drosophila stocks , culture conditions  the 
  sod1 and ry strains 
  are described in ref , 10 . 
 
hsod1 was expressed using lines carrying uas - hsod1 and the gal4 driver line , 
  dagal4 as described 
  ( 14 ) .
stocks were maintained 
  at 25 c on standard cornmeal and agar medium unless otherwise stated .
the copper chaperone coding region was amplified by pcr ( primers ccs5 + 
  ccs7 ) using dna extracted from a drosophila canton s adult cdna 
  library ( stratagene catalog number 936603 ) .
the 930-bp pcr product was 
  blunt - ended and subcloned into the smai site of pbluescriptiisk ( stratagene ) 
  to generate the vector pbcc .
the transformation vector pucc was constructed by 
  subcloning the ecori - noti fragment of pbcc into puast 
  ( 15 ) .
yeast strains , growth conditions , plasmids  yeast strains 
  used in this study were derived from eg103 ( mat , 
  leu2 - 3,112 , his31 , trp1 - 289 , ura3 - 52 ) 
  ( 16 ) and include ks107 
  ( sod1 ) ( 17 ) 
  and ls101 ( sod1 ccs1 ) 
  ( 18 ) .
cells were propagated at 
  30 c either in enriched yeast extract , peptone - based medium supplemented 
  with 2% glucose ( ypd ) , or in synthetic complete ( sc ) medium 
  ( 19 ) .
solid medium was 
  supplemented with 15 mg / liter ergosterol and 0.5% tween 80 to enhance growth 
  in anaerobic conditions .
yeast expression plasmids for dsod1 and dccs were produced using cdna clone 
  re52090 ( open biosystems ) for dsod1 and plasmid pucc for dccs .
the coding 
  sequences for dsod1 and dccs were pcr0-amplified introducing 5 bglii 
  and 3 snabi sites in dsod1 and 5 mlui and 3 rsrii sites 
  in dccs .
the dsod1 pcr product was digested with the appropriate enzymes and 
  ligated into pls108 ( 18 ) cut 
  with bglii / snabi , replacing the yeast sod1 coding sequence with that of dsod1 
  resulting in plasmid plj373 .
the digested dccs pcr product was inserted into 
  plj366 ( a derivative of pls113 
  ( 18 ) with an mlui site just 
  prior to the yccs1 start codon and an rsrii site immediately after the stop 
  codon ) , replacing the yccs1 coding sequence with that of dccs , generating 
  plasmid plj375 .
both dsod1 and dccs were under control of their corresponding 
  s. cerevisiae regulatory sequences .
the sequence integrity of these 
  plasmids was ensured by double - stranded dna sequencing ( dna analysis facility , 
  the johns hopkins university ) .
yeast ccs1 plasmids phal-413 , pls008 ( c229s , 
  c231s ) , and pls010 ( k136e , g137e ) have been described previously 
  ( 18 ) .
domain i mutation in 
  yccs1 ( c17s , c20s ) was introduced using the quikchange mutagenesis kit 
  ( stratagene ) using phal-413 as a template 
  ( 4 ) resulting in plasmid 
  pps031 .
sod1 in - gel activity assay  to analyze sod1 activity from 
  drosophila , five males were homogenized in 50 l of 1% triton 
  x-100 .
10 l of supernatant was transferred to a tube containing an equal 
  volume of loading buffer ( 125 mm tris - hcl , ph 6.8 , 20% glycerol , 
  0.01% bromphenol blue ) .
the gel was incubated in nitro blue tetrazolium ( 2 mg / ml ) for 5 min 
  followed by incubation in 36 mm k2hpo4 , 0.035 
  mm riboflavin , 0.3% temed for 7 min and then washed several times 
  in h2o as described 
  ( 20 ) .
sod enzymatic activity was assayed from yeast grown shaking in selected sc 
  medium to an a600 of 1.5 .
yeast lysates were generated by 
  glass bead homogenization in a buffer containing 0.6 m sorbitol , 10 
  mm hepes , ph 7.2 , and protease inhibitors .
analysis of sod activity 
  by nondenaturing gel electrophoresis using 12% pre - cast gel ( invitrogen ) and 
  staining with nitro blue tetrazolium was performed as described previously 
  ( 21 ) except that 10 
  mm edta was added to the native running buffer .
sod1 spectrophotometric assay2448-h - old males were 
  homogenized in b1 ( 50 mm sodium phosphate , ph 7.4 , 0.1 
  mm edta ) .
the supernatant was extracted twice with chloroform ( 0.15 
  v ) , ethanol ( 0.25 v ) with a 15-min incubation time for each extraction .
sod1 
  activity was determined spectrophotometrically by monitoring the autooxidation 
  of 6-hydroxydopamine ( 6-hd ) at 490 nm at 37 c in 500 l of b1 
  containing 0.1 mm 6-hd 
  ( 22 ) .
aconitase activity assay30 adult males were homogenized in 
  120 l of extraction buffer ( 0.6 mm mncl2 , 2 
  mm citric acid , 50 mm tris - hcl , ph 8.0 ) and centrifuged 
  at 13,000  g. samples were electrophoresed on sepraphore iii 
  membranes ( pall corp . ) .
aconitase was detected by incubation of the membrane 
  in 100 mm potassium phosphate , ph 6.5 , 1 mm nadph , 2 
  mm cis - aconitic acid , 1.2 mm 
  3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide , 0.3 
  mm phenazine methosulfate , 25 mm mgcl2 , and 5 
  units / ml isocitrate dehydrogenase 
  ( 23 , 
  24 ) .
western blotting  for analysis of sod1 from 
  drosophila , males were homogenized in 1% triton x-100 .
the extracts 
  were centrifuged at 13,000  g for 5 min at 4 c , and the 
  supernatant was transferred to a tube containing an equal volume of loading 
  buffer ( 125 mm tris - hcl , ph 7.2 , 4% sds , 10% mercaptoethanol , 20% 
  glycerol , 0.01% bromphenol blue ) .
samples were boiled for 5 min , and 10 g 
  of total protein was separated on a sds - polyacrylamide gel ( 4% stacking , 15% 
  separating ) .
the protein was transferred to nitrocellulose membrane ( ecl ) and 
  probed with sheep anti - human cu , zn superoxide dismutase ( cedarlane catalog 
  number k9007c ) and rabbit anti - sheep igg horseradish peroxidase conjugate 
