a. nidulans  palb gene replacements were carried out 
  using mad1764 ( pyrg89 pyroa4 inob2 nkua::bar pacc900 
  palb::pyroa ) , a palb strain in 
  which the complete coding region of palb had been substituted by 
  pyroa this strain carries pacc900 encoding 
  myc3-pacc 
  ( 16 , 
  18 ) . palb::(ha)3::pyrg , 
  palb::ha3::pyrg , 
  and vps24 : : 
  palb::ha3::pyrg 
  transgenes , flanked by 5-utr and 3-utr palb sequences 
  to drive homologous recombination , were obtained as noti - clai linear fragments 
  from plasmids p1725 , p1727 , and p1786 , respectively . the resulting 
  palb gene - replaced alleles were denoted palb800 [ palb : : 
  ( ha)3::pyrg ] , palb801 
  [ palb::ha3::pyrg ] , 
  and vps24::palb 
  [ vps24::palb::ha3::pyrg ] . gene - replaced transformants were identified by their pyrimidine - independent , 
  pyridoxine - dependent growth and verified by southern . the palb801 
  phenotype ( or the wild - type phenotype for palb800 and 
  vps24::palb ) co - segregated 
  with the recombinant allele , ruling out the possibility of the presence of 
  spurious mutations . ph shift experiments and a. nidulans protein 
  extracts were as described 
  ( 18 ) . recombinant strains were 
  confirmed to carry the expected single - copy integration events by southern 
  analysis . subcellular fractionation experiments  these were carried out 
  at 4 c following a s. cerevisiae protocol 
  ( 48 , 
  49 ) . mycelia cultured in 
  minimal medium ( 50 ) were 
  protoplasted with glucanex ( novo ch-4243 ) . protoplasts were resuspended in 
  lysis buffer ( 0.2 m sorbitol , 50 mm potassium acetate , 2 
  mm edta , 20 mm hepes ph 7.2 and protease inhibitor 
  mixture ( roche applied science ) ) and lysed using a dounce homogenizer . the 
  supernatant was then centrifuged at 13,000  g for 15 min , 
  which resulted in a p13k pellet fraction and a supernatant . this supernatant 
  was centrifuged at 100,000  g for 60 min , using a beckman 
  tla100 micro - ultracentrifuge , which resulted in a p100k pellet fraction and 
  s100 supernatant soluble fraction . the s100 , as well as the p13k and p100k 
  pellets , which were resuspended in lysis buffer , were trichloroacetic 
  acid - precipitated and resuspended in sds - page loading buffer . physiological levels of ( flag)3-pep12 were driven by the 
  pep12 promoter from a single - copy 
  flag3::pep12::pyrg transgene integrated into 
  the pep12 locus . two - hybrid analyses and gst pull - down assays using a. nidulans 
  extracts  yeast two - hybrid assays and plasmids encoding gst - gfp and 
  gst - vps32 fusion protein have been described 
  ( 28 ) . pgex-2t - derivatives 
  encoding wild type and mutant ( l222d , l223d ) gst - vps24 and gst - didb were 
  constructed by standard techniques and quikchange ( stratagene ) mutagenesis . gst fusion proteins were expressed in e. coli dh1 for 24 h at 20 
  c after induction with 
  isopropyl-1-thio--d - galactopyranoside and purified from 
  lysates clarified by centrifugation for 20 min at 13,000 rpm and 4 c in a 
  sorvall ss-34 rotor as described 
  ( 51 ) . gst baits were mixed 
  with 1 mg of total soluble protein extract from palb800 or 
  palb801 strains in boa buffer ( 10 mm tris - hcl ph 8.0 , 150 
  mm nacl , 1 mm edta , 5 mm dithiothreitol , 
  0.05% ( v / v ) triton x-100 , and roche 's protease inhibitor mixture ) , using 
  0.8-ml handee spin columns ( pierce ) , and rotated at 4 c in the presence 
  of 25 l of glutathione - sepharose ( amersham biosciences ) . beads were washed 
  six times with boa buffer , and the bound proteins were eluted after boiling in 
  sds - page loading buffer . replicate gels were stained with coomassie blue and 
  analyzed by western blot , using anti - ha antibody . gst pull - down assays using purified 
