cell culture and reagents293 t cells were maintained in 
  dulbecco 's modified eagle 's medium supplemented with 10% fetal bovine serum 
  and transfected in 6- or 12-well plates by using the calcium phosphate method 
  leading usually to > 70% transfection efficiency . 0.25 g of 
  htt73 encoding plasmid were transfected together with 0.125 , 0.25 , and 0.5 
  g of plasmid encoding 19s subunits except when indicated otherwise . where 
  indicated , transfections were performed after 8 days of culture in 6-well plates with 0.5 
  g of htt73-gfp plasmid with or without 0.5 g of rpt6 plasmid and 
  lipofectamine 2000 ( invitrogen ) . rpt6 was kindly 
  provided by r. losson and subcloned in the hindiii and xhoi restriction sites 
  of pxj41 . rpt6 was generated by pcr and cloned hindiii and 
  xhoi restriction sites of pxj41 . the cdnas encoding rpt4 was amplified by 
  reverse transcription - pcr from hela cell total rna flanked with ecori and xhoi 
  sites and cloned into pxj41 . to generate gfp fusion , htt73 was pcr - amplified , 
  flanked with bamhi and kpni sites , and cloned in the bglii and kpni sites of 
  pegfp - n2 ( bd biosciences ) . to produce gst fusions , rpt6 and 
  rpt6 were subcloned into the bamhi and xhoi restriction sites 
  of pgex-4t3 vector in - frame with gst ( ge healthcare ) . the cdna sequence coding 
  for the last 52 amino acids of ornithine decarboxylase ( odc ) was pcr - amplified 
  using pzs proteasome sensor ( clontech ) as a template , flanked with xhoi and 
  xbai restriction sites , and cloned in pcdna1/amp vector ( invitrogen ) 
  containing htt73 to produce htt73odc . in the odc - encoding sequence , cysteine 
  441 was mutated in alanine to generate htt73odcc441a . the cdna 
  encoding the first 108 amino acids of huntingtin with an expansion of 50 cag 
  was cloned in pgex-4 t vector in - frame with gst ( ge healthcare ) generating 
  gst - htt50 , similar to gst - hd51 described in scherzinger et al . ( 38 ) . expression of 
  recombinant proteins was performed as described in scherzinger et al . small interfering rnas80 pmol of double - stranded rna 
  oligonucleotides directed against the target rat rpt6 ( rpt6 sirna_1 , 
  ggattgacaggaaaattga ; rpt6 sirna_2 , ggagatcaaagaagtgatt ) or rpt4 ( rpt4 sirna_1 , 
  tggacagattgtaggcgaa ; rpt4 sirna_2 , tgaggtatctgccgagaga ) or control sirna 
  silencer negative control # 1 ( ambion ) were transfected in primary 
  hippocampal neurons 8 days after their plating in a 6-well plate using 
  lipofectamine 2000 ( invitrogen ) . micrographs were taken at 100 magnification on a leica tcs 
  sp2aobs confocal microscope or leica dmrb fluorescence microscope . frozen brain samples ( parietal cortex , 
  brodmann area number 7 ) from two individuals with juvenile hd were obtained 
  from the harvard brain tissue resource center . the two cases were a 
  14-year - old , grade 3 female with 97 cag repeats and a 18-year - old grade 4 
  female with 75 cag repeats , previously described in hoffner et al . sections were fixed with 
  4% paraformaldehyde and labeled with a polyclonal antibody directed against 
  the first 17 amino acids of human htt ( 1:400 dilution ) 
  ( 39 ) , 7 ( pw8110 biomol , 
  1:1000 dilution ) , and rpt6 antibody ( sug-1b8 , euromedex , 1:5000 dilution ) . 100 
  inclusions were examined , 50 nuclear and 50 cytoplasmic , in the two tissues . micrographs were taken at 63 magnification combined with digital 
  zooming on a zeiss confocal microscope ( carl zeiss , inc . , routinely , 70% 
  confluent cells from a well of a 12-well plate were lysed in 140 l of 
  boiling laemmli buffer ( 25 mm tris - hcl , ph 6.8 , 1% sds , 25 
  mm dithiothreitol , 7.5% glycerol , 0.05% bromphenol blue ) for 
  immunoblot analysis or 140 l of ice - cold nonidet p-40 buffer ( 25 
  mm tris - hcl ph 7.5 , 1% nonidet p-40 , 300 mm nacl , 5 
  mm edta , 1 mm phenylmethylsulfonyl fluoride , 5 g / ml 
  pepstatin , leupeptin , and aprotinin ) for fractionation experiments . 18 l 
  of protein extracts were loaded on 10% sds - page and transferred to optitran 
  ba - s 83 reinforced nitrocellulose membrane ( whatman and schleicher & 
  schuell ) . equal loading of protein extracts analyzed by immunoblot was 
  controlled by ponceau red staining ( data not shown ) and vimentin . membranes were saturated 
  in 5% dried skimmed milk in phosphate - buffered saline and probed with htt 2b4 
  antibody unless specified . the appropriate secondary antibody coupled to 
  peroxidase was revealed using the supersignal west pico chemiluminescent kit 
  ( pierce ) . chemiluminescent images were acquired using the chemi - smart 5000 
  ( vilber - lourmat ) allowing quantitative detection of chemiluminescence . antibodies  for immunocytochemistry , primary antibodies were 
  diluted as follows : 1:1000 dilution for tau-1 antibody ( chemicon ) , 7 
  ( pw8110 biomol ) , rpt1 ( ab3322 , abcam ) , rpt2 ( ab3317 ) , rpt3 ( ab22634 ) , and rpt5 
  ( ab22635 ) ; 1:5000 dilution for rpt6 antibody ( sug-1b8 , euromedex ) or rpt4 
  ( ab22639 ) . for immunoblots , antibodies were used as follows : 1:5000 for htt 
  2b4 antibody and 1:1000 for antibodies against proteasome subunits . vimentin 
  antibody v 6630 ( sigma ) was used at a 1:500 dilution , p21 antibody ( bd 
  pharmingen 556431 ) at a 1:500 dilution , and e2f-1 ( santa cruz ) sc-251 at a 
  1:200 dilution . immunoprecipitation293 t cells were plated 24 h before 
  transfection in 10-cm dishes at a density of 80000 cells / ml . transfection were 
  performed with 2.5 g of htt73 alone or with 5 g of rpt6-flag - encoding 
  plasmid . 48 h post - transfection , cells were washed with ice - cold 
  phosphate - buffered saline , lysed in 1 ml of 0.2% nonidet p-40 buffer ( 0.2% 
