also revealed that tunicamycin increased rp2cp ( figures 6 g and 6h ) . in contrast to wild - type cells , the ability of adc17 cells to increase the levels of rp2cp following tunicamycin treatments was compromised ( figures 6e6h ) . furthermore , adc17 cells accumulated free cp ( figures 6e6h ) , revealing a defect in proteasome assembly . importantly , the levels of the rp subunits rpt6 or rpt5 or the core subunit 7 were undistinguishable in wild - type and adc17 cells , both in untreated cells and following tunicamycin treatments ( figure  6i ) . thus the reduced levels of rp2cp in adc17 cells compared to wild - type cells following tunicamycin treatment were caused by a proteasome assembly defect . similar experiments were carried out using heat shock as a different stress inducer ( figure  s6a ) . as seen in tunicamycin - treated cells ( figure  6e ) , heat shock increased the levels of 26s proteasomes detected by in - gel peptidase assays , and this increase was compromised in adc17 cells ( figures s6b  s6e ) . together , these results establish that adc17 is crucial for biogenesis of adequate proteasome levels during stress . with the discovery of a stress - inducible atpase dedicated chaperone , this study reveals how cells control homeostatic levels of proteasomes . cells have evolved numerous and often overlapping homeostatic pathways to adapt to changes in their environment and to survive under adverse conditions . the proteasome controls the degradation of a large number of cellular proteins and thereby regulates essentially all cellular processes . as cell survival depends on adequate levels of functional proteasome , cells must have evolved multiple mechanisms to adapt to the adverse conditions that compromise proteasome function or overwhelm its capacity . such conditions include protein misfolding stress , which represents an important problem for cells and organisms , where failure to remove misfolded , aggregation - prone proteins is associated with a broad range of pathological conditions . one adaptive response to increased demands on proteasomes is well established and relies on the transcription factor rpn4 ( xie and varshavsky , 2001 ) . when the load of proteins to be degraded exceeds the capacity of the proteasome , the otherwise rapidly degraded transcription factor rpn4 accumulates in cells , and this in turn increases expression of proteasome subunits ( xie and varshavsky , 2001 ) . these subunits then need to be faithfully assembled to generate a functional proteasome . while the mechanisms governing the assembly of the proteasome have been largely elucidated , how cells accomplish such a challenging task under stress conditions is unknown . since the levels of proteasomes are known to increase under stress ( lee et  al . , 2004 ) , cells must have additional mechanisms to overcome the difficult challenge that proteasome assembly represents during stress . through an unbiased screen in yeast , we have discovered that a previously uncharacterized protein is an effector of a stress response pathway that couples proteasome assembly to the rpn4-mediated increased expression of its subunits . adc17 facilitates the pairing of rpt6 and rpt3 , an early step in proteasome biogenesis ( figures 5 and 7 ) . adc17 is inducible upon stress , thereby providing an opportunity to adapt proteasome assembly to the increased needs , a function that is crucial for cells to adapt to stresses ( figure  6 ) . importantly , unlike the proteasome subunits , adc17 s expression is not under the control of rpn4 . therefore , two pathways control proteasome homeostasis : one depending on rpn4 and one depending on adc17 . while the rp assembly chaperones nas2 , nas6 , hsm3 , and rpn14 have been studied in many different laboratories , there is no evidence that their expression might be regulated , unlike adc17 . thus , adc17 defines a distinct paradigm in proteasome stress