  proteins[s]palb or [ s]pala preys were 
  synthesized in vitro using the promega tnt system ( 20 
  ci of [ s]met per reaction ) . 20 l of glutathione - sepharose 
  beads loaded with the corresponding gst bait were incubated at 4 c with 
  the s - labeled prey in a total volume of 0.5 ml of 1% triton x-100 
  boa buffer . beads were washed , and the bound material eluted as above was run 
  in 10% polyacrylamide gels , which were coomassie blue - stained , dried , and 
  autoradiographed , using a kodak biomax amplifying screen . igg sepharose pull - downs with purified 
  proteins  zz - palb is a peptide consisting of two 
  protein a igg binding z domains fused to the palb n - terminal 129 residues 
  containing the mit domain . ( his6)-vps24 was 
  purified by ni affinity chromatography using nta his - bind resin 
  ( novagen ) . zz - palb was purified from cells lysed in the french 
  press in a buffer containing 20 mm tris - hcl , ph 7.9 , 300 
  mm nacl , 5 mm imidazole , 0.1% triton x-100 , 1 
  mm pfa - block , and roche applied science 's edta - free protease 
  inhibitor mixture . clarified bacterial lysates were loaded onto an igg 
  sepharose 6 fast flow column in 50 mm tris - hcl , ph 7.6 , 150 
  mm nacl , and 0.02% tween 20 ( tst ) . zz - palb was eluted 
  with 0.5 m acetic acid , adjusted to ph 3.5 with ammonium acetate , 
  lyophilized , resuspended in tst buffer , and stored at -20 c . purified 
  proteins ( or buffer in the case of controls ) were mixed in 0.8-ml handee 
  columns ( pierce ) with 30 l of igg sepharose slurry and rotated for 2 h at 
  4 c . after five washes with tst , the bound material was eluted with 50 
  l of sds - page loading buffer . proteins were resolved using 12% 
  polyacrylamide sds - page and detected with coomassie blue . western blot analyses  the following primary monoclonal 
  antibodies were used : mouse anti - myc 9e10 ( santa cruz biotechnology , 1/1000 ) 
  for detection of ( myc)3::pacc ; rat anti - ha 3f10 ( roche applied science , 
  1/1000 ) for palb::(ha)3 ; mouse anti - flag m2 ( sigma , 1/80,000 ) for 
  ( flag)3::pep12 ; and mouse anti - actin c4 ( icn biomedicals ) for -actin . rabbit anti - yeast hexokinase antiserum ( chemicon , 1/80,000 ) was used for 
  hexokinase . these were combined with peroxidase - coupled sheep anti - mouse igg 
  ( amersham , 1/4,000 ) , goat anti - rat igm+igg ( goat , 1/4,000 ) or donkey 
  anti - rabbit igg as secondary antibodies , which were reacted with ecl ( amersham 
  biosciences ) . palb contains a central calpain - like thiol protease catalytic domain , a 
  calpain iii domain toward the c terminus and an n - terminal mit domain 
  ( fig . palb is an interactor of escrt - iii vps24 : the palb mit domain is 
  necessary and sufficient for binding vps24to explore the possible 
  association of palb with escrt - iii , we performed two - hybrid assays with 
  full - length palb . palb did not interact at all with a. nidulans 
  escrt - iii components vps32 , vps2 , or vps20 , with the aaa atpase vps4 or with 
  escrt - ii vps25 ( data not shown ) ( only one of the two possible bait / prey 
  combinations was tested for vps20 and vps4 , which inhibited growth when fused 
  to the gal4 dbd ) . however , palb showed two - hybrid interaction with vps24 in 
  either possible orientation ( fig . 1 , 
  b and c ) . in sharp contrast , interaction of palb 
  with didb was barely above detection 
  ( fig . 1b , lane 
  4 ) whereas , in agreement with previous reports with s. 
  cerevisiae and human proteins , vps4 interacted strongly with 
  didb ( fig . 
  1c , lane 15 ) . as mutant palb , deleted for the mit domain , palb-(77847 ) did not 
  interact with vps24 ( fig . 
