the formation of gap 
 junction channels between two adjacent cells 
 allows for direct cell  cell communication . functional diversity is dictated by their structural subunits , 
 a family of 21 connexin proteins in humans . each pore , known 
 as a connexon or hemichannel , makes up one - half of a gap junction 
 channel . in nonjunctional plasma membranes , connexons allow for regulated 
 passage of ions and small molecules up to 1.8 kda to and from the 
 extracellular space . in junctional plasma membranes , end - to - end docking of two apposing hemichannels enables gap junction 
 intercellular communication ( gjic ) . between adjacent membranes of 
 the same cell , connexon alignments create reflexive junctions acting 
 as conduits between membrane compartments ( e.g. , allowing for molecular 
 traffic from perinuclear to adaxonal cytoplasm between processes of 
 axon - ensheathing oligodendrocytes ) . signaling 
 specificity is achieved by  mixing and matching assemblies of identical ( homotypic channels ) or diverse 
 pairings ( heteromeric channels ) define membrane channels with different 
 permeabilities , gating properties , and docking compatibilities . only recently have connexin - specific interacting 
 partners been 
 considered part of the gap junction protein signaling nexus . connexin43 
 ( cx43 ) , the most abundant and ubiquitously expressed connexin , has 
 received the most attention with approximately 25 intracellular interactors 
 identified ( reviewed in refs ( 7,8 ) ) . transient extracellular cx43cx43 ( and cx26cx26 ) interactions 
 along extracellular docking domains also act as adhesive contacts , 
 directing the migration of neural progenitor cells along radial glia . finally , connexin complexes can also form functional protein complexes 
 in intracellular organelles . using selected ms / ms ion monitoring validated 
 by immunofluorescence , cx43 has been identified in the inner mitochondrial 
 membrane of cardiac myocytes . using classic 
 biochemical methods , cx32 has also been reported in hepatic mitochondria , 
 but without further suborganelle localization . ms methodologies , however , have fallen short of the level 
 of sensitivity required to explore these mitochondrial interactomes , 
 given difficulties associated with purifying sufficient amounts of 
 membrane - integrated connexin protein . mutations in cx32 are 
 the genetic determinant of x - linked charcot - marie 
 tooth disease , a debilitating demyelinating peripheral neuropathy . loss of cx32 ( or depletion from the plasma membrane ) 
 enhances tumorigenesis in liver . here , to identify potential 
 cx32 interacting partners that may prevent hepatic disorders when 
 complexed with cx32 , we enriched for connexin - specific membrane microdomains 
 by subcellular fractionation , and adapted 
 our ip - high throughput mass spectrometry ( ip - htms ) methodology to study endogenous integral membrane protein complexes . we show 
 that cx32 can be detected at both the plasma membrane and the inner 
 mitochondrial membrane of healthy hepatocytes and interacts with plasma 
 membrane , cytosolic , endoplasmic reticulum , and mitochondrial - resident 
 proteins . bioinformatic analysis , supported by confocal microscopy , 
 provides further support for an intriguing role for cx32 in transiently 
 tethering mitochondria to plasma membrane microdomains . klaus willecke ( universitat bonn , germany ) , were backcrossed for 15 generations onto a c57bl/6 background 
 in the bennett laboratory . male mice used in this study were between 3 and 4 months 
 of age . dna , isolated from tail snips , was amplified using 
 primers a , b , and c ( a : 5-tcattctgcttgtattcaggtgagaggcgg-3 , 
 b : 5-atacaccttgctcagtggcgtgaatcggca-3 , 
 c : 5-tcttactccacacaggcatagagtgtctgc-3 ) . primer a ( forward ) and b ( reverse ) generate a 750 bp wt amplicon . 