  ( chemicon catalog number ap147p ) .
the ecl western blotting detection reagents 
  ( rpn 2109 , amersham biosciences ) was used for detection .
immunoblot analysis of cu , zn - sod expressed in yeast was carried out with 
  cell lysates prepared as described above and a polyclonal antibody that 
  displays strong reactivity with cu , zn - sods from several species ( jh765 ) at a 
  1:5000 dilution ( 8) .
detection 
  utilized an alexa fluor 680 goat anti - rabbit secondary antibody ( invitrogen ) , 
  and immunoblots were visualized using the odyssey infrared imagining system 
  ( licor biosciences ) .
a , genomic architecture of 
  ccs , the drosophila copper chaperone for superoxide 
  dismutase 1 ( fly base annotation cg17753 , fbgn0010531 ) .
29e is the mutant chromosome 
  containing a 1907-bp deletion extending upstream from bp 124 of the 2nd exon 
  through the first intron , the first exon and 1717 bp of the 5-noncoding 
  region .
drosophila ccs . a , genomic architecture of 
  ccs , the drosophila copper chaperone for superoxide 
  dismutase 1 ( fly base annotation cg17753 , fbgn0010531 ) .
29e is the mutant chromosome 
  containing a 1907-bp deletion extending upstream from bp 124 of the 2nd exon 
  through the first intron , the first exon and 1717 bp of the 5-noncoding 
  region .
drosophila life span determination024-h - old males 
  were collected , 25 males per vial , and kept on cornmeal food containing 
  tegosept .
drosophila paraquat resistance024-h - old males were 
  collected and kept on cornmeal food for 24 h , 10 males in each vial .
they were 
  then transferred to vials containing 3-mm paper filter disks saturated with 
  250 l of 1% sucrose , 1% sucrose containing 2 mm paraquat 
  ( sigma ) or 1% sucrose and 5 mm paraquat .
the vials were stored at 
  25 c in the dark , and flies were enumerated after 24 h as originally 
  described ( 10 ) .
dccs , the copper chaperone of sod1 gene of drosophila  the 
  drosophila genomic homologue of yeast and mammalian ccs was 
  initially identified by isolation of a sod1-like sequence from a 
  drosophila genomic library , later verified by sequence annotation in 
  flybase ( cg17753 , fbgn0010531 , and confirmed by amplification of a 
  ccs cdna from a d. melanogaster cdna library 
  ( fig .
the 
  architecture of the native drosophila gene includes a 795-bp coding 
  sequence interrupted by two small introns of 51 and 61 bp , respectively .
the 
  nearest open reading frame ( cg11867 ) is 5 kb upstream from the 
  5-transcription start site .
the encoded sequence contains the expected 
  region of high homology to sod1 
  ( 2 ) in the central domain ii of 
  ccs ; the domain iii cxc is conserved .
unexpectedly , the domain i 
  cxxc copper - binding motif is absent 
  ( fig .
a 
  comparison to all known ccs - like molecules in the data base revealed that the 
  absence of a cxxc motif in drosophila ccs is unique .
of 20 
  ccs sequences analyzed from fungi to humans , only d. melanogaster and 
  anopheles gambiae lack the mxcxxc copper - binding 
  site ( table 1 )
. however , all 20 
  ccs molecules retain the cxc copper site in domain iii .
table 1drosophila ccs lacks domain 1 mtcxxcthe presence of the domain i mxcxxc and domain iii 
  cxc sequences was examined in ccs proteins from fungi , plants , and 
  animals .
drosophila ccs lacks domain 1 mtcxxc the presence of the domain i mxcxxc and domain iii 
  cxc sequences was examined in ccs proteins from fungi , plants , and 
  animals .
ccs deletion mutation  a loss of function mutation of 
  ccs ( ccs ) , 
  generated by an apparent imprecise excision of a p element located near 
  ccs in the autosomal enhancer - detector parent strain , h340 
  ( 25 ) , was identified by 
  recovery of a strain , 29e , with reduced sod1 activity and a set of phenotypes 
  exhibited by known sod1 deficiency mutants ( see below ) .
sequence analysis of 
  ccs , restriction analysis by southern hybridization , and sod1 
  activity all indicated that ccs is functionally and structurally 
  intact in h340 .
the genomic deletion in 
  ccs includes 1617 bp of 
  5-upstream region and 1907 bp downstream from the transcription start 
  site , including all of the first exon , the first intron , and 124 bp of the 
  second exon ( fig .
are relatively rare throughout normal development in 
  h340 and are undetectable by northern blot analysis in 29e ( data not 
  shown ) . loss of sod1 enzymatic activity in ccs - null flies  because 
  the only known assay for ccs function is via its role in the activation of 
  sod1 , we assayed sod1 activity in extracts of h340 and 29e using two 
  functionally different methods , the in - gel with nitro blue tetrazolium 
  reduction interference assay and the spectrophotometric 6-hydroxydopamine 
  reduction interference assay ( fig .
2 , 
  a and b ) . using both methods , we were unable to 
  detect significant sod1 activity in 29e .
loss of ccs in 29e appears to have no 
  effect on the activity of the mitochondrial manganese - containing sod2 ( see 
  fig .
4a ) . these 
  results by themselves would suggest that ccs in drosophila , as in 
  yeast and mammals , is specifically required for activating sod1 . however ,
analysis of sod1 protein levels revealed that the steady state 
  level of sod1 polypeptide in 29e is reduced to about one - quarter of its normal 
  level in h340 ( fig . 
  2c ) .
thus , the loss of sod1 activity in 29e probably 
  arises primarily from the reduced level of sod1 protein in the absence of ccs . 
 