  1b , lane 2 ) whereas bait palb-(196 ) and 
  prey palb-(1130 ) constructs encompassing the n - terminal mit domain 
  showed strong interaction ( fig . 1 , 
  b and c ) , we conclude that the palb - vps24 
  interaction is mit domain - mediated . vps4 contains an intensively investigated escrt - iii - interacting mit domain 
  ( 37 , 
  38 , 
  42 , 
  52 ) . however , in sharp 
  contrast with its strong interaction with didb , vps4 interacted 
  very inefficiently with vps24 in two - hybrid assays , as reflected by the 
  30-fold lower -galactosidase activity 
  ( fig . 1c , lanes 
  14 and 15 ) . these and the above palb data strongly support the 
  contention that vps24 specifically recruits the palb mit domain whereas 
  didb specifically recruits the vps4 mit domain . in agreement , the palb mit domain prey construct containing the n - terminal 130 palb residues 
  interacted 10 times more efficiently with vps24 than with didb in terms of 
  -galactosidase activity ( fig . 1c , lanes 13 and 16 ) whereas the 
  palb(196 ) bait construct interacted strongly with vps24 and not at all 
  with didb ( fig . a gene replacement allele encoding wild - type palb::(ha)3 was constructed as 
  outlined in supplemental fig . s1 and denoted palb800 . to confirm the 
  vps24-palb interaction , we used gst pull - down assays with a. nidulans 
  extracts expressing physiological levels of palb::(ha)3 . unlike the unrelated 
  gst - gfp fusion protein , gst - vps24 efficiently pulled - down palb::(ha)3 
  ( fig . s2 ) . of note , several ( ha)3 immunoreactive bands showing 
  faster electrophoretic mobility than the full - length protein , almost certainly 
  representing n - terminal palb degradation products ( ha3 is attached to the palb 
  c terminus ) , were detectable in the input extract . however , only the 
  full - length protein co - purified with gst - vps24 beads , in agreement with the 
  vps24-palb interaction being mediated by the n - terminal mit domain 
  ( fig . 1d ) . indeed , 
  mutant palb-(ha)3 lacking the mit domain was not pulled - down by gst - vps24 
  ( fig . figure 1.palb interacts with vps24 of escrt - iii . a , schemes of the 
  domain organizations of palb and of vps24 ; b and c , 
  two - hybrid analyses : the indicated gal4 dna binding domain ( pgbkt7 ) and 
  activation domain ( pact2ad ) constructs were tested in s. cerevisiae 
  y187 , using quantitative -galactosidase assays . d , gst - vps24 
  pull - down assays of a. nidulans extracts expressing physiological 
  levels of palb or palb , as indicated . a , schemes of the 
  domain organizations of palb and of vps24 ; b and c , 
  two - hybrid analyses : the indicated gal4 dna binding domain ( pgbkt7 ) and 
  activation domain ( pact2ad ) constructs were tested in s. cerevisiae 
  y187 , using quantitative -galactosidase assays . d , gst - vps24 
  pull - down assays of a. nidulans extracts expressing physiological 
  levels of palb or palb , as indicated . palb interacts directly with vps24the mit domain dependence 
  displayed by the vps24-palb interaction detected by two - hybrid and gst 
  pull - down experiments strongly indicates that this interaction is direct . definitive evidence that palb interacts directly with vps24 was obtained from 
  two types of experiments . firstly , we used in vitro labeled 
  [ s]palb and [ s]pala baits , rather than a. 
  nidulans extracts , in pull - down assays with gst - vps32 , gst - didb , and 
  gst - vps24 . gst - vps32 interacted directly with pala and did not pull - down palb . in contrast , gst - vps24 efficiently pulled - down palb whereas gst - didb did not 
  ( fig . 2a ) . secondly , 
  we confirmed that vps24 interacts with a zz - palb mit domain polypeptide using 
  proteins purified from recombinant bacteria . purified his6-vps24 
  was retained on igg - sepharose beads only after preincubation with purified 
  zz - palb peptide ( supplemental fig . we conclude that palb 
  interacts specifically and directly with vps24 in a mit domain - dependent 
  manner . the vps24 mim motif is necessary and sufficient for interaction with 
  palb  a . nidulans vps24 is a 228-residue protein containing 
  an n - terminal ( residues 12197 ) snf7 domain and a c - terminal potential 
  mim motif ( residues 214228 ) , conserved in vps2 and didb ( figs . two - hybrid assays 
  demonstrated that , in agreement with crystallographic yeast and human protein 
  data , these c - terminal residues mediate the interaction with palb . a bait 
  construct containing the n - terminal 197 resides of vps24 did not interact with 
  a palb prey ( data not shown ) . however , that containing residues 196228 
  interacted similarly to full - length vps24 
  ( fig . 1c , lanes 
  11 and 12 ) . structural studies demonstrated the key involvement that one arg and one 
  leu residue , located at positions 0 and + 1 , respectively , of yeast and human 
  mim motifs play in binding to mit domains 
  ( 38 , 
  42 ) . these residues correspond 
  to arg-222 and leu-223 in a. nidulans vps24 
  ( fig . in 
  agreement with the mim motif mediating the interaction with palb , pull - down 
  experiments demonstrated that a double r222d / l223d substitution prevents palb 
  binding ( fig . 2c ) . vps24 is virtually essential in a. nidulans  to address the 
  role of vps24 in ph signaling , we constructed a null allele by reverse 
  genetics , by exploiting that , in multinucleated cells of a. nidulans , 
  lethal or severely debilitating recessive mutations can be maintained in 
  heterokaryosis ( 53 ) 
  ( fig . we constructed a 
  vps24 dna fragment containing the 5- and 3-utr flanking 
  regions , in which the complete coding region had been replaced by the a. 