response pathway . in the recent years , there has been much progress toward understanding the assembly of the proteasome ( saeki and tanaka , 2012 ; tomko and hochstrasser , 2013 ) . the hexameric aaa atpase ring of the regulatory particle ( rp ) forms upon assembly of three modules , each composed of a specific pair of atpases , rpt1-rpt2 , rpt3-rpt6 , or rpt5-rpt4 , bound to one or two rp assembly chaperones , hsm3 , nas2 , nas6 , and rpn14 ( funakoshi et  al . , 2009 ; le tallec et  al . , 2009 ; . however , the mechanisms governing the formation of the three modules remain to be elucidated . the four previously known rp assembly chaperones share a common function that is now well defined ( funakoshi et  al . , 2009 ; le tallec et  al . , 2009 ; park et  al . hsm3 , nas2 , nas6 , and rpn14 bind to the carboxyl termini of rpts , thereby preventing interactions of immature complexes with the core particle ( cp ) ( park et  al . , 2013 ) . in contrast to all known rp assembly chaperones , nas2 , nas6 , hsm3 , and rpn14 , we found that adc17 binds to rpt6 amino terminus . thus , the mapping of the interaction between rpt6 and adc17 immediately revealed that the function of adc17 was mechanistically distinct from the known rp assembly factors , although functionally related . since adc17 interaction maps to the amino terminus of rpt6 , a region predicted to be important for the selective pairing of rpts ( beck et  al . , 2012 ) , we suspected that adc17 might be involved in the formation of the atpase pairs . an unbiased assay with recombinant proteins revealed that productive pairing of rpt6 and rpt3 amino termini depends on adc17 ( figures 5 g and s4 ) . these findings are relevant to the full - length rpt6 protein for the following reasons . we found that adc17 not only binds the amino terminus of rpt6 but also forms a complex with rpt6 full - length that we recovered from cell lysates by immunoprecipitation and confirmed this interaction in a two - hybrid assay . we also established that a point mutation in a conserved leucine in the coiled - coil region of adc17 abolishes its ability to interact with rpt6-nt ( figure  5 g ) as well as its function ( figure  5f ) , thereby demonstrating that the  function of adc17 is mediated by its interaction with the rpt6 amino terminus . interestingly , coexpression of adc17 with rpt6-nt and rpt3-nt led to the formation of a heterotrimer rpt6-rpt3-adc17 complex , in addition to an rpt6-adc17 dimer ( figure  5h ) , while immunoprecipitations from yeast lysates only  recovered rpt6-adc17 complex ( figure  4c ) . this  suggests that in cells , an rpt6-rpt3-adc17 complex might be short - lived and rapidly incorporated in higher order modules . supporting this hypothesis , rpt5 module was detectable in wild - type cells ,  and the abundance of this assembly intermediate increased in the cim3 - 1 mutant ( figure  3a ) . thus , the formation of the rpt3-rpt6 module might be a rate - limiting step in proteasome assembly . adc17 , being stress inducible , provides a mechanism to finely tune the first commitment step in proteasome assembly . although a proteasome regulatory complex can be produced by coexpression of base subunits and the four known proteasome assembly chaperones nas2 , nas6 , rpn14 , and hsm3 in e.  coli ( beckwith et  al . , 2013 ) , in absence of adc17 , our results demonstrate that in  vivo , adc17 confers a selective advantage for proteasome assembly . the deletion of adc17 compromises cell fitness ( figure  2a ) , and this is associated with increased levels of free 20s proteasome ( figure  6h ) , a hallmark of proteasome assembly defects . furthermore , the requirements of adc17 are exacerbated under conditions of proteasome deficiencies or when proteasomes are insufficient ( figures 2c , 2d , and 6d ) . the recent study ( beckwith et  al . , 2013 ) suggests that it should be possible to reconstitute rp assembly with recombinant