 primer a ( forward ) and c ( reverse ) generate a 1300 bp ko amplicon . cycling 
 parameters were : 95 c for 10 min , followed by 30 cycles of 95 
 c for 60 s , 67 c for 60 s , and 72 c for 60 s. for 
 all studies , liver was dissected and gall bladders removed from both 
 wt and ko mice . for fractionation studies , tissue was frozen in liquid 
 nitrogen and stored at 80 c until required . for 
 measurements of ferritin , transferrin , iron , and total iron binding 
 capacity , blood was collected by cardiac puncture from wt and ko mice 
 ( 7 animals per genotype ) between 1 and 8 months of age . samples were 
 clotted at rt for 1 h and centrifuged for 10 min at 1500 g. recovered serum supernatant was sent on ice to the charles 
 river research animal diagnostic services laboratory ( wilmington , 
 ma ) . transferrin saturation was calculated as the percentage of iron 
 to total iron binding capacity . for each experiment , one liver lobe was homogenized in 1 ml ptn buffer ( 50 mm sodium phosphate , 
 1% triton x-100 [ t-8787 , sigma - aldrich ] , 50 mm nacl , ph 7.4 , fresh 
 protease inhibitor cocktail ( 10 mm dtt [ promega # v3151 ] , 1 mm pmsf 
 [ omnipure # 7110 ] , 5 g leupeptin , [ pierce 78435 ] , and 1 g 
 pepstatin a [ bioshop # pep 605 ] ) using a teflon potter - elvehjem homogenizer 
 fitted to a 30 ml glass tube . homogenates were incubated on ice for 
 30 min and centrifuged at 12000 rpm for 3 min at 4 c to remove 
 nuclei . the triton x-100 supernatant ( containing the insoluble membrane 
 fraction ) was reserved on ice and diluted with an equal volume of 
 80% sucrose . resulting homogenates ( 40% sucrose ) were overlaid with 
 equal volumes of 30% and 5% sucrose in beckman polyallomer centrifuge 
 tubes , ( 14  89 mm , # 331372 ) and centrifuged at 32085 rpm ( 130000 gave ) in an sw40 ti swinging - bucket rotor overnight 
 ( 18 h ) at 4 c . gradients were carefully aliquoted ( 10 fractions 
 of 500 l ) with fraction 1 being the uppermost , lightest fraction . protein quantification was performed using the bio - rad dc protein 
 assay kit ( bio - rad , # 5000112 ) . four livers per 
 genotype ( wt and ko ) were dissected in ice - cold 
 pbs ( 10 mm phosphate , 154 mm nacl ) . pbs was decanted following collection 
 and tissue weighed prior to being minced with scissors until homogeneous . tissue pieces were washed with three changes of ice - cold solution 
 b ( 0.25 m sucrose , 1 mm edta , 2 mm mgcl2 , 20 mm hepes - naoh , 
 ph 7.4 ) . five milliliters of solution b with fresh protease inhibitors 
 ( 1 mm sodium fluoride [ sigma # s-1504 ] , 50 g / ml aprotinin [ sigma 
 # a-6279 ] , 1 mm sodium orthovanadate [ sigma # s-6508 ] , 1 mg / ml pmsf ) 
 was added to the crude lysate at twice the volume per wet weight ( g ) 
 of liver tissue ( e.g. , 5 ml buffer a per 2.5 g liver tissue ) . lysates 
 were homogenized using a tight - fitting dounce homogenizer and teflon 
 pestle ( approximately 30 strokes ) . lysates were transferred to 15 
 ml falcon tubes and solution b was added to fill the tubes prior to 
 centrifugation at 3100 rpm ( 1561 gave ) in a gh-3.8a rotor ( beckman - coulter ) at 4 c for 10 min . the 
 supernatant was collected with a pasteur pipet , transferred to 1.5 
 ml polyallomer centrifuge tubes ( beckman # 357448 , with adaptors # 355919 ) , 
 and centrifuged at 48,537 rpm ( 100000 gave ) in a tla100.3 rotor for 50 min . the supernatant ( containing 
 cytosolic proteins ) was discarded , and the pellets were combined and 
 resuspended in in a 35% op solution ( sigma # d15556 ) . the 35% op solution 
 was prepared by diluting a 50% working solution of op in solution 
 b ( 50% working solution of op : 5 parts op with 1 part diluent [ 0.25 
 m sucrose , 6 mm edta , 12 mm mgcl2 , 120 mm hepes - naoh , ph 
 7.4 ] ) . subsequent dilutions of op were also prepared by diluting the 
 50% working solution with solution b as above . a discontinuous 
 op gradient was prepared on ice by slowly overlaying 2 ml of the resuspended 
 pellets with 1 ml 30% op , 2.5 ml each of 25% and 17.5% op , and 2 ml 
 each of 10% , and 2.5% op solutions into 13.2 ml ( 14  89 mm ) 
 polyallomer centrifuge tubes . solution b was added to a point one 
 mm below the surface of the tube ( 1 ml ) . tubes were carefully 
 balanced and adjusted with solution b , if necessary , before assembly 
 into an sw41 ti rotor ( beckman coulter ) . gradients were centrifuged 