  fumigatus pyrg gene ( 54 ) . the construct was transformed into a non - homologous recombination - deficient , 
  pyrimidine - requiring ( nkua pyrg89 ) double mutant 
  strain . type i transformants 
  showed wild - type growth on transfer to pyrimidine - deficient medium and 
  subsequent genotyping showed these to be heterozygous aneuploids or diploids 
  carrying one copy of the vps24 wild - type allele and one 
  copy of the null vps24 allele 
  ( fig . type ii transformants 
  showed typically heterokaryotic growth , indicating that their coenocytic 
  hyphal cells were carrying vps24 , pyrimidine - requiring 
  pyrg89 nuclei and vps24::pyrg , 
  pyrimidine prototrophic nuclei . southern blot analyses demonstrated that these 
  transformants were indeed heterokaryotic and that they contained a relatively 
  higher proportion of wild - type nuclei than of 
  vps24::pyrg nuclei 
  ( fig . 3 , a and 
  b ) , as reported previously for other lethal or severely 
  debilitating alleles ( 53 , 
  55 , 
  56 ) . as individual nuclei 3a , scheme ) , conidiospores of these 
  heterokaryons were streaked on pyrimidine - sufficient and deficient media . conidial inoculations led to barely detectable growth in the absence of 
  pyrimidines , which contrasted with the normal growth in the presence of 
  pyrimidines ( the latter condition allowing growth of pyrg 
  nuclei ) ( fig . the 
  fact that growth of pyrimidine - independent 
  vps24::pyrg homokaryotic clones is 
  nearly nil led us to conclude that vps24 is a nearly essential gene . 
  a similar situation has been reported for aspergillus oryzae vps2 and 
  vps24 ( 57 , 
  58 ) . key residues in the vps24 mim domain 
  are essential for palb binding . a , autoradiographs of gst pull - down 
  assays with the indicated a. nidulans gst fusion proteins and 
  s - labeled pala and palb preys synthesized in vitro using 
  a coupled transcription / translation system . input lanes contained 2% of the 
  material used in the pull - downs . note that gst - didb does not pull - down palb 
  under these conditions . b , amino acid sequence comparison of the mim 
  motifs of fungal vps24 and vps2 proteins and their respective chmp3 and chmp2a 
  human orthologues is shown on the left . shaded residues correspond to 
  the consensus mim motif recognizing the vps4 mit domain 
  ( 38 , 
  42 ) . c , 
  gst pull - down assays using wild - type or mutant ( r222d and l223d substituted ) 
  a. nidulans gst::vps24 fusion proteins as baits and 
  s - labeled wild - type palb as prey . key residues in the vps24 mim domain 
  are essential for palb binding . a , autoradiographs of gst pull - down 
  assays with the indicated a. nidulans gst fusion proteins and 
  s - labeled pala and palb preys synthesized in vitro using 
  a coupled transcription / translation system . note that gst - didb does not pull - down palb 
  under these conditions . b , amino acid sequence comparison of the mim 
  motifs of fungal vps24 and vps2 proteins and their respective chmp3 and chmp2a 
  human orthologues is shown on the left . shaded residues correspond to 
  the consensus mim motif recognizing the vps4 mit domain 
  ( 38 , 
  42 ) . anvps24 arg-222 and 
  leu-223 correspond to positions 0 and 1 in the consensus motif . c , 
  gst pull - down assays using wild - type or mutant ( r222d and l223d substituted ) 
  a. nidulans gst::vps24 fusion proteins as baits and 
  s - labeled wild - type palb as prey . a proportion of palb associates with membranes the above 
  data strongly supported the conclusion that palb is associated with escrt - iii . 