further analysis confirmed that the lack of sod1 activity in 29e is not 
  because of a mutation in the sod1 
  gene.4 deficiency of ccs reduces the functional level of specific iron - sulfur 
  protein sentinels  the cytosolic and mitochondrial aconitases both 
  contain cubane [ 4fe-4s ] centers that are sensitive targets of inactivation by 
  superoxide ( 23 , 
  26 ) .
steady state levels of 
  these activities in drosophila are significantly reduced in the 
  absence of sod1 and sod2 , respectively 
  ( 24 ) .
assay of cacon and macon 
  activities in 29e shows that cacon activity is selectively depleted by about 
  50% ( h340 1.6  29e ) , with no detectable affect on the activity of 
  macon ( fig .
these data indicate that at the biochemical level , the absence of 
  dccs is equivalent to a deficiency in sod1 .
the important question then 
  arises , does this parallel extend to whole organism phenotypes ?
genetic ablation of ccs elicits broad phenotypic 
  consequences  at a minimum , the mutational loss of ccs should 
  generate a set of phenotypes predicted by the loss of sod1 , including reduced 
  adult life span with little or no overt effect on pre - adult development and 
  hypersensitivity to applied oxidative stress .
3a shows that the absence of ccs confers early onset 
  adult mortality with an 30% reduction in the median adult life span . in 
  contrast
, 29e displays the extreme toxic hypersensitivity to the redox cycling 
  agent , paraquat , exhibited by sod1-null 
  ( fig .
based on previous studies with rna interference - mediated knockdown of 
  sod1 ( 27 ) , and transgenic 
  rescue of sod1-null mutants 
  ( 28 ) , which show that paraquat 
  toxicity is a more sensitive indicator of sod1 deficiency than life span , the 
  phenotype of 29e is consistent with the presence in 29e of a very small 
  residual level of sod1 activity which , although below the level of detection 
  by conventional assays of whole - fly extracts , is sufficient to extend median 
  adult life span ( 30 days ) beyond the 10-day median life span of sod1-null 
  mutants .
such activity , if it were to occur , would have to arise via 
  ccs - independent activation of apo - sod1 ( see below ) .
human sod1 is active in ccs - null drosophila and restores their adult 
  life span  in earlier work 
  ( 14 ) , we described the 
  functional expression of human sod1 ( hsod1 ) in drosophila in a 
  ccs genetic background .
since then , the ccs - independent 
  activation of hsod1 has been described 
  ( 7 , 
  9 ) .
here we investigated the 
  activation of hsod1 in flies in the absence of functional dccs .
as would be 
  expected from this sod1 capable of ccs independent activation , hsod1 is quite 
  active in ccs - null flies ( fig . 
 
