the reported sequence of hsm3 in the saccharomyces genome database differs from the sequence in our strain background at the c - terminus ( see supplementary information ) . plasmids and antibodies used are described in supplementary table 3 and 4 respectively .
recombinant proteins were expressed in and purified from rosetta 2 cells ( novagen ) as detailed in supplementary methods .
cell lysates were made in an atp - containing buffer , cleared by centrifugation , and loaded on a 3.6% or 5.25% polyacrylamide gel . to transfer proteins from native gels to pvdf , the gels were pre - soaked for 10 min in 1x sds - page running buffer and transferred according to standard protocols . for 2d - immunoblotting
, a lane was excised and loaded on top of an sds - page gel .
proteins were transferred from the second dimension gel to pvdf and probed with indicated antibodies .
the reported sequence of hsm3 in the saccharomyces genome database differs from the sequence in our strain background at the c - terminus ( see supplementary information ) . plasmids and antibodies used are described in supplementary table 3 and 4 respectively .
recombinant proteins were expressed in and purified from rosetta 2 cells ( novagen ) as detailed in supplementary methods . for pulldown experiments
for native gels , cell lysates were made in an atp - containing buffer , cleared by centrifugation , and loaded on a 3.6% or 5.25% polyacrylamide gel . to transfer proteins from native gels to pvdf , the gels were pre - soaked for 10 min in 1x sds - page running buffer and transferred according to standard protocols . for 2d - immunoblotting
, a lane was excised and loaded on top of an sds - page gel .
proteins were transferred from the second dimension gel to pvdf and probed with indicated antibodies .
 the proteasome regulatory particle ( rp ) initiates the degradation of ubiquitin - protein conjugates by unfolding the substrate and translocating it into the proteasome core particle ( cp ) to be degraded1 . 
 here we show that in yeast three proteins are found associated with rp but not rp - cp holoenzyme : nas6 , rpn14 , and hsm3 . 
 mutations in these genes confer proteasome loss of function phenotypes , despite their virtual absence from holoenzyme . 
 these effects result from deficient rp assembly . 
 thus , nas6 , rpn14 , and hsm3 are rp chaperones . 
 the rp contains six atpases  the rpt proteins  and each rp chaperone binds to the c - terminal domain of a specific rpt . 
 we show in an accompanying study2 that rp assembly is templated through the rpt c - termini , apparently by their insertion into binding pockets in the cp . 
 thus , rp chaperones may regulate proteasome assembly by directly restricting the accessibility of rpt c - termini to the cp . 
 in addition , competition between the cp and rp chaperones for rpt engagement may explain the release of rp chaperones as proteasomes mature . 
Methods summary
Yeast strains, plasmids and antibodies
Immunoprecipitations and protein purifications
Native gels
Supplementary Material