  to determine whether palb:(ha)3 , expressed at physiological levels , is 
  associated with membranes , we performed subcellular fractionation studies . protoplast lysates were separated into 13,000  g pellet ( p13 ) 
  and supernatant fractions , and the supernatant further separated into 100,000 
   g soluble ( s100 ) and insoluble ( p100 ) fractions , as described 
  for yeast ( 35 , 
  48 , 
  49 ) . in agreement with data in 
  s. cerevisiae , flag - tagged endosomal syntaxin pep12 largely localized 
  to p13 membranes , whereas the soluble enzyme hexokinase localized to the s100 
  fraction . palb::(ha)3 was distributed between membrane - associated and soluble 
  fractions in approximately equal proportions 
  ( fig . this would be consistent with the view that palb is 
  associated with endosomal membranes via escrt - iii . unlike the pep12p syntaxin or the escrt - iii - associated protein bro1p , the 
  s. cerevisiae escrt - iii components vps32p or vps24p form high order 
  oligomers that can not be extracted from membranes by detergent ( 1% triton 
  x-100 ) treatment ( 35 , 
  59 ) . the p13-associated 
  fraction of palb can be solubilized after resuspending the p13 pellet in 1% 
  triton x-100-containing buffer ( supplemental fig . this association appears to be 
  independent of the pal pathway , as the distribution between p13 and 
  s100 fractions was unaffected by the palh17 loss - of - function mutation 
  in the gene encoding the 7-tmd receptor ( data not shown ) . deletion of the palb mit domain impairs but does not prevent the 
  signaling proteolysis step  in view that vps24 is 
  essential , we addressed the physiological role of the mit domain - dependent 
  palb - vps24 interaction by constructing a 
  palb801 mutant allele by 
  gene replacement ( supplemental fig . in contrast to palb800 , 
  which encodes wild - type palb::(ha)3 , 
  palb801 encodes a mutant 
  palb::(ha)3 protein that lacks the n - terminal 76 residues 
  of palb and thus is deleted for the mit domain . in diagnostic plate tests of ph signaling , palb800 is 
  phenotypically indistinguishable from the wild - type ( data not shown ) . in 
  contrast , palb801 shows a 
  weak loss - of - function phenotype compared with a null palb 
  mutation ( fig . 4c ) demonstrated that deletion of the mit domain has no 
  effect on the levels of palb , we concluded that deletion of the mit domain 
  impairs but does not prevent palb function . in wild - type palb800 cells cultured under acidic 
  conditions , pacc is the only detectable form of pacc . upon 
  shifting cells to alkaline ph , pacc is converted to 
  pacc within 30 min , and pacc becomes the predominant 
  form 3060 min after the ph shift 
  ( fig . 4d ) 
  ( 10 , 
  16 , 
  18 , 
  22 , 
  28 ) . ( after 1 h at 
  alkaline ph , newly synthesized pacc becomes detectable again , 
  possibly as a result of a negative feedback loop that regulates its 
  processing ) . palb801 
  reduced , but did not prevent , pacc processing to 
  pacc ( fig . the most conspicuous effect of the mit deletion was 
  the impairment of the signaling proteolysis step , such that pacc 
  was the predominating form at all sampled times after the ph shift . pacc remained almost undetectable throughout the experiment , 
  whereas pacc was only very slowly formed 
  ( fig . these data 
  indicate that palb801 
  reduces the rate at which pacc is converted into 
  pacc , such that the pacc processing activity is in 
  excess and the intermediate does not accumulate at any time point 
  ( 22 ) . as a 
  palb allele ( fig . 4d ) as well as pacc signaling protease 
  box - deficient mutants prevent pacc processing 
  ( 10 , 
  16 ) , these data indicate that 
  deletion of the mit domain results in partial loss of palb function , in 
  agreement with diagnostic plate tests . this partial loss - of - function phenotype 
  correlates with the shifting of a large proportion of the membrane - associated 
  palb pool toward the soluble fraction ( fig . most importantly , these data demonstrate that palb 
  specifically associates with vps24-containing membranes and that the mit 
  domain mediates this association . however , a proportion of the mutant protein 
  remained in the p13 fraction , strongly suggesting that mit domain - dependent 
  association to vps24 is not the sole mechanism for palb recruitment to 
  membranes . a , scheme of a conidiophore of an a. nidulans heterokaryotic 
  strain carrying untransformed vps24 , pyrimidine - requiring 
  haploid nuclei ( blue ) and transformed vps24 , 
  pyrimidine - independent nuclei ( red ) ( see also the color scheme in 
  b ) . during conidiophore development , individual nuclei segregate into 
  conidia , thereby resolving the heterokaryotic situation . thus , heterokaryotic 
  pyrg strains carrying nuclei where an essential gene has 
  been replaced by a pyrg allele can be propagated from 
  conidiospores in the presence but not in the absence of pyrimidines , as the 
  auxotrophic conidia corresponding to the untransformed nuclei required uracil 