moreover , hsod1 functions biologically to rescue the 
  early mortality and reduced median life span of ccs - null flies 
  ( fig .
these 
  results demonstrate the presence in drosophila of a ccs - independent 
  pathway capable of robust activation of heterologous hsod1 while giving only 
  weak activation of homologous dsod1 .
ccs - independent activation of drosophila sod1 revealed in yeast 
  expression system  based on amino acid sequence alone , dsod1 is 
  predicted to acquire copper independently of ccs .
studies with yeast , human , 
  and c. elegans sod1 have identified a pair of prolines near the c 
  terminus that can prohibit ccs - independent activation 
  ( 7 , 
  8) .
yeast sod1 naturally 
  contains these prolines and is incapable of ccs - independent activation , 
  whereas mammalian and c. elegans sod1 with nonproline residues at 
  these positions can be activated independently of ccs .
5a ) leading us to predict that dsod1 should not exhibit 
  dependence on ccs for activity .
and although from the phenotype of dccs - nulls 
  we inferred the presence of a residual level of sod1 activity , the instability 
  of dsod1 in the absence of ccs obscures any measurable ccs - independent 
  activation of dsod1 that might occur in the drosophila model .
we 
  therefore turned to a yeast expression system to further examine 
  drosophila ccs and sod1 .
figure 2.loss of ccs affects sod1 and maconitase . a , in - gel activity 
  assay of sod1 .
h340 is the dccs parent stock from which 
  the ccs - null mutant , 
  ccs , was derived .
extracts 
  were treated with triton x-100 to eliminate interference from mitochondrial 
  superoxide dismutase ( sod2 ) .
note , the  background activity  found in 29e extracts is 
  also present in sod1-null mutant extracts .
extracts were prepared from h340 and 29e adult males , and sod1 
  protein was detected in western immunoblots with anti - sod1 antibody . 
 
top , immunoblot analysis ; bottom , densitometric quantitation 
  of immunoblot where 100 = amount of dsod1 polypeptide in h340 .
d , selective loss of 
  cytosolic aconitase ( cacon ) activity in the absence of ccs .
aconitase 
  activities in extracts of 23-day - old adult males were assayed after 
  electrophoretic separation .
top , zymogram analysis ; 
  bottom , densitometric quantitation of zymogram where 100 = level of 
  cacon activity in h340 .
loss of ccs affects sod1 and maconitase . a , in - gel activity 
  assay of sod1 .
h340 is the dccs parent stock from which 
  the ccs - null mutant , 
  ccs , was derived .
extracts 
  were treated with triton x-100 to eliminate interference from mitochondrial 
  superoxide dismutase ( sod2 ) .
note , the  background activity  found in 29e extracts is 
  also present in sod1-null mutant extracts .
extracts were prepared from h340 and 29e adult males , and sod1 
  protein was detected in western immunoblots with anti - sod1 antibody . 
 
top , immunoblot analysis ; bottom , densitometric quantitation 
  of immunoblot where 100 = amount of dsod1 polypeptide in h340 .
d , selective loss of 
  cytosolic aconitase ( cacon ) activity in the absence of ccs .
aconitase 
  activities in extracts of 23-day - old adult males were assayed after 
  electrophoretic separation .
top , zymogram analysis ; 
  bottom , densitometric quantitation of zymogram where 100 = level of 
  cacon activity in h340 .
all strains are homozygous for sod1 . to test the comparative functionality of dsod1 in the absence of ccs ,
the 
  coding sequences for human , yeast , and drosophila sod1 were all 
  placed under control of the s. cerevisiae sod1 gene promoter and 
  analyzed for sod1 activity and protein levels in 
  ccs1 versus ccs1 yeast . as in ccs - null 
  flies ( fig .
5b ) , and dsod1 
  activity was initially difficult to discern 
  ( fig .
yet when increasing levels of cell lysate were analyzed , the dsod1 
  polypeptide was readily detected , and ccs - independent activation of dsod1 
  became apparent ( fig . 
 
5b , lane 9 ) , similar to levels seen with human 
  sod1 ( lane 4 ) .
hence , dsod1 does have the capacity to be activated by 
  the ccs - independent pathway , ostensibly explaining the less severe phenotypes 
  of a ccs - null fly compared with a sod1-null mutant .
role of ccs in helping to stabilize dsod1the dramatic loss 
  of the dsod1 polypeptide in both ccs1 null yeast 
  ( fig .
2c ) 
  is atypical , and it is not observed with yeast and human sod1 analyzed in 
  parallel ( fig .
this loss in dsod1 can be rescued by expressing 
  either yeast ccs or drosophila ccs in the ccs1 yeast 
  ( fig .
this apparent stabilization of dsod1 requires physical 
  interactions between ccs and sod1 , as a k136e , g137e derivative of yeast ccs 
  that can not dock with sod1 
  ( 29 ) fails to increase dsod1 
  levels ( fig .
6a , 
  lane 4 ) . to test whether sod1-ccs interactions are by themselves 
  sufficient , we employed a c229s , c231s mutant of yeast ccs .
the highly 
  conserved cysteines cys-229 and cys-231 in domain iii of ccs are needed for 
  copper transfer and disulfide oxidation in sod1 , and a c229s , c231s mutant of 
  ccs can dock with sod1 but can not activate the enzyme 
  ( 29 , 
  30 ) . as seen in 
  fig .
6a , lane 
  3 , c229s , c231s yccs failed to stabilize dsod1 , indicating that copper 
  transfer and/or disulfide oxidation are required . to directly test the 
  requirement for copper
, yeast cells were starved for copper by treatment with 
  the cu(i ) chelator , bathocuproine sulfonate 
  ( 31 ) . under such 
  copper - limiting conditions , levels of the dsod1 polypeptide
were lowered even 
  in cells expressing wild type ccs from yeast or drosophila 
  ( fig .
together , 
  these studies indicate that apo - dsod1 is unusually unstable and that ccs 
  affords stability to dsod1 by activating the enzyme through copper insertion 
  and/or disulfide oxidation .
unique n terminus of drosophila ccs  as mentioned above , 
  drosophila ccs lacks the mxcxxc copper - binding 
  motif that is well conserved in ccs molecules from phylogenetically distant 
  taxa .
in fact , an inspection of ccs molecules across diverse species reveals 
  that with the exception of drosophila and mosquito ccs , all ccs 
  molecules identified to date harbor these cysteines 
  ( table 1 ) .
interestingly , we 
  observed that drosophila ccs is very poor at activating yeast sod1 
  compared with the homologous yeast ccs expressed from the identical yeast 
  ccs1 gene promoter ( fig . 
 