  nonidet p-40 , 50 mm tris - hcl , ph 7.5 , 300 mm nacl , 5 
  mm edta , 1 mm phenylmethylsulfonyl fluoride , 5 g / ml 
  pepstatin , leupeptin , and aprotinin ) , left 10 min on ice , and clarified by 
  centrifugation at 16,000  g for 10 min . soluble proteins were 
  immunoprecipitated with 2 l of the anti - rpt6 , anti - gfp , or anti - htt 
  monoclonal antibodies together with 10 l of protein g - sepharose overnight 
  at 4 c . beads were then washed 3 times in 0.2% nonidet p-40 buffer and 
  resolved by 10% sds - page under reducing conditions , transferred to 
  nitrocellulose membranes , and revealed with corresponding antibodies . gst pulldown assay  gst , gst - rpt6 , and 
  gst - rpt6 were expressed in escherichia coli strain 
  bl21 ( de3 ) plyss and purified by glutathione - sepharose chromatography . for 
  each pulldown experiment , about 0.1 g of gst or gst - rpt6 fusion protein 
  was incubated with htt73 produced in 293 t cells and extracted in 1% nonidet 
  p-40 buffer . after 3 h of incubation at room temperature , beads were washed 3 
  times for 5 min in 1% nonidet p-40 buffer , and complexes were analyzed by 
  sds - page followed by immunoblots . figure 2.overexpressed and endogenous rpts are subunits of two distinct 
  complexes , the 26 s proteasome and apis . a - c , lysates of 
  mock - transfected cells or cells overexpressing rpt6-flag or rpt4-flag were 
  analyzed by glycerol gradients sedimentation . an aliquot of each fraction was 
  analyzed by immunoblots and for peptidase activity with 
  n - succinyl - leu - leu - val - tyr - aminomethylcoumarin as indicated below the 
  immunoblots ( 41 ) . d , fractions 
  2 - 4 ( apis ) and 8 - 9 ( 26 s ) of glycerol gradients similar to those shown in 
  ( a and c ) were pooled , immunoprecipitated , and revealed with 
  indicated antibodies . horseradish peroxidase - conjugated goat anti - mouse 
   were used to reveal the immunoblots , as rpts co - migrate with the heavy 
  chain of the immunoglobulins . overexpressed and endogenous rpts are subunits of two distinct 
  complexes , the 26 s proteasome and apis . a - c , lysates of 
  mock - transfected cells or cells overexpressing rpt6-flag or rpt4-flag were 
  analyzed by glycerol gradients sedimentation . an aliquot of each fraction was 
  analyzed by immunoblots and for peptidase activity with 
  n - succinyl - leu - leu - val - tyr - aminomethylcoumarin as indicated below the 
  immunoblots ( 41 ) . d , fractions 
  2 - 4 ( apis ) and 8 - 9 ( 26 s ) of glycerol gradients similar to those shown in 
  ( a and c ) were pooled , immunoprecipitated , and revealed with 
  indicated antibodies . horseradish peroxidase - conjugated goat anti - mouse 
   were used to reveal the immunoblots , as rpts co - migrate with the heavy 
  chain of the immunoglobulins . glycerol - gradient sedimentation225000 cells were plated in 
  a 5-cm dish and transfected with empty vector or the indicated constructs 24 h 
  after plating . cells were lysed 40 h post - transfection in 200 l of 
  proteasome lysis buffer ( 50 mm tris - hcl , ph 8.0 , 1% triton x-100 , 1 
  mm edta , 1.5 mm mgcl2 , 1 mm 
  dithiothreitol , and 5 mm atp ) . extracts were clarified by 
  centrifugation and fractionated through linear 20 - 40% glycerol gradients in 
  polyallomer tubes of 11  60 mm for 13 h at 40,000 rpm in a sw60 rotor 
  ( beckman coulter ) . note that all the proteasome complexes sedimented below their 
  expected molecular weights , as previously reported 
  ( 25 ) . assays for proteasome activity were 
  performed using the substrate 
  n - succinyl - leu - leu - val - tyr - aminomethylcoumarin as described in 
  kisselev and goldberg 
  ( 41 ) . aggregation assays  gst - htt50 ( 0.5 m ) 
  was incubated with or without purified 19s proteasome ( 70 nm ; 
  boston biochem ) in the presence of 10 units of thrombin ( amersham biosciences ) 
  at 37 c with 300 rpm shaking in 50 mm tris - hcl , ph 7.5 , 150 
  mm nacl , 10% glycerol , 20 mm mgcl2 with or 
  without 5 mm atp or 15 mm atps . reactions were 
  stopped at the indicated times by adding an equal volume of laemmli buffer and 
  boiling for 5 min . aliquots corresponding to 50 ng of gst fusion protein were 
  analyzed by sds - page stained with coomassie brilliant blue , and filter 
  retardation assays revealed with htt antibody . a , lysates of cells expressing increasing amounts of htt73 were 
  either boiled in 1% sds or fractionated into np40 supernatant and pellet and 
  analyzed by immunoblotting with htt antibody . b - d , htt73 was 
  co - expressed with increasing amounts of rpt4 or rpt6 , and whole cell lysates 
  or supernatants and pellets were analyzed by immunoblotting with htt , rpt6 , or 
  rpt4 antibodies . confocal micrographs of cells expressing htt73-gfp 
  either alone or together with rpt6 or rpt4 are shown . arrows show cells with small inclusions , and 
  asterisks show cells with large inclusions . f , micrographs 
  of primary neurons expressing htt17-gfp and htt73-gfp alone or together with 
  rpt6 . neuronal identity was confirmed by staining with tau-1 antibody , and 
  nuclei are revealed with h33258 . a , lysates of cells expressing increasing amounts of htt73 were 
  either boiled in 1% sds or fractionated into np40 supernatant and pellet and 
  analyzed by immunoblotting with htt antibody . b - d , htt73 was 
  co - expressed with increasing amounts of rpt4 or rpt6 , and whole cell lysates 
  or supernatants and pellets were analyzed by immunoblotting with htt , rpt6 , or 
  rpt4 antibodies . confocal micrographs of cells expressing htt73-gfp 
  either alone or together with rpt6 or rpt4 are shown . arrows show cells with small inclusions , and 
  asterisks show cells with large inclusions . f , micrographs 