  supplementation for growth . transformants 1 through 5 , 7 , and 8 are 
  heterokaryotic , whereas transformant 6 is a spontaneous heterozygous diploid 
  or aneuploid for vps24 . b , southern blot analysis of the 
  transformants with indication of diagnostic bands . note that , in 
  heterokaryotic transformants 15 , the intensities of the 
  vps24 bands relative to the vps24 band 
  are lower than in the diploid / aneuploid transformant 6 , indicating that the 
  heterokaryons contain fewer vps24 than 
  vps24 nuclei . a , scheme of a conidiophore of an a. nidulans heterokaryotic 
  strain carrying untransformed vps24 , pyrimidine - requiring 
  haploid nuclei ( blue ) and transformed vps24 , 
  pyrimidine - independent nuclei ( red ) ( see also the color scheme in 
  b ) . during conidiophore development , individual nuclei segregate into 
  conidia , thereby resolving the heterokaryotic situation . thus , heterokaryotic 
  pyrg strains carrying nuclei where an essential gene has 
  been replaced by a pyrg allele can be propagated from 
  conidiospores in the presence but not in the absence of pyrimidines , as the 
  auxotrophic conidia corresponding to the untransformed nuclei required uracil 
  supplementation for growth . transformants 1 through 5 , 7 , and 8 are 
  heterokaryotic , whereas transformant 6 is a spontaneous heterozygous diploid 
  or aneuploid for vps24 . b , southern blot analysis of the 
  transformants with indication of diagnostic bands . note that , in 
  heterokaryotic transformants 15 , the intensities of the 
  vps24 bands relative to the vps24 band 
  are lower than in the diploid / aneuploid transformant 6 , indicating that the 
  heterokaryons contain fewer vps24 than 
  vps24 nuclei . vps24-mediated association of palb with endosomes is important for ph 
  signaling  to demonstrate that vps24-mediated recruitment of palb to 
  endosomal membranes plays a role in ph processing and to circumvent the 
  problems associated with the fact that vps24 is virtually essential , 
  we fused the complete vps24 coding sequence inframe to the sequence 
  encoding residues 77847 of palb . the resulting gene , which encodes a 
  vps24::palb fusion protein , was introduced into the 
  a. nidulans genome using the palb gene replacement 
  procedure . thus , vps24::palb , expressed at physiological 
  levels from the palb promoter ( scheme in 
  fig . 5a ) , was the only 
  source of palb in strains with this transgene , which contain a wild - type 
  vps24 gene to ensure viability . ( c - terminal fusion of palb to vps24 
  almost certainly precludes vps24 function 
  ( 60 ) ) . western blots demonstrated that , at acidic , neutral and alkaline ph , levels 
  of vps24::palb were markedly similar to those of palb and 
  palb , encoded by the palb800 and 
  palb801 alleles described above ( data not shown ) . thus n - terminal 
  attachment of vps24 to palb does not lead to protein 
  destabilization / degradation . in fact , diagnostic plate tests demonstrated that 
  vps24 attachment reversed the partial loss - of - function phenotype associated 
  with palb ( fig . subcellular fractionation studies demonstrated that , 
  while a major proportion of palb is cytosolic , 
  vps24::palb is mostly associated with p13 membranes 
  ( fig . finally , 
  sensitive pacc - processing experiments after an alkaline ph shift 
  demonstrated that vps24 attachment alleviates the 
  palb - associated impairment in pacc 
  processing , markedly increasing levels of pacc at late time 
  points after the shift and , most notably , leading to detection of 
  pacc ( fig . however , perhaps due to spatial constrains on pacc 
  signaling protease cleavage resulting from the direct attachment of the palb 
  catalytic domain to vps24 , the fusion protein did not show fully wild - type 
  pacc processing ( compare fig . thus , the impairment of palb function resulting from 
  absence of the mit domain can be alleviated by direct fusion with vps24 . this 
  correlates with the nearly complete association of vps24 : : 
  palb with membranes . we demonstrate that palb is recruited to escrt - iii - containing endosomal 
  membranes via specific interaction with vps24 and that this recruitment is 
  required for normal ph signaling . palb is the founding member of the family of 
  calpain - like proteases containing a pbh ( palb 
 homology ) domain . although it was 
  originally reported that calpain 7 is nuclearly localized 
  ( 61 ) , more extensive studies 
  demonstrated that calpain 7 localizes in fact to the cytosol and to endosomes 
  and that this protein , which contains an n - terminal tandem repeat of mit 
  domains , interacts in vitro with chmp1b 
  ( 62 ) . however , these studies were unable to demonstrate 
  any physiological involvement for the recruitment of calpain 7 to endosomes 
  and therefore this report is first in demonstrating that mit domain - mediated 
  recruitment of a calpain - like protease to escrt - iii plays a physiological 
  role . figure 4.deletion of the palb mit domain impairs its association with membranes 
  and the processing of pacc . wild - type palb800 and mutant 