6c , lanes 2 and 3 ) . by comparison , fly 
  ccs was nearly as effective as yeast ccs in activating human sod1 
  ( fig .
6c , lanes 
  5 and 6 ) , and drosophila sod1 exhibited no apparent 
  preference for ccs and showed strong activation by both ccs molecules 
  ( lanes 8 and 9 ) . to address whether the yeast sod1 
  preference for yeast ccs reflected loss of the conserved 
  mxcxxc cysteines , we tested the effects of a c17s , c20s 
  substitution in yeast ccs .
6d , this mutant retains the ability to fully activate 
  yeast sod1 . despite the well conserved nature of domain i cxxcin ccs , 
  these cysteines are not essential for activating sod1 .
these results also 
  demonstrate that the apparent poor reactivity between fly ccs and yeast sod1 
  is not simply explained by the absence of these cxxc cysteines . in 
  any case , sod1 molecules from phylogenetically distant organisms exhibit 
  unique preferences in copper activation pathways .
figure 3.ccs deficiency confers early adult mortality and enhanced sensitivity to 
  the redox cycling agent , paraquat .
survival of at least 125 males of each genotype on standard cornmeal 
  food was followed at 25 c with enumeration and transfer of survivors to 
  fresh bottles every 23 days .
ccs is h340 , the 
  ccs parent stock from which 
  ccs was derived . 
 
sod1 is 
  sod1/sod1 , 
  sod1 is the wild type stock , ry . 
 
ccs is h340 , the 
  ccs parent stock from which 
  ccs was derived . 
 
young adult males ( at 
  least 200 flies per genotype , 10 flies per vial ) were exposed to 2 
  mm paraquat .
ccs deficiency confers early adult mortality and enhanced sensitivity to 
  the redox cycling agent , paraquat .
survival of at least 125 males of each genotype on standard cornmeal 
  food was followed at 25 c with enumeration and transfer of survivors to 
  fresh bottles every 23 days .
ccs is h340 , the 
  ccs parent stock from which 
  ccs was derived . 
 
sod1 is 
  sod1/sod1 , 
  sod1 is the wild type stock , ry . 
 
ccs is h340 , the 
  ccs parent stock from which 
  ccs was derived . 
 