  of primary neurons expressing htt17-gfp and htt73-gfp alone or together with 
  rpt6 . neuronal identity was confirmed by staining with tau-1 antibody , and 
  nuclei are revealed with h33258 . aggregation of peptides with a long poly(q ) stretch can occur spontaneously 
  in vitro , but in yeast aggregation of the amino - terminal fragment of 
  htt requires the presence of the chaperone hsp104 
  ( 42 ) . although chaperones are 
  widely conserved throughout evolution , there is no mammalian orthologue of 
  hsp104 . the typical feature of hsp104 and the bacterial atpase clpb proteins 
  is the presence of two aaa domains and a middle region or m - domain , adopting a 
  coiled - coil structure essential for the hsp104/clpb protein remodeling 
  function ( 43 , 
  44 ) . aaa atpases in available 
  databases were examined aiming to identify human proteins containing both a 
  coiled - coil and an aaa domain . we found that only the atpase subunits of the 
  19s proteasome , rpt6 , rpt4 , rpt3 , rpt2 , and rpt1 , exhibit these specific 
  features . interestingly , an immunohistochemical study of brain tissue from 
  patients with spinocerebellar ataxia type 3 ( sca-3 ) has revealed that poly(q ) 
  inclusions co - localize with subunits of the 19s proteasome but rarely of the 
  20s catalytic particle ( 45 ) . these observations together with the unfoldase function of the 19s atpases in 
  protein degradation prompted us to investigate whether rpts contribute to 
  inclusion formation . to determine whether co - localization of regulatory subunits of the 
  proteasome with poly(q ) inclusions was a generic feature of distinct poly(q ) 
  disorders , we analyzed the localization of several proteasome subunits in 
  nerve cells containing mutant htt inclusions . in primary neurons , rpt6 
  staining was extremely intense around poly(q ) aggregates , in contrast to its 
  diffuse and mostly nuclear localization in untransfected cells 
  ( fig . the other 19s atpases were 
  also enriched around poly(q ) inclusions 
  ( fig . in 
  contrast , the 20s proteasome was not enriched around the poly(q ) inclusions , 
  as revealed by immunostaining with antibodies directed against the 20s 
  subunit , 7 . these observations reveal that rpt subunits co - localize 
  with mutant htt inclusions in neuronal cells independently of the 20s 
  proteasome . to determine whether these observations were relevant to hd , we 
  stained cortical sections of post - mortem hd brains with htt and rpt6 or 
  7 ( 20s ) antibodies . we performed this analysis on brain tissue from two 
  juvenile hd patients to examine a large number of cytoplasmic and nuclear 
  inclusions . like primary neurons expressing mutant htt , all the inclusions 
  from both hd patients were intensely labeled with rpt6 regardless of their 
  subcellular localization ( fig . in contrast , only a small 
  fraction of htt inclusions ( 13% ) exhibited a light staining for the 20s 
  subunit ( fig . these results indicate that the chaperone subunits co - localize with hd 
  inclusions largely independently of the 20s subunits . figure 4.the coiled - coil region is essential for the activity of rpt6 on 
  htt73 . a , schematic representation of rpt6 domain organization 
  and coiled - coil mutant analyzed in this study . b , lysates of cells 
  expressing htt73 alone or together with increasing amounts of rpt6 wild type 
  or rpt6 deletion mutant were analyzed by immunoblots and 
  filter retardation assays . the coiled - coil region is essential for the activity of rpt6 on 
  htt73 . a , schematic representation of rpt6 domain organization 
  and coiled - coil mutant analyzed in this study . b , lysates of cells 
  expressing htt73 alone or together with increasing amounts of rpt6 wild type 
  or rpt6 deletion mutant were analyzed by immunoblots and 
  filter retardation assays . seeking further evidence for the existence of rpt subunits independently of 
  the proteasome , we analyzed the distribution of proteasome subunits from cell 
  lysates by glycerol gradient fractionation . 19s and 20s subunits as well as 
  the proteolytic activity of the proteasome were localized in high molecular 
  weight fractions 8 and 9 , indicating that these fractions contained the 26s 
  proteasome ( fig . 2a ) . in addition to the 26s proteasome , 19s subunits were also found in lower 
  molecular weight fractions devoid of 20s subunits and proteolytic activity 
  ( fig . this sedimentation profile is reminiscent of the gel filtration profile 
  of the free 19s - like complex 
  ( 25 ) , also called apis complex 
  ( atpase proteins independent of 20s ) 
  ( 22 ) . thus , in mammalian cells 
  rpts are subunits of 2 distinct complexes , the 26s proteasome and apis or free 
  19s - like complex . the observation that inclusions of expanded poly(q)-containing proteins 
  co - localize with rpts independently of the proteasome 
  ( fig . 1 and 
  ( 45 ) suggested that apis could 
  be involved in inclusion formation . we , therefore , attempted to increase the 
  abundance of this complex by overexpressing some of its subunits . the 
  sedimentation profile of overexpressed rpt4 and rpt6 was similar to the 
  sedimentation profile of the endogenous subunits . in fractions 8 and 9 of the 
  glycerol gradients , rpt6 antibody revealed 2 bands corresponding to the 
  endogenous protein and the overexpressed flag - tagged rpt6 , indicating that 
  overexpressed rpt6 was a subunit of the 26s proteasome complex 
  ( fig . a large 
  fraction of overexpressed rpt6 was detected in fractions 2 - 4 of the gradient 
  together with the apis complex . similarly , a fraction of overexpressed rpt4 
  entered the 26s proteasome , whereas most of the protein was found in the 
  smaller apis complex ( fig . 