proteins and assembly factors . such a system would be ideal to fully characterize the sequential steps in the ordered assembly of the rp and the release of proteasome chaperones from assembly intermediates , and to isolate intermediates that in cells may be too evanescent to isolate , such as the adc17-rpt6-rpt3 intermediate . adc17 defines a paradigm of rp chaperones , which binds to the amino terminus of an rpt to facilitate assembly with its partner ( figure  s7 ) . we have shown that adc17 is essential to assemble the amino - terminal domains of rpt6 and rpt3 ( figure  5 g ) . in the context of the full - length proteins , the function of adc17 is also important since adc17 is important for cell fitness and proteasome integrity but conditionally essential , as adc17 s function partly overlaps that of rpn14 ( figure  4d ) . the interaction between adc17 and rpt6 seems stronger with the isolated amino - terminal domain of rpt6 than with the full - length protein ( figure  5b ) , raising the possibility that this interaction might take place cotransnationally . ( tma17 ) was previously isolated in a systematic screen for ribosome - associated proteins ( fleischer et  al . , 2006 ) . while no sequence homologs of adc17 have been identified in mammals so far , the conservation of proteasome assembly across species ( funakoshi et  al . , 2009 ; kaneko et  al . , 2009 ; le tallec et  al . , 2009 ; roelofs et  al . , 2009 ; saeki et  al . , , no sequence homologs were found in higher eukaryotes for the yeast hac1 , the target of the evolutionarily conserved ire1 , a key component of the unfolded protein response , while a functional homolog , xbp-1 , exists ( walter and ron , 2011 ) . the high sequence similarities between rpt1rpt6 raises the possibility that other atpase dedicated chaperones might exist to facilitate the pairing of rpt1 with rpt2 and rpt4 with rpt5 . alternatively , the rpt6-rpt3 module may require a higher number of chaperones than the other modules , as the formation of this module appears to be the first and rate - limiting step in rp assembly . the identification of adc17 and its functional characterization reveals that cells have mechanisms to adjust proteasome assembly to increased demands ( figure  7 ) . as shown here , failure to adapt proteasome assembly when the demands on proteasomes increase is deleterious . identifying pathways to survive protein quality control failure may ultimately be of therapeutic value to ameliorate the large  number of pathologies associated with proteasome deficiencies . all yeast manipulations were conducted according to standard protocols ( gietz and woods , 2006 ) . to assess growth phenotypes , exponentially growing liquid cultures expressing the indicated genes were equilibrated to an od600 of 0.2 , and 5  l were spotted in serial dilutions ( 1/6 ) onto ypd or selective media as required . plates were incubated at the indicated temperature for 23  days . to assess canavanine sensitivity , cells were spotted on plates lacking arginine supplemented with canavanine ( 1  g / ml ) . ( p416 gpd ) , rpn14 ( p416 gpd ) , or wild - type rpt6 ( pfl44 ) . the identification of suppressors was carried out in two different screening setups . in one screening setup , cim3 - 1 was transformed with the library pfl44 , plated onto ura plates and the plates were incubated at 37c for seven days . were heat shocked at 37c for 24  hr and then grown at 30c for 24  hr . during the second incubation , clones were isolated . the plasmid of each isolated clone was extracted using a standard yeast dna extraction procedure , and the dna was transformed into e.  coli dh5 ( life technologies ) . five to ten milliliters of exponentially growing cells were harvested by centrifugation at 4,000  rpm for 3  min at 4c , washed with ice - cold water , and subsequently frozen on dry ice . where indicated , cells were cultured in the presence of 1  g / ml or 5  g / ml canavanine in medium lacking arginine or in ypd supplemented with 5  g / ml tunicamycin . cells were resuspended in 200  l rapid protein preparation buffer ( foiani et  al . , 1991 ) ( 10  mm tris - hcl [ ph 7.4 ] , 100  mm nacl , 30  mm mgcl2 , 1  mm dtt , two complete protease inhibitor cocktail tablets [ pic , roche ] per 50  ml , 200  m pmsf ) and transferred into clean tubes containing 200  l acid - washed glass beads ( 0.5  mm ; sigma ) and frozen on dry ice . subsequently , 200  l rapid protein preparation buffer was added , and samples were centrifuged for 1  min at 15,000  rpm at 4c . the supernatant was transferred to a clean tube and the remaining cell debris was removed by centrifugation for 10  min at 15,000  rpm at 4c . protein concentrations were measured by monitoring od280 , and 75  g of total protein extract was loaded on nupage 4%12% bis - tris gels ( life technologies ) and resolved in mes buffer . membranes were incubated with antibodies to ubiquitin ( 1:2,000 ; dako z0458 ) , phosphoglycerate kinase ( pgk ; 1:5,000 ) ( life technologies ) , flag ( 1:1,000 , m2 , sigma a8592 ) , 7 ( 1:1,000 ; biomol pw8110 ) , 20s ( 1:2,000 ; biomol pw9355 ) , rpt5 ( 1:1,000 ; biomol pw8245 ) , rpt6 ( 1:50 ; carl mann ) , and adc17 ( 1:1,000 ; this study ) . , cells were resuspended in 200  l lysis buffer ( 50  mm tris - hcl [ ph 7.4 ] , 1  mm edta , 1.5  mm mgcl2 , 1  mm dtt , pic , 1  mm pmsf , and 50  mm n - ethylmaleimide ) . extracts from frozen pellets were prepared by heating the resuspended pellets for 10  min at 90c . subsequently 10  l of 4  m acetic acid was added and samples were vortexed for 30 s. after a second incubation for 10  min at 90c , the samples were centrifuged for 15  min at 15,000  rpm at room temperature and the supernatant was recovered . for native - page , total yeast extracts were prepared using a freezer mill ( spex sampleprep llc ) . exponentially growing cells were harvested , washed in ice - cold water , resuspended in lysis buffer ( 50  mm tris - hcl [ ph  7.4 ] , 1  mm edta , 5  mm mgcl2 , 1  mm dtt , 2  mm atp , and pic ) , and flash - frozen in liquid nitrogen . yeast extracts were produced freshly by resuspending 1.5  g powder in 1.5  ml lysis buffer for 30  min at 4c . subsequently , the samples were centrifuged at 4,000  rpm for 10  min and the supernatants were transferred to ultracentrifuge tubes and spun in an tla100.4 rotor ( beckman coulter ) for 27  min at 4c at 50,000  rpm . native - page of 4.2% or 5.25% acrylamide were prepared as described in ( elsasser et  al . , 2005 ; wild - type and endogenously tagged adc17-flag were grown to exponential phase and lysed in flag immunoprecipitation buffer ( 50  mm hepes - koh [ ph 6.8 ] , 150  mm koac , 5  mm mgoac , 15% glycerol , 1  mm edta , 1  mm dtt , pic , and 2  mm atp ) . yeast cell extracts were subjected to anti - flag immunoprecipitation using anti - flag m2 magnetic beads ( sigma - aldrich ) , as described in brandman et  al . , 2012 . immunoprecipitated proteins were eluted with 3xflag peptides , and eluates were analyzed by mass spectrometry . yeast two - hybrid assays were performed as described in fleischer et  al . , 2006 and saeki et  al . , 2009 . constructs were transformed into bl21-gold competent cells and selected on ampicillin- and/or kanamycin - containing plates ( 100  mg / l and 50  mg / bacteria were grown in luria - bertani ( lb ) medium containing the appropriate antibiotics at 37c up to an od600 of approximately 0.6 . expression was induced by addition of 0.1  mm iptg for 3  hr at 30c . cultures were harvested and cell pellets were resuspended in lysis buffer ( 25  mm tris - hcl [ ph 8.0 ] , 300  mm nacl , 10% glycerol , pic , and 0.2  mm pmsf ) , frozen in liquid nitrogen , thawed slowly on ice , and lysed by sonication . bacterial lysates were centrifuged at 15,000 g at 4c for 20  min and the supernatant was collected . the proteins