young adult males ( at 
  least 200 flies per genotype , 10 flies per vial ) were exposed to 2 
  mm paraquat .
figure 4.expression of hsod1 in ccs - null drosophila . a , 
  hsod1 expressed in drosophila is enzymatically active in the absence 
  of ccs .
hsod1 
  was expressed in ccs 
 and ccs 
  flies using the gal4/uas system .
genotypes ( by lane ) are as follows : lane 
  1 , ccs / ccs ; 
  dsod1/dsod1 , 
  dagal4 ; lane 2 , 
  ccs / ccs ; 
  dsod1/dsod1 ; lane 3 , 
  ccs / ccs ; 
  dsod1/dsod1 ; 
  dagal4 ; lane 4 , 
  ccs / ccs uas - hsod1 ; 
  dsod1/dsod1 ; lane 5 , 
  ccs / ccs , uas - hsod1 ; 
  dsod1/dsod1 ; lane 6 , 
  ccs / ccs , uas - hsod1 ; 
  dsod1/dsod1 , 
  dagal4 ; lane 7 , 
  ccs / ccs , uas - hsod1 ; 
  dsod1/dsod1 , 
  dagal4 .
the uas - hsod1 transgene is on 
  recombinant 2nd chromosomes carrying ccs or 
  ccs .
genotypes are as follows : 
  ccs , ccs / ccs ; 
  dsod1/dsod1 , 
  dagal4 .
ccs , hsod1(a ) , 
  ccs / ccs , 
  uas - hsod1(a ) ; 
  dsod1/dsod , 
  dagal4 .
ccs , hsod1(a ) , 
  ccs / ccs , 
  uas - hsod1(a ) ; 
  dsod1/dsod1 , 
  dagal4 .
ccs , hsod1(b ) : 
  ccs / ccs , 
 uas - hsod1(b ) ; 
  dsod1/dsod1 , 
  dagal4 .
ccs , hsod1(b ) : 
  ccs / ccs , 
  uas - hsod1(b ) ; 
  dsod1/dsod1 , 
  dagal4 .
note that all strains are homozygous for 
  dsod1 and hemizygous for 
  dagal4 , the latter of which is known to 
  impose a mild dominant reduction of adult life span ( compare with 
  fig .
hsod1(a ) , 
  hsod1(b ) are independently selected recombinant 2nd chromosomes carrying both 
  ccs and 
  uas - hsod1 .
expression of hsod1 in ccs - null drosophila . a , 
  hsod1 expressed in drosophila is enzymatically active in the absence 
  of ccs .
hsod1 
  was expressed in ccs 
 and ccs 
  flies using the gal4/uas system .
genotypes ( by lane ) are as follows : lane 
  1 , ccs / ccs ; 
  dsod1/dsod1 , 
  dagal4 ; lane 2 , 
  ccs / ccs ; 
  dsod1/dsod1 ; lane 3 , 
  ccs / ccs ; 
  dsod1/dsod1 ; 
  dagal4 ; lane 4 , 
  ccs / ccs uas - hsod1 ; 
  dsod1/dsod1 ; lane 5 , 
  ccs / ccs , uas - hsod1 ; 
  dsod1/dsod1 ; lane 6 , 
  ccs / ccs , uas - hsod1 ; 
  dsod1/dsod1 , 
  dagal4 ; lane 7 , 
  ccs / ccs , uas - hsod1 ; 
  dsod1/dsod1 , 
  dagal4 .
the uas - hsod1 transgene is on 
  recombinant 2nd chromosomes carrying ccs or 
  ccs .
genotypes are as follows : 
  ccs , ccs / ccs ; 
  dsod1/dsod1 , 
  dagal4 .
ccs , hsod1(a ) , 
  ccs / ccs , 
  uas - hsod1(a ) ; 
  dsod1/dsod , 
  dagal4 .
ccs , hsod1(a ) , 
  ccs / ccs , 
  uas - hsod1(a ) ; 
  dsod1/dsod1 , 
  dagal4 .
ccs , hsod1(b ) : 
  ccs / ccs , 
 uas - hsod1(b ) ; 
  dsod1/dsod1 , 
  dagal4 .
ccs , hsod1(b ) : 
  ccs / ccs , 
  uas - hsod1(b ) ; 
  dsod1/dsod1 , 
  dagal4 .
note that all strains are homozygous for 
  dsod1 and hemizygous for 
  dagal4 , the latter of which is known to 
  impose a mild dominant reduction of adult life span ( compare with 
  fig .
hsod1(a ) , 
  hsod1(b ) are independently selected recombinant 2nd chromosomes carrying both 
  ccs and 
  uas - hsod1 .
mutational inactivation of ccs generates a set of 
  phenotypes that are equivalent to those from a weakly expressing sod1 
  hypomorphic mutation , and are consistent with the notion that ccs appears to 
  have a single function in drosophila , which is to mediate the 
  activation of apo - sod1 by copper .
compared with the severity of phenotypes 
  caused by sod1 deficiency ( either genomic null mutation or rna interference 
  knockdown ) , the ccs - null phenotypes are unexpectedly mild .
this likely arises 
  from a vanishingly small amount of ccs - independent sod1 activity that is at or 
  just below the threshold of detectability in fly extracts and could only be 
  discerned in concentrated lysates from yeast cells expressing dsod1 . 
 
considering the rather severe phenotype of sod1-null flies with respect to 
  adult life span and oxidative stress resistance 
  ( 10 ) , these findings suggest 
  that only a very small fraction of wild type sod1 is actually required for 
  adult viability in the absence of oxidative stress , whereas the bulk of sod1 
  is utilized in defense against oxidative stress .
this view is supported by the 
  contrasting modest and severe effects of ccs deficiency on life span and 
  paraquat resistance , respectively .
thus , the age of onset and severity of 
  early adult mortality in ccs - nulls is markedly less than in sod1-nulls , 
  whereas the hypersensitivity of ccs - nulls to paraquat is essentially 
  equivalent to that of sod1-nulls .
a , 
  an alignment of the c - terminal region of sod1 molecules from s. 
  cerevisiae , human , and drosophila , highlighting the proline 
  residues known to preclude ccs - independent activation of yeast sod1 
  ( 5 ) .
b , coding 
  sequences for s. cerevisiae , human , and drosophila sod1 were 
  placed under control of the s. cerevisiae sod1 gene promoter and used 
  to transform a yeast strain that was sod1 and either 
  ccs1 ( yccs:+ ) or ccs1 ( yccs: ) as indicated . 
 
sod1 lysates were analyzed for sod1 activity ( top ) or sod1 protein 
  levels using an antibody directed against c. elegans cu , zn - sod1 that 
  cross - reacts well with diverse sod1 molecules 
  ( 6 ) .
amounts of lysate protein 
  analyzed are as follows : lanes 16,5 g ; lane 7,10 
  g ; lane 8,25 g ; lane 9,50 g .
strains analyzed 
  include ks107 ( sod1 ) and ls101 ( sod1 
  ccs1 ) .
a , 
  an alignment of the c - terminal region of sod1 molecules from s. 
  cerevisiae , human , and drosophila , highlighting the proline 
  residues known to preclude ccs - independent activation of yeast sod1 
  ( 5 ) .
b , coding 
  sequences for s. cerevisiae , human , and drosophila sod1 were 
  placed under control of the s. cerevisiae sod1 gene promoter and used 
  to transform a yeast strain that was sod1 and either 
  ccs1 ( yccs:+ ) or ccs1 ( yccs: ) as indicated . 
 