  2c ) . note that overexpression of rpt4 or rpt6 did not 
  alter the sedimentation of the endogenous proteasome nor its proteolytic 
  activity ( fig . 2 , a - c , 
  and data not shown ) . to confirm these observations , immunoprecipitations were 
  carried out with rpt6 antibodies on glycerol gradient fractions containing 
  either apis ( fractions 2 - 4 ) or the 26s complex ( fractions 8 - 9 ) . both 
  endogenous and overexpressed rpt4 were detected in rpt6 immunoprecipitates 
  performed on fractions 2 - 4 as well as fractions 8 - 9 of the glycerol gradients 
  ( fig . the 
  amounts of rpt3 immunoprecipitated in the endogenous complexes were probably 
  below the detection limit of the rpt3 antibody . altogether , these findings 
  establish that endogenous and overexpressed rpt4 and rpt6 are engaged in two 
  complexes , the 26s proteasome and apis . most of the overexpressed rpt6 and 
  rpt4 co - sedimented with apis and recruit other subunits of the complex . we , 
  thus , used this experimental setting to analyze whether rpts modulated htt 
  misfolding and aggregation . figure 5.rpt6 , but not rpt6 lacking the coiled - coil region , binds to htt73 . 
 a , lysates of cells transfected with empty vector and htt73 alone or 
  together with rpt6 analyzed by immunoblot ( left panel ) or 
  immunoprecipitated ( ip ) with htt or gfp antibody followed by rpt6 
  immunoblot . b , gst , gst - rpt6 , and gst - rpt6 fusion 
  proteins were expressed in e. coli , purified on glutathione - sepharose 
  beads , separated by sds - page , transferred onto nitrocellulose , and stained 
  with ponceau red . c , htt73 containing cell lysates ( input ) were 
  incubated on beads containing gst , gst - rpt6 , or gst - rpt6 and 
  washed , and complexes bound to beads were revealed with htt antibody . rpt6 , but not rpt6 lacking the coiled - coil region , binds to htt73 . 
 a , lysates of cells transfected with empty vector and htt73 alone or 
  together with rpt6 analyzed by immunoblot ( left panel ) or 
  immunoprecipitated ( ip ) with htt or gfp antibody followed by rpt6 
  immunoblot . b , gst , gst - rpt6 , and gst - rpt6 fusion 
  proteins were expressed in e. coli , purified on glutathione - sepharose 
  beads , separated by sds - page , transferred onto nitrocellulose , and stained 
  with ponceau red . c , htt73 containing cell lysates ( input ) were 
  incubated on beads containing gst , gst - rpt6 , or gst - rpt6 and 
  washed , and complexes bound to beads were revealed with htt antibody . nucleation is the rate - limiting step of poly(q ) aggregation in 
  vitro ( 31 ) . to determine 
  whether similar features could be observed in cells , we expressed large 
  amounts of htt73 and performed biochemical fractionation . we found that a 
  10 - 25-fold increase in the sds - soluble protein leads to a very modest increase 
  of nonidet p-40-insoluble , presumably misfolded htt73 
  ( fig . thus , cells 
  can accumulate a large amount of nonidet p-40-soluble htt73 , suggesting that 
  aggregation requires not only a high protein concentration but also additional 
  events . because the function of rpts is to unfold proteins and because they 
  co - localize with poly(q ) inclusions , we asked whether these unfoldases might 
  assist the conversion from soluble to aggregated mutant htt . increasing the 
  levels of rpt6 or rpt4 led to a remarkable and dose - dependent increase of 
  htt73 when low amounts of htt73 were co - transfected with rpt6 or rpt4 
  ( fig . 3 , b - d , 
  lanes 1 , 4 , and 8 , compared with 
  fig . biochemical fractionation studies revealed that 
  nearly all the sds - soluble htt73 that accumulated upon rpt6 or rpt4 
  overexpression was nonidet p-40-insoluble , suggesting that it was in a 
  pre - aggregated misfolded state ( fig . in contrast , when large amounts of htt73 were 
  transfected alone , cells contained very high levels of soluble htt73 
  ( fig . these 
  experiments revealed that rpt6 and rpt4 convert soluble htt into an insoluble 
  species . to further characterize the effect of rpt6 and rpt4 on htt73 , htt73 
  fused to gfp was overexpressed either alone or together with rpt6 or rpt4 
  ( fig . although 
  cells expressing htt73-gfp alone contained either diffuse fluorescence or 
  small fluorescent foci , cells co - expressing htt73-gfp and rpt6 or rpt4 
  contained large inclusions ( fig . this was also observed in primary neuronal cells 
  expressing htt73-gfp alone or together with rpt6 , 3f ) . northern blot analyses were carried out with cells co - expressing rpt6 
  with htt73 and revealed that the effects of rpt6 overexpression on htt 
  accumulation were not due to changes at the level of htt mrna ( supplemental 
  fig . previous studies have indicated that rpt subunits of the 19s have 
  distinct functions ( 46 ) . to 
  determine whether the effect on mutant htt aggregation was a general property 
  of all rpts or was restricted to rpt6 and rpt4 , htt 73 was overexpressed with 
  the other rpts . increasing the levels of rpt6 or rpt4 led to a remarkable and 
  dose - dependent increase of aggregated htt73 , as revealed by immunoblots and 
  filter retardation assays ( supplemental fig . 2 ) . overexpression of rpt3 
  modestly enhanced aggregation of htt73 , in contrast to rpt2 , rpt1 , and rpt5 , 
  which had no significant effect ( supplemental fig . this indicated that 