  palb801 protoplast lysates 
  were fractionated into 13,000  g insoluble ( p13 ) and 100,000 
   g insoluble ( p100 ) and soluble ( s100 ) fractions , which were 
  analyzed by western blotting . b , 
  palb801 results in weak 
  loss - of - function in diagnostic plate tests of ph signaling 
  ( 16 , 
  74 ) . , 
  palb801 confers some 
  neomycin resistance ( neo ) , weakly impairs growth on alkaline ph ( oh ) 
  and molybdate ( mo ) plates , and decreases tolerance of licl 
  ( li ) , indicating that deletion of the mit domain results in weak 
  loss - of - function ; sc is synthetic complete medium without any addition . palb800 and palb801 
  cells cultured at acidic ambient ph were shifted to alkaline ph . deletion of the palb mit domain impairs its association with membranes 
  and the processing of pacc . a , mit domain - mediated association of 
  palb with membrane fractions . wild - type palb800 and mutant 
  palb801 protoplast lysates 
  were fractionated into 13,000  g insoluble ( p13 ) and 100,000 
   g insoluble ( p100 ) and soluble ( s100 ) fractions , which were 
  analyzed by western blotting . b , 
  palb801 results in weak 
  loss - of - function in diagnostic plate tests of ph signaling 
  ( 16 , 
  74 ) . in contrast , 
  palb801 confers some 
  neomycin resistance ( neo ) , weakly impairs growth on alkaline ph ( oh ) 
  and molybdate ( mo ) plates , and decreases tolerance of licl 
  ( li ) , indicating that deletion of the mit domain results in weak 
  loss - of - function ; sc is synthetic complete medium without any addition . palb800 and palb801 
  cells cultured at acidic ambient ph were shifted to alkaline ph . we show that palb binds 
  selectively to vps24 , which suggests that the binding specificity determined 
  by the compound mit domain differs from that in the single palb mit domain . this selectivity for vps24 is rather suggestive , as work in s. 
  cerevisiae indicated that endosomal , escrt - iii - containing mvb pathway 
  complexes and ph signaling complexes may be functionally different entities 
  ( 26 ) and a recent report 
  strongly implicates vps24 as a negative regulator of vps32 oligomerization on 
  endosomal membranes ( 63 ) . unfortunately , our attempts to determine the subcellular localization of n- or 
  c - terminally tagged gfp - palb using different promoters have failed , apparently 
  due to instability of the fusion proteins , and immunolocalization studies of 
  palb::(ha)3 were inconclusive . our work unambiguously places palb as a member of the fungal endosomal 
  ambient ph signaling complex , associated with the bro1-domain scaffold pala 
  ( 25 ) and , perhaps transiently , 
  with the bro1 domain - like containing protein palc 
  ( 28 ) . as pala - dependent 
  recruitment of the zinc - finger transcription factor pacc to this complex is essential for ph signaling and palb is almost certainly the 
  signaling protease , one implication is that the signaling proteolysis step 
  takes place in association with escrt - iii . finally , by demonstrating that 
  palb , like the signaling proteolysis substrate pacc 
  ( 20 , 
  25 ) , is connected to 
  escrt - iii , this work constitutes yet further evidence that palb is the 
  signaling protease . analyses of null class e ( escrt ) mutant ph signaling phenotypes in yeasts 
  ( 6469 ) 
  and our unpublished data in a. 
  nidulans4 
  unambiguously demonstrate the essential involvement of escrt - iii in ph 
  signaling . here we show that tailored recruitment to endosomal membranes of a 
  largely cytosolic mutant palb protein impaired in pacc proteolytic processing 
  significantly restores function , strongly supporting the current view that 
  ambient ph - dependent proteolytic cleavage of pacc and rim101p takes place in 
  the context of endosomal membranes 
  ( 8 , 
  9 , 
  20 , 
  25 ) . the interaction between palb and escrt - iii vps24 reported here is specific , 
  as pull - down assays failed to detect palb interaction with didb 
  or with vps32 under conditions in which vps24 very efficiently pulled down 
  palb . the palb mit domain and the vps24 mim - containing c - terminal region are 
  necessary and sufficient for the vps24-palb interaction . in pull - down assays 
  carried out with purified proteins , the interaction is strictly dependent on 
  the crucial , adjacent arg and leu residues of the vps24 mim 
  ( 38 , 
  42 ) , which represents direct 
  evidence that the atypical mim motif in vps24 ( and , by extension , in chmp3 ) is 
  able to bind mit domains . our data strongly indicate that this mit - mediated interaction can not be the 
  sole mechanism by which palb is recruited to endosomal membranes in 
  vivo . this is perhaps not unexpected , as mit - mim interactions show 
  relatively weak affinities ( kd in the m 
  range ( 34 , 
  38 , 
  42 ) ) . the precedent of the 
  de - ubiquitinase amsh is most relevant . like palb , however , the interaction of amsh 
  with escrt membranes is further reinforced through simultaneous escrt-0 stam 
  binding ( 45 ) . moreover , recent 
