strains carrying proa - tev - rpt1 were grown in 6 l of sd medium to od600 0.8 to 1.2 .
cells were harvested , washed once with ice - cold water and drop - frozen in liquid nitrogen .
frozen yeast samples were then ground using an mm301 grinding mill ( restch ) under liquid nitrogen following manufacturer s instructions , or using a mortar and a pestle as previously described24 .
ground powder was hydrated in proteasome buffer ( 50 mm tris - hcl [ ph7.5 ] , 5 mm mgcl2 , 1 mm edta , and 10% glycerol ) supplemented with 2 mm atp , protease inhibitor tablets ( complete , roche ) , 2 mm pmsf , 1 mm benzamidine , 10 g / ml pepstatin a , and 1 g / ml antipain .
cell extracts were cleared at 30,000  g for 30 min at 4c , and the supernatants were mixed with rabbit igg resin ( cappel , mp biomedicals ) for 90 min at 4c .
resins were collected at 900  g for 2 min at 4c and washed with proteasome buffer containing 50 mm nacl three times , followed by a final wash with proteasome buffer alone .
bp1 was then released from the resin by incubating with actev protease ( invitrogen ) at 2.5 unit / l culture in proteasome buffer containing 2 mm atp and protease inhibitors for 1 hr at 30c .
eluates were concentrated using ultrafree-0.5 centrifugal filter device with 30 kda nmwl ( millipore ) .
strains carrying proa - tev - rpt1 were grown in 6 l of sd medium to od600 0.8 to 1.2 .
cells were harvested , washed once with ice - cold water and drop - frozen in liquid nitrogen .
frozen yeast samples were then ground using an mm301 grinding mill ( restch ) under liquid nitrogen following manufacturer s instructions , or using a mortar and a pestle as previously described24 .
ground powder was hydrated in proteasome buffer ( 50 mm tris - hcl [ ph7.5 ] , 5 mm mgcl2 , 1 mm edta , and 10% glycerol ) supplemented with 2 mm atp , protease inhibitor tablets ( complete , roche ) , 2 mm pmsf , 1 mm benzamidine , 10 g / ml pepstatin a , and 1 g / ml antipain .
cell extracts were cleared at 30,000  g for 30 min at 4c , and the supernatants were mixed with rabbit igg resin ( cappel , mp biomedicals ) for 90 min at 4c .
resins were collected at 900  g for 2 min at 4c and washed with proteasome buffer containing 50 mm nacl three times , followed by a final wash with proteasome buffer alone .
bp1 was then released from the resin by incubating with actev protease ( invitrogen ) at 2.5 unit / l culture in proteasome buffer containing 2 mm atp and protease inhibitors for 1 hr at 30c .
eluates were concentrated using ultrafree-0.5 centrifugal filter device with 30 kda nmwl ( millipore ) .
 substrates of the proteasome are recognized and unfolded by the regulatory particle ( rp ) , then translocated into the core particle ( cp ) to be degraded1 . 
 a hetero - hexameric atpase ring , containing subunits rpt1-rpt6 , is situated within the base subassembly of the rp1 . 
 the atpase ring sits atop the cp , with the rpt c - termini inserted into pockets in the cp26 . 
 we have identified a novel function of the rpt proteins in proteasome biogenesis through deleting the c - terminal residue from each rpt . 
 our results indicate that assembly of the hexameric atpase ring is templated on the cp . 
 we have also identified an apparent intermediate in base assembly , bp1 , which contains rpn1 , three rpts , and hsm3 , a chaperone for base assembly . 
 the rpt proteins with the strongest assembly phenotypes , rpt4 and rpt6 , were absent from bp1 . 
 we propose that rpt4 and rpt6 form a nucleating complex to initiate base assembly , and that this complex is subsequently joined by bp1 to complete the rpt ring . 
 our studies show that assembly of the proteasome base is a rapid yet highly orchestrated process . 
Methods summary
Purification of BP1
Pulse-chase analysis of BP1, native PAGE and immunoblot
Supplementary Material