  overexpression of an individual rpt was not sufficient to trigger htt 
  aggregation but , rather , that the effects of rpt6 and rpt4 on htt73 were 
  specific . because cellular protein concentration varies with cell density and 
  equal cell numbers were seeded in each experiment , the fact that the levels of 
  the cellular protein vimentin did not vary in the different conditions 
  revealed that none of the transfected constructs elicited cell death 
  ( supplemental fig . this is in agreement with previously studies that 
  established that mutant htt is not toxic 2 days post - expression 
  ( 47 , 
  48 ) . we next sought to 
  determine which domains were important for the activity of rpt6 . the amino 
  terminus of rpt6 contains a coiled - coil domain , a region which was found 
  important for the chaperone function of other aaas 
  ( 43 ) . therefore , the 
  amino - terminal 79 amino acids of rpt6 containing the coiled - coil region were 
  deleted , generating a protein fragment , rpt6 . overexpression 
  of increasing doses of rpt6 together with htt73 augmented the levels of htt73 , 
  detected on immunoblots , by 1.3- , 3- , and 7-fold and the levels of aggregated 
  htt73 , detected on filter retardation assays by 2- , 5- , and 7-fold . in contrast , rpt6 lacking the coiled - coil region , expressed at a level similar 
  to the full - length protein , was devoid of any activity on htt73 
  ( fig . 4 ) . this result reveals 
  that the coiled - coil region of rpt6 is essential for its function on htt 
  aggregation . figure 6.rpt6 and rpt4 enhance misfolding of proteins containing expanded poly(q ) 
  independently of proteolysis . a , immunoblots and filter 
  retardation assays of cells overexpressing sca3 22q or 82q alone or together 
  with rpt6 and either left untreated or treated with 1 m 
  epoxomycin ( epoxo ) for 14 h. b , 293 t cells were transfected 
  with htt73 alone or together with rpt6 and rpt4 encoding plasmids and either 
  left untreated or treated with 0.5 or 1 m epoxomycin for 14 h 
  and analyzed by immunoblots and filter retardation assays . rpt6 and rpt4 enhance misfolding of proteins containing expanded poly(q ) 
  independently of proteolysis . a , immunoblots and filter 
  retardation assays of cells overexpressing sca3 22q or 82q alone or together 
  with rpt6 and either left untreated or treated with 1 m 
  epoxomycin ( epoxo ) for 14 h. b , 293 t cells were transfected 
  with htt73 alone or together with rpt6 and rpt4 encoding plasmids and either 
  left untreated or treated with 0.5 or 1 m epoxomycin for 14 h 
  and analyzed by immunoblots and filter retardation assays . the results presented so far indicate that rpt6 and rpt4 assist mutant htt 
  misfolding and aggregation . we next tested whether this effect involved a 
  direct interaction between rpt6 and htt73 , as suggested by co - localization 
  studies ( fig . coimmunoprecipitation experiments were carried out using cells expressing 
  htt73 alone or together with rpt6 . 5a ) . to determine whether rpt6 and htt73 interact 
  directly , recombinant rpt6 , either wild type or lacking the coiled - coil 
  region , was produced in e. coli as a fusion with gst and immobilized 
  on glutathione beads . nonidet p-40 lysates containing htt73 produced in 293 t 
  cells were then incubated with gst and gst - rpt6 . 5b ) . deleting rpt6 coiled - coil region abolished its 
  ability to bind htt . these interaction data suggest that the effect of rpt6 on 
  htt aggregation is mediated through a direct interaction and requires the 
  coiled - coil region . because a fraction of rpts were found independently of the proteasome in 
  sca-3 ( 45 ) , we tested whether 
  overexpressing rpt6 affected accumulation of ataxin-3 , a protein unrelated to 
  htt , which contains a poly(q ) expansion in sca-3 . rpt6 overexpression 
  dramatically enhanced mutant ataxin-3 aggregation but had no effect on 
  wild - type ataxin ( fig . previous studies have reported that proteasome 
  inhibition increases accumulation and aggregation of htt 
  ( 6 - 8 ) . therefore , we next analyzed whether rpt6 or rpt4 overexpression generally 
  impaired proteasomal degradation , thereby enhancing accumulation of expanded 
  poly(q ) . epoxomycin , which blocks the catalytic activity of the 20s 
  proteasome , provoked accumulation of both sds - soluble and aggregated htt73 or 
  aggregated mutant ataxin-3 ( fig . a and b ) . similarly to epoxomycin treatment , 
  rpt6 or rpt4 overexpression enhanced htt73 accumulation and aggregation 
  ( fig . 6b ) . in 
  contrast , we found that overexpressing these 19s subunits did not stabilize 
  the short - lived proteins e2f-1 and p21 
  ( fig . 6b ) , whereas 
  inhibition of the proteolytic activity of the proteasome markedly stabilize 
  these two short - lived proteins ( fig . these results indicate that rpt6 or rpt4 
  overexpression does not impair proteasomal degradation . having shown that increasing the levels of rpt4 and rpt6 promotes mutant 
  htt misfolding , we next tested whether we could interfere with the endogenous 
  proteins in primary neurons . two sirnas targeting rpt6 were found to 
  significantly reduce the levels of rpt6 by 25 and 40% , respectively . similarly , two sirnas designed against rpt4 reduced the levels of the protein 
  by 50% ( fig . 7a ) . 