and protein complexes were purified either on ni - nta sepharose ( qiagen ) or glutathione sepharose 4b ( ge healthcare ) and analyzed on nupage sds - page 4%12% bis - tris gels ( life technologies ) stained with coomassie blue . the ni - nta resin was equilibrated in lysis buffer plus 20  mm imidazole and proteins were eluted with 25  mm tris - hcl ( ph 8.0 ) , 100  mm nacl , 10% glycerol , 250  mm imidazlole . the glutathione resin was equilibrated in lysis buffer and proteins were eluted with 25  mm tris - hcl ( ph 8.0 ) , 100  mm nacl , 10% glycerol , 5  mm glutathione . rpt6-nt - mbp and rpt3-nt - gst were coexpressed in e.  coli with or without his6x - adc17 using the popc / popt polycistronic plasmids ( frster et  al . , 2009 ; ghislain et  al . , 1993 ; the protein complexes were purified on amylose resin ( new england biolabs ) equilibrated in lysis buffer and eluted with 25  mm tris - hcl ( ph 8.0 ) , 100  mm nacl , 10% glycerol , 20  mm maltose , followed by gel filtration analysis using a 24  ml superdex 200 10/300 gl column ( ge healthcare ) . one hundred microliters of each complex was loaded onto the column at a flow rate of 0.4  ml / min , and 0.3  ml fractions were eluted in column buffer ( 25  mm tris - hcl [ ph 8.0 ] , 100  mm nacl ) . an equal volume ( 10  l ) from each fraction was loaded onto nupage sds - page 4%12% bis - tris gels ( life technologies ) . for the production of anti - adc17 antibody , his - adc17 was expressed in e.  coli , purified using ni - nta affinity chromatography . purified proteins were injected to two rabbits , and antisera were used at dilutions of 1:1,000 . spectra ( 190260  nm ) of 18  g of adc17 protein in 5  mm tris - hcl ( ph 8.0 ) , 50 mm nacl were recorded on a jasco j-810 spectropolarimeter . yeast cells were harvested during exponential growth phase and cell wall was digested with zymolyase ( zymo research ) . rna was extracted by high pure rna isolation kit ( roche ) according to the manufacturer s instructions . rna quality and quantity were assessed by nanodrop 2000c spectrophotometer ( thermo fisher scientific ) , and 500  ng of mrna was reverse - transcribed to cdna using an iscript cdna synthesis kit ( bio - rad ) . quantitative pcr was performed with primers ( rpt6 forward , ttccattggctctactcgtg ; rpt6 reverse , aaacccgtccaattggttta ; taf10 forward , atattccaggatcaggtcttccgtagc ; taf10 reverse , gtagtcttctcattctgttgatgttgttgttg ) using sybr select master mix ( applied biosystems ) on a rotor - gene 6000 and analyzed using corbett rotor gene software ( corbett life science ) . expression of rpt6 was normalized to housekeeping gene taf10 , and fold change was measured using the pfaffl equation and expressed relative to wild - type control . a.h . performed the screen , discovered adc17 , and characterized the cim3 - 1 strain and adc17 in yeast . z.z . elucidated the function of adc17 .
summarythe proteasome is essential for the selective degradation of most cellular proteins . to survive overwhelming demands on the proteasome arising during environmental stresses , cells increase proteasome abundance . 
 proteasome assembly is known to be complex . 
 how stressed cells overcome this vital challenge is unknown . in an unbiased suppressor screen aimed at rescuing the defects of a yeast rpt6 thermosensitive proteasome mutant 
 , we identified a protein , hereafter named adc17 , as it functions as an atpase dedicated chaperone . adc17 
 interacts with the amino terminus of rpt6 to assist formation of the rpt6-rpt3 atpase pair , an early step in proteasome assembly . 
 adc17 is important for cell fitness , and its absence aggravates proteasome defects . 
 the abundance of adc17 increases upon proteasome stresses , and its function is crucial to maintain homeostatic proteasome levels . 
 thus , cells have mechanisms to adjust proteasome assembly when demands increase , and adc17 is a critical effector of this process .