sod1 lysates were analyzed for sod1 activity ( top ) or sod1 protein 
  levels using an antibody directed against c. elegans cu , zn - sod1 that 
  cross - reacts well with diverse sod1 molecules 
  ( 6 ) .
amounts of lysate protein 
  analyzed are as follows : lanes 16,5 g ; lane 7,10 
  g ; lane 8,25 g ; lane 9,50 g .
strains analyzed 
  include ks107 ( sod1 ) and ls101 ( sod1 
  ccs1 ) .
a sod1 ccs1 yeast strain expressing the 
  designated sod1 and ccs molecules was analyzed for sod1 activity and sod1 
  polypeptide levels using 10 g of cell lysate protein for each sample . 
 
a , strains expressing dsod1 and either wild type ( wt ) or the 
  indicated mutant versions of s. cerevisiae ccs1 or empty vector were 
  analyzed for steady state levels of the dsod1 polypeptide .
top , 
  immunoblot analysis ; bottom , quantitation of immunoblot by odyssey 
  quantitation software ( version 1.2 ) where 100 = amount of dsod1 polypeptide 
  accumulated in cells expressing wt ccs1 .
b , cells expressing dsod1 
  and either s. cerevisiae ccs1 or drosophila ccs driven by 
  the s. cerevisiae ccs1 promoter were grown in the presence of 100 
  m bathocuproine sulfonate where indicated ( + ) , and dsod1 
  polypeptide levels measured as in a where 100 = amount of dsod1 
  polypeptide with no addition of bathocuproine sulfonate .
c , cells 
  expressing either yeast , human , or drosophila sod1 in combination 
  with either yeast or drosophila ccs or with vector as indicated were 
  analyzed for sod1 activity and sod1 protein levels as 
  fig .
d , 
  cells expressing yeast sod1 and either wild type or the indicated mutant 
  versions of s. cerevisiae ccs1 were analyzed for sod1 activity and 
  protein levels as in fig . 
  5b .
a sod1 ccs1 yeast strain expressing the 
  designated sod1 and ccs molecules was analyzed for sod1 activity and sod1 
  polypeptide levels using 10 g of cell lysate protein for each sample . 
 
a , strains expressing dsod1 and either wild type ( wt ) or the 
  indicated mutant versions of s. cerevisiae ccs1 or empty vector were 
  analyzed for steady state levels of the dsod1 polypeptide .
top , 
  immunoblot analysis ; bottom , quantitation of immunoblot by odyssey 
  quantitation software ( version 1.2 ) where 100 = amount of dsod1 polypeptide 
  accumulated in cells expressing wt ccs1 .
b , cells expressing dsod1 
  and either s. cerevisiae ccs1 or drosophila ccs driven by 
  the s. cerevisiae ccs1 promoter were grown in the presence of 100 
  m bathocuproine sulfonate where indicated ( + ) , and dsod1 
  polypeptide levels measured as in a where 100 = amount of dsod1 
  polypeptide with no addition of bathocuproine sulfonate .
c , cells 
  expressing either yeast , human , or drosophila sod1 in combination 
  with either yeast or drosophila ccs or with vector as indicated were 
  analyzed for sod1 activity and sod1 protein levels as 
  fig .
d , 
  cells expressing yeast sod1 and either wild type or the indicated mutant 
  versions of s. cerevisiae ccs1 were analyzed for sod1 activity and 
  protein levels as in fig . 
 
it is also possible that the residual sod1 activity in ccs - nulls reflects 
  the presence of enzymatically active sod1 in just one or a small number of 
  cell types in which the ccs - independent activation pathway is particularly 
  active .
although we know of no direct evidence in support of this possibility , 
  the ectopic expression of the ccs - independent hsod1 in motor neurons , which 
  confers robust rescue of the sod1-null adult life span 
  ( 14 ) , suggests that motor 
  neurons do possess an active ccs - independent activation pathway ; consequently , 
  motor neurons could potentially be one such cell type that could activate 
  dsod1 in the absence of ccs . because drosophila life span is 
  sensitive to sod1 activity in motor neurons , the functioning of the 
  ccs - independent activation pathway in motor neurons could also help explain 
  why ccs deficiency has such a modest effect on adult life span relative to the 
  severe life span reduction conferred by sod1 deficiency . alternatively , and as 
  discussed later , our results are consistent with the possibility that although 
  most cells can survive under normal conditions with little sod1 , some cells 
  may require more than the residual level of sod1 activity present in ccs - nulls 
  under conditions of applied oxidative stress ( paraquat ) or increased metabolic 
  activity ( motor neurons ) . finally , it is also plausible that cell type 
  variation in copper availability could , in principle , underpin such cell type 
  specificity in ccs - independent activation of sod1 .
drosophila sod1 can now be added to the growing list of sod1 
  molecules that can obtain copper independently of ccs .
currently , the only 
  cu , zn - sod known to solely rely on ccs is the sod1 of s. cerevisiae . 
 