  we found that reducing the levels of rpt6 or rpt4 substantially reduced the 
  number of cells containing poly(q ) inclusions when we examined cells 
  expressing mutant htt together with sirna targeted against rpt6 or rpt4 
  ( fig . 70% of neurons expressing mutant htt contained 
  inclusions , and 30% of cells exhibited diffuse fluorescence . this ratio was 
  inverted in cells transfected with rpt4 or rpt6 sirnas 
  ( fig . the number 
  of cells expressing mutant htt was very similar when the levels of rpt4 and 
  rpt6 were reduced and in control cells , indicating that the sirnas were not 
  toxic under our experimental conditions ( data not shown ) . this analysis 
  demonstrates that reducing the levels of rpt4 or rpt6 in primary neurons 
  significantly decrease inclusion formation . a , immunoblots showing rpt6 , rpt4 , and vimentin in 
  extracts of primary neurons transfected with control , rpt6 , or rpt4 sirnas . ratio of rpt6 and rpt4 proteins , normalized to vimentin , in protein extracts 
  of primary neurons transfected with control , rpt6 , or rpt4 sirnas . 
  quantifications were performed on three independent experiments . sirna_2 
  together with htt73-gfp , fixed 24 h post - transfection , and labeled with tau-1 
  antibody . c , ratio of primary neurons transfected with control , 
  rpt6 , or rpt4 sirnas together with htt73-gfp , expressed as percentage of 
  transfected cells . data were scored from 10 independent experiments 
  corresponding to more than 4000 cells in total . , p < 
  0.01 ; , p < 0.02 . a , immunoblots showing rpt6 , rpt4 , and vimentin in 
  extracts of primary neurons transfected with control , rpt6 , or rpt4 sirnas . ratio of rpt6 and rpt4 proteins , normalized to vimentin , in protein extracts 
  of primary neurons transfected with control , rpt6 , or rpt4 sirnas . sirna_2 
  together with htt73-gfp , fixed 24 h post - transfection , and labeled with tau-1 
  antibody . c , ratio of primary neurons transfected with control , 
  rpt6 , or rpt4 sirnas together with htt73-gfp , expressed as percentage of 
  transfected cells . data were scored from 10 independent experiments 
  corresponding to more than 4000 cells in total . , p < 
  0.01 ; , p < 0.02 . overexpressed rpt6 and rpt4 recruited other rpts 
  ( fig . 2d ) , suggesting 
  that their stimulatory activity on htt misfolding was mediated by a complex 
  containing rpts . to directly examine this hypothesis , we next tested whether 
  purified 19s proteasome could modulate htt aggregation in vitro . aggregation of mutant htt was monitored by filter retardation assay as 
  described in scherzinger et al . cleavage of gst - htt 
  released the gst moiety and initiated htt aggregation 
  ( fig . the addition of substoichiometric amounts of purified 
  19s proteasome and atp strongly enhanced aggregation of mutant htt 
  ( fig . the 
  stimulatory effect of 19s proteasome on mutant htt aggregation in 
  vitro was significantly decreased by atp-s , a non - hydrolysable atp 
  analogue ( fig . the addition of 19s proteasome with or without atps did not affect the 
  cleavage of gst - htt ( fig . this suggests that the stimulatory activity of the 
  19s proteasome on htt aggregation is mediated by its unfoldase function . altogether , the results presented here reveal that atpases of the 19s 
  proteasome function non - proteolytically in facilitating misfolding and 
  aggregation of proteins with a poly(q ) expansion . because htt inclusions are 
  ubiquitinated , htt is likely to be addressed to the proteasome by a 
  ubiquitin - dependent pathway ( 3 , 
  4 ) . we next investigated what 
  would be its fate if targeted to the proteasome by the degradation pathway 
  utilized by the evanescent protein odc 
  ( 49 ) . the 37 carboxyl - terminal 
  amino acids of odc have been found to be an efficient and autonomous 
  destabilizing sequence when fused to heterologous proteins 
  ( 49 ) . htt73 and 
  htt73odc were expressed in 293 t cells . although both soluble and aggregated 
  htt73 were readily detected by immunoblots and filter retardation assays , 
  htt73odc was hardly detectable , even when large amounts of dna encoding this 
  protein were transfected , suggesting that the protein was very rapidly and 
  efficiently degraded ( fig . we confirmed that htt73odc was fully degraded and 
  did not escape our detection ( supplemental fig . 3 ) . when the proteolytic 
  capacity of the proteasome was compromised , htt73odc was stabilized , 
  confirming that htt73odc is a proteasome substrate 
  ( fig . the fact that htt73odc is insensitive to rpt6 overexpression but 
  is stabilized by inhibition of the catalytic activity of the proteasome 
  further supports the conclusion that rpt6 overexpression does not perturb 
  proteasomal degradation . when the ability of the proteasome to degrade 
  proteins is compromised , small but detectable amounts of htt73odc aggregates 
  were found by means of their retention on a cellulose acetate filter 
  ( fig . these 
  observations suggest that , when the catalytic activity of the proteasome is 
  compromised , a small amount of htt73odc recovers its ability to aggregate . to 
  obtain further insight into the mechanism by which the odc carboxyl terminus 
  destabilizes mutant htt , we mutated cysteine 441 to alanine , as this point 
  mutation has been shown to stabilize odc 
  ( 49 ) . in contrast to htt73odc , 
  htt73odcc441a was readily detectable , and its aggregation increased 
  upon rpt6 overexpression ( fig . 9 , 
  c and d ) . the finding that the odc destabilizing 
  sequence converts htt73 into an evanescent protein indicates that the 
  proteolytic capacity of the proteasome is not the limiting factor for mutant 
  htt degradation . figure 8.the 19s enhances mutant htt aggregation in vitro . 