  work strongly indicated that regions of chmp3 other than its acidic c - terminal 
  region contribute to the interaction with amsh , markedly increasing the 
  strength of the interaction 
  ( 34 ) . we have not yet 
  determined which , if any , additional escrt components contribute to palb 
  recruitment . we can not formally rule out either a role for undetected 
  vps24-dependent , palb mit - independent interactions in its in vivo 
  recruitment to escrt - iii , as deletion of a. nidulans vps24 nearly 
  abolishes growth ( fig . a , transgene expressing a 
  vps24 : : palb fusion protein was constructed by gene 
  replacement as described in supplemental fig . while 
  palb predominates in the soluble fraction , n - terminal 
  attachment of vps24 redirects the protein to p13 membranes . b , 
  attachment of vps24 to the n terminus of palb results in 
  a marked increase in the two - step processing of pacc , such that 
  pacc becomes clearly detectable in a ph shift experiment . attachment of vps24 to the n terminus of palb 
  significantly restores function . a , transgene expressing a 
  vps24 : : palb fusion protein was constructed by gene 
  replacement as described in supplemental fig . s1 . while 
  palb predominates in the soluble fraction , n - terminal 
  attachment of vps24 redirects the protein to p13 membranes . b , 
  attachment of vps24 to the n terminus of palb results in 
  a marked increase in the two - step processing of pacc , such that 
  pacc becomes clearly detectable in a ph shift experiment . the possibility that a second interactor reinforces palb recruitment to 
  escrt - iii is strongly suggested by data from s. cerevisiae , where the 
  rim13p calpain - like protease presumably interacts with one or more escrt 
  protein(s ) other than vps24p because rim13p does not contain a mit domain and 
  vps24 does not prevent rim101p processing 
  ( 64 ) . moreover , while s. 
  cerevisiae escrt - iii vps32p and vps20p are essential for ph signaling , 
  the absence of vps2p or vps24p results in partially constitutive signaling 
  ( 67 ) , possibly by increasing 
  the availability of other escrt proteins to ph signaling pathway interactors . vps32 and/or vps20 are the most obvious candidates to mediate rim13p 
  recruitment to escrt - iii , and indeed rim13p interacts with vps32 in two - hybrid 
  assays ( 30 ) , although it 
  remains to be determined whether this interaction is direct and whether it 
  plays a physiological role similar to that demonstrated here for vps24 and 
  palb . thus , palb and rim13p would share one anchor in escrt - iii whereas palb 
  would have acquired ( or rim13p would have lost ) the mit domain - mediated 
  attachment to vps24 . non - compartmentalized intracellular proteases would be harmful for the cell 
  if their activities were not regulated precisely . calpain - like proteases lack 
  the calmodulin - like ca binding domain characterizing calpains but 
  contain one calpain iii domain , which is structurally related to c2 domains 
  ( 70 ) . indeed , the calpain iii 
  domain has been shown to bind ca and , notably , 
  phosphoinositide - containing liposomes 
  ( 71 ) . this suggests a highly 
  speculative mechanism by which vps24-mediated recruitment of palb to endosomal 
  membranes might lead to protease activation in the proximity of endosomal , 
  pala - recruited pacc substrate . vps24-dependent 
  recruitment of palb might bring the calpain iii domain in close proximity to 
  endosomal membrane domains enriched in ptins3,5p2 , 
  facilitating phospholipid binding , which would lead to an activating 
  conformational change in palb . we note that a similar calpain iii 
  domain - mediated , phosphoinositide - dependent mechanism activates m - calpain 
  after its epidermal growth factor - dependent recruitment to the plasma membrane 
  ( 73 ) .
activation of the aspergillus nidulans transcription factor pacc , 
  which mediates ambient ph regulation of gene expression and is recruited to 
  escrt - iii by the vps32-interacting scaffold pala , involves its ambient 
  ph - dependent c - terminal proteolysis . 
 this reaction is almost certainly 
  catalyzed by the palb calpain - like protease . here 
 we show that palb associates 
  with membranes and interacts specifically and directly with escrt - iii vps24 . 
 the palb n - terminal mit domain and the vps24 c - terminal mim motif are 
  necessary and sufficient for this interaction . 
 palbmit , a 
  mutant palb lacking the mit domain is inefficiently recruited to membranes and 
  impaired in pacc proteolytic processing . 
 notably , membrane recruitment is 
  promoted and pacc processing largely restored by covalent attachment of vps24 
  to mutant palbmit . 
 this is the first reported evidence that 
  calpain - like recruitment to escrt - iii lattices plays a physiological role . 
 it 
  unambiguously positions the calpain - like protease palb within the 
  escrt - iii - associated ph signaling complex , underlines the positive role of 
  escrt - iii in ambient ph signal transduction , and suggests a possible mechanism 
  for palb activation .