it is curious that yeast does not need a ccs independent pathway , yet retains 
  the ability to activate heterologous sods independent of ccs .
a likely 
  explanation is that the factors for ccs independent activation serve another 
  function in the cell .
for example , ccs independent activation may involve 
  metal binding or thiol - reactive molecules that participate in more general 
  metal homeostasis and/or redox control .
drosophila sod1 appears unique in that the apo - form is very 
  unstable in the absence of the copper chaperone .
the apparent instability is 
  not because of the drosophila host , as human sod1 expressed in 
  ccs - null flies shows no polypeptide loss , and in the heterologous yeast 
  expression system , ccs1 mutations affect loss of dsod1 but not 
  yeast or human sod1 examined in parallel .
moreover , in 
  ccs mouse models , the mammalian sod1 polypeptide is 
  stable ( 32 ) .
this loss of 
  dsod1 in the absence of ccs is in fact reminiscent of what has been observed 
  with certain amyotrophic lateral sclerosis mutants of human sod1 associated 
  with amyotrophic lateral sclerosis 
  ( 33 ) .
because ccs both inserts 
  copper and oxidizes the sod1 disulfide , either one or both of these 
  post - translational modifications must be important stabilizing factors for the 
  dsod1 molecule . prior to these studies , apo - inactive sod1 was generally thought to stably 
  accumulate in cells , allowing for rapid enzyme activation by ccs without the 
  need for new sod1 synthesis 
  ( 31 , 
  34 ) . yet drosophila 
  sod1 does not stably accumulate in the inactive apo - form .
consequently , the 
  high level of enzyme activity that is required to defend against oxidative 
  challenge would require de novo sod1 synthesis .
the inability of 
  ccs - null adults , which are constitutively deficient in sod1 activity , to mount 
  a successful defense against paraquat - induced oxidative stress is consistent 
  with this scenario .
it is also possible that available copper is limiting in 
  drosophila , and it would be disadvantageous to produce more 
  copper - binding sod1 than is needed . with the exception of certain insects , ccs molecules from diverse 
  eukaryotes
harbor an mxcxxc near the n terminus that is a 
  well characterized cu(i ) site 
  ( 35 ) . although earlier studies 
  with human ccs indicated that the mxcxxc cysteines may be 
  necessary for sod1 activation in vivo 
  ( 36 ) ,
studies with recombinant 
  ccs in vitro indicate that only the c - terminal cxc cysteines 
  are needed to bind and transfer the metal 
  ( 37 , 
  38 ) .
we observed that the 
  mxcxxc motif is not required for activation of yeast sod1 
  in vivo ; copper binding and transfer must occur exclusively via the 
  c - terminal cxc motif of ccs and the same must be true for dccs 
  lacking the n - terminal cysteines .
it remains possible that in certain species , 
  the n - terminal mxcxxc site becomes critical for maintaining 
  sod1 activity when oxygen and/or copper are limiting reagents for ccs .
if 
  true , the absence of such a motif in drosophila ccs would minimize 
  copper loading of sod1 when pools of the metal are sparse .
however , the 
  biological consequences of transient fluctuations in copper availability may 
  be minimized by the relative stability of metalated sod1 coupled with the low 
  cell turnover in the post - mitotic drosophila adult .
 copper , zinc superoxide dismutase ( sod1 ) in mammals is activated principally 
  via a copper chaperone ( ccs ) and to a lesser degree by a ccs - independent 
  pathway of unknown nature . in this study , we have characterized the 
  requirement for ccs in activating sod1 from drosophila . 
 a ccs - null 
  mutant ( ccsn29e ) of 
  drosophila was created and found to phenotypically resemble 
  drosophila sod1-null mutants in terms of reduced adult life span , 
  hypersensitivity to oxidative stress , and loss of cytosolic aconitase 
  activity . however , the phenotypes of ccs - null flies were less severe , 
  consistent with some ccs - independent activation of drosophila sod1 
  ( dsod1 ) . 
 yet sod1 activity was not detectable in 
  ccsn29e flies , due largely 
  to a striking loss of sod1 protein . 
 in contrast , human sod1 expressed in 
  ccs - null flies is robustly active and rescues the deficits in adult life span 
  and sensitivity to oxidative stress . 
 the dependence of dsod1 on ccs was also 
  observed in a yeast expression system where the dsod1 polypeptide exhibited 
  unusual instability in ccs - null ( ccs1 ) yeast . 
 the residual 
  dsod1 polypeptide in ccs1 yeast was nevertheless active , 
  consistent with ccs - independent activation . 
 stability of dsod1 in 
  ccs1 cells was readily restored by expression of either yeast 
  or drosophila ccs , and this required copper insertion into the 
  enzyme . 
 the yeast expression system also revealed some species specificity for 
  ccs . 
 yeast sod1 exhibits preference for yeast ccs over drosophila 
  ccs , whereas dsod1 is fully activated with either ccs molecule . 
 such variation 
  in mechanisms of copper activation of sod1 could reflect evolutionary 
  responses to unique oxygen and/or copper environments faced by divergent 
  species . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Supplementary Material