  a , time course of proteolysis of gst - htt50c with or without 
  19s and atps . 0.5 m gst - htt50 was cleaved with 10 
  units of thrombin in the absence or presence of 70 nm 19s and 15 
  mm atps . aliquots of the reaction were analyzed after 3.5 , 
  6 , or 8 h at 37 c by sds - page stained with coomassie brilliant blue . b , samples shown in a 
  were analyzed by filter retardation assays and revealed with htt 
  antibodies . the 19s enhances mutant htt aggregation in vitro . 
  a , time course of proteolysis of gst - htt50c with or without 
  19s and atps . 0.5 m gst - htt50 was cleaved with 10 
  units of thrombin in the absence or presence of 70 nm 19s and 15 
  mm atps . aliquots of the reaction were analyzed after 3.5 , 
  6 , or 8 h at 37 c by sds - page stained with coomassie brilliant blue . b , samples shown in a 
  were analyzed by filter retardation assays and revealed with htt 
  antibodies . in this study we report on the finding that in cells aggregation of htt 
  amino - terminal fragments with a pathogenic poly(q ) expansion is dependent on a 
  remodeling event mediated by rpt6 and rpt4 . proteasomal chaperones are 
  subunits of 2 complexes , the 26s proteasome and the previously reported apis 
  or 19s - like complex ( this study and refs . 
  22 and 
  25 ) . although the function of 
  the proteasome in protein degradation is well recognized , there is also 
  growing evidence indicating that 19s atpases , in particular rpt6 , function 
  independently of degradation 
  ( 50 ) . in hd patients , 
  inclusions are often found in the nucleus , where a non - proteolytic function of 
  the 19s atpases has been extensively reported . a remodeling activity of rpts 
  independently of degradation provides a unifying mechanism to reconcile their 
  diverse functions . conditions provoking the dissociation of the 19s from the 
  20s have been reported previously . binding 
  of alternative rp such as pa28 to the cp also provokes dissociation of the 
  atpases ( 17 ) . intriguingly , 
  pharmacological inhibition of the catalytic function of the proteasome 
  stimulates binding of pa28 to the proteasome 
  ( 17 ) , suggesting that free 19s 
  particles may be released by this treatment . what precisely modulates the 
  relative abundance of 26s proteasome and apis ( or free 19s - like complex ) 
  remains to be determined . however , it is noteworthy that aging was reported to 
  promote proteasome disassembly in flies 
  ( 51 ) . in neurons of hd ( this study ) and sca-3 
  ( 45 ) brains as well as in 
  primary neurons expressing mutant htt and rpts , but not 20s subunits , 
  co - localize with poly(q ) inclusions , indicating that rpts are not associated 
  with the catalytic particles in poly(q ) inclusions . this suggests that rpts 
  may be involved in inclusion formation independently of the proteolytic 
  function of the proteasome . in support of this hypothesis , we found that 
  overexpression of rpt6 , rpt4 , and more modestly rpt3 enhance mutant htt and 
  sca-3 aggregation . because the majority of overexpressed rpt4 and rpt6 
  co - sediment with apis , we propose that rpt4 and rpt6 remodel proteins with a 
  poly(q ) expansion and thereby elicit their aggregation independently of 
  degradation . overexpressing rpt6 or rpt4 has no effect on the levels of 
  vimentin or the short - lived e2f-1 , p21 , and htt7odc , showing unambiguously 
  that rpt6 or rpt4 overexpression does not impair proteasome degradation . more 
  importantly , reducing the levels of rpt6 or rpt4 in primary neurons decreases 
  inclusion formation . together , these results indicate that endogenous rpt6 and 
  rpt4 enhance htt misfolding , and this effect is likely mediated through an 
  unfoldase function of these proteins . in vitro reconstitution 
  experiments support this model ; substoichiometric amounts of 19s complex 
  promote mutant htt aggregation , and this activity is markedly attenuated by 
  atps . htt supplied in the in vitro aggregation assay is not 
  ubiquitinated , suggesting that the 19s does not required ubiquitination of htt 
  to recognize and remodel htt . thus , ubiquitination does not seem to be 
  required for the aggregation process per se . however , it is possible 
  that in cells ubiquitination of htt plays a role in targeting htt to the 
  proteasome . this is in agreement with previous in vitro studies on 
  the chaperone function of the proteasome 
  ( 52 ) . when the domain 
  composition of rpts was carefully examined , we found that the three atpases 
  enhancing poly(q ) aggregation , rpt6 , rpt4 , and rpt3 , possess a coiled - coil 
  domain of nearly 50 amino acids . in contrast , the 19 s atpases unable to 
  enhance poly(q ) aggregation have either no coiled - coil region ( rpt5 ) or only a 
  short one ( rpt1 , rpt2 ) according to domain predictions with the web - based tool 
  smart . the importance of the equivalent m - region of the bacterial 
  atpase clpb and the yeast hsp104 suggested that this finding might not be 
  fortuitous . we , thus , deleted the coiled - coil region in rpt6 and found that 
  although rpt6 was expressed at levels similar to the 
  full - length protein , this mutant lacking the coiled - coil domain was inactive 
  toward poly(q ) aggregation ( fig . 
  4 ) . in agreement with this result , rpt6 lacking the coiled - coil 
  region was no longer able to bind mutant htt 
  ( fig . , our findings suggest that in the disease 
  process , the formation of misfolded and presumably toxic species may be 
  assisted by rpt6 and rpt4 . a recent study has revealed that tau inclusions , 
  the pathological hallmark of alzheimer disease , pick disease , and other 
  tauopathies , are immunoreactive for rpt3 , raising the question of whether the 
  findings reported here for htt and sca-3 may be a generic feature of many 
  neurodegenerative diseases 
  ( 53 ) . a , lysates of cells transfected with increasing amounts 
  of htt73 or htt73odc encoding plasmids analyzed by immunoblots . b , 
  cells were transfected with htt73 ( left panel ) or htt73odc ( right 
  panel ) alone or together with increasing amounts of rpt6 encoding plasmid 
  and left untreated or treated with 0.25 , 0.5 and 1 m epoxomycin 
  ( epoxo ) for 14 h. lysates were analyzed by immunoblots or by filter 
  retardation assays . c , cells transfected with increasing amounts of 
  htt73odc or htt73odcc441a were analyzed by immunoblots . d , 
  cells were transfected with htt73odcc441a alone or together with 
  increasing amounts of rpt6 encoding plasmid and left untreated or treated with 
  0.25 , 0.5 , and 1 m epoxomycin for 14 h. lysates were analyzed 
  by immunoblots or by filter retardation assays revealed with htt antibody . e , possible routes followed by htt : degradation by a proteasome 
  complex ( this study and ref . 
  13 ) or misfolding and 
  aggregation by apis or 19 s - like complex . a , lysates of cells transfected with increasing amounts 
  of htt73 or htt73odc encoding plasmids analyzed by immunoblots . b , 
  cells were transfected with htt73 ( left panel ) or htt73odc ( right 
  panel ) alone or together with increasing amounts of rpt6 encoding plasmid 
  and left untreated or treated with 0.25 , 0.5 and 1 m epoxomycin 
  ( epoxo ) for 14 h. lysates were analyzed by immunoblots or by filter 
  retardation assays . c , cells transfected with increasing amounts of 
  htt73odc or htt73odcc441a were analyzed by immunoblots . d , 
  cells were transfected with htt73odcc441a alone or together with 
  increasing amounts of rpt6 encoding plasmid and left untreated or treated with 
  0.25 , 0.5 , and 1 m epoxomycin for 14 h. lysates were analyzed 
  by immunoblots or by filter retardation assays revealed with htt antibody . e , possible routes followed by htt : degradation by a proteasome 
  complex ( this study and ref . 
  13 ) or misfolding and 
  aggregation by apis or 19 s - like complex . monomeric pure poly(q ) peptides are in a random coil conformation 
  ( 54 ) , but the addition of the 
  polyproline domain present in the carboxyl terminus of the poly(q ) stretch in 
  htt imposes structural constrain to the protein , which adopts a proline type 
  ii helical conformation ( 36 ) . this explains why the proline - rich region of htt antagonizes the toxicity of 
  the poly(q ) expansion ( 34 , 
  35 ) . thus , a remodeling event 
  ought to be required to alleviate the inhibitory function of the proline - rich 
  region and to elicit the conformational transition into the -sheet 
  conformation . in agreement with biophysical data , we found that in cells large 
  amounts of htt fragments with expanded poly(q ) can be expressed in a soluble 
  state ( this study and ref . this implies that 
  aggregation is not just a consequence of an increase in concentration but is 
  dependent in the cellular milieu on additional events . the data presented here 
  indicate that proteasomal chaperones facilitate the transition from soluble to 
  aggregated proteins with a poly(q ) expansion . because inhibition of the proteolytic activity of the proteasome increases 
  htt aggregation , it has been proposed that aggregates form as a consequence of 
  an impairment of the proteolytic function of the proteasome . however , two 
  conflicting studies question whether purified proteasomes can degrade poly(q ) 
  peptides in vitro ( 9 , 
  13 ) . here we show that the 
  addition of wild type but not mutant odc destabilizing sequence to htt with a 
  poly(q ) expansion profoundly destabilizes this aggregation - prone protein . it 
  is noteworthy that under certain conditions odc can be degraded by the 20s 
  proteasome ( 56 ) . although the 
  mechanism by which wild - type but not mutant odc carboxyl terminus destabilizes 
  htt remains to be determined , it is likely that htt - odc is degraded by the 
  same ubiquitin - independent pathway as odc . thus , the route by which htt is 
  targeted to the proteasome determines its fate 
  ( fig . furthermore , the fact that the addition of the odc destabilizing sequence 
  converts mutant htt into a short - lived protein shows unambiguously that , in 
  cells , the proteolytic capacity of the 20s proteasome is not a factor limiting 
  mutant htt degradation . we propose that misfolding and aggregation of proteins 
  with poly(q ) expansion arise as a consequence of a remodeling event elicited 
  by a non - proteolytic function of proteasomal chaperones . in conclusion , the data presented here reveal that aggregation of proteins 
  with poly(q ) expansion requires a transition from a benign to an 
  aggregation - prone conformation , a process which in cells is facilitated by 
  proteasomal chaperones . aggregation of proteins with a poly(q ) expansion is , 
  thus , reminiscent of a typical folding reaction , requiring the assistance of 
  chaperones .