 at 36555 rpm ( 165000 gave ) at 4 
 c for 3.5 h ( acceleration = 1 , deceleration = 0 ) . the interfaces 
 between the gradients were collected in 0.5 ml volumes by tube puncture . the collected interfaces were diluted with two volumes of solution 
 b and centrifuged in a tla100.3 rotor for 50 min at 48500 rpm to collect 
 the pellet . pellets were resuspended in ripa buffer ( 10 mm pbs , 1% 
 np-40 , 0.5% sodium deoxycholate , 0.1% sds ) with fresh protease inhibitors 
 ( 1 mm sodium fluoride , 50 g / ml aprotinin , 1 mm sodium orthovanadate , 
 1 mg / ml pmsf ) and assayed for protein concentration using the bio - rad 
 dc protein assay kit . two volumes of buffer a ( 20 mm hepes , 220 mm mannitol , 68 
 mm sucrose , 80 mm kcl , 0.5 mm egta , 2 mm magnesium acetate , 1 mm dtt , 
 ph 7.4 , and fresh protease inhibitors ( 1 mm naf , 50 g / ml aprotinin , 
 1 mm sodium orthovanadate , 1 mg / ml pmsf ) were added and tissue homogenized 
 in a dounce homogenizer with teflon pestle until very smooth ( about 
 30 strokes ) . the homogenate was centrifuged at 3000 rpm ( 738 gave)in a ja25.50 rotor ( 50 ml tube ) at 4 c 
 for 20 min . the supernatant was collected with a pasteur pipet and 
 transferred to a new tube for centrifugation at 8000 rpm ( 5251 gave ) at 4 c for 15 min . this supernatant 
 was discarded and the pellet was washed with 30 ml of buffer a at 
 8000 rpm at 4 c for 15 min as before . the mitochondrial pellet 
 was resuspended in 1 ml buffer a. crude mitochondria were subjected 
 to sucrose gradient fractionation 
 with the following modifications : mitochondrial samples were mixed 
 with 80% sucrose to achieve a final concentration of 60% sucrose ( 4 
 ml total ) , and were overlaid with 50% , 40% , and 30% sucrose layers 
 ( 2 ml each ) , followed by 2 ml homogenization buffer ( minus dtt ) , and 
 centrifuged at 35000 rpm ( 151263 gave ) for 16 h at 4 c . wt and ko fractions ( wt , fractions 1822 ; ko , 
 fractions 1721 ) were pooled and protein concentrations assayed 
 using the bio - rad dc protein assay kit . each set of pooled mitochondrial 
 fractions were diluted 2:1 with cold buffer a. mitochondria were pelleted 
 by centrifugation at 8000 rpm for 15 min . pellets were resuspended 
 in cold buffer a and 0.6% digitonin ( # d141 , sigma - aldrich ) and incubated 
 on ice for 10 min to selectively permeabilize the outer mitochondrial 
 membrane . mitochondria were again collected by centrifugation at 10000 g for 10 min at 4 c . mitoplasts were resuspended in 150 
 mm kcl with fresh protease inhibitors and incubated on ice for 10 
 min prior to centrifugation at 10000 g for 
 10 min at 4 c . anti - cx32-coupled 
 protein g agarose beads were prepared as follows : one ml of a protein 
 g agarose bead slurry ( 1:1 pbs ; roche # 20399 ) was incubated with 30 
 g monoclonal anti - cx32 antibody ( cx32c13-m , alpha diagnostics ) 
 overnight at 4 c . beads were washed with 10 ml of 0.1 m borate 
 buffer ( ph 9.0 ) and resuspended in 10 ml of the same buffer . anti - cx32 
 antibody was chemically coupled to the protein g beads by the addition 
 of solid dimethyl pimelimidate dmp ( pierce # d8388 ) to a final concentration 
 of 20 mm . the 
 cross - linking reaction was stopped by washing twice with 0.2 m ethanolamine 
 ( sigma # e-9508 , ph 8.2 ) . beads were resuspended in 10 ml of 0.2 m 
 ethanolamine and incubated at room temperature for 2 h , followed by 
 two washes with 10 ml of pbs . beads were resuspended in pbs to a final 
 volume of 1 ml and stored at 4 c . one milligram of total protein 
 of pooled sucrose fractions 4 , 5 , and 6 was used as input material 
 for the 1 reaction . the pooled fractions were diluted 1:2 in 
 fresh ripa buffer with protease inhibitors ( 1 mm sodium flouride , 
 50 g / ml aprotinin , 1 mm sodium orthovanadate , 1 mg / ml pmsf ) 
 and precleared with 100 l of uncoupled protein g agarose beads 
 per 1 ml of protein lysate for 1 h at 4 c . precleared lysates 
 ( or an equal volume of ripa buffer for the no lysate ( nl ) control ) 
 were added to either 400 l ( 1 reaction ) or 2 ml ( 5 
 reaction ) anti - cx32-coupled beads and incubated overnight at 4 c . beads were washed once in ripa buffer with 250 mm nacl , once in ripa 
 buffer with 125 mm nacl , and three times in pbs ( 10 mm ) for two minutes 
 with gentle inversion . proteins were eluted at room temperature for 
 30 min with inversion in one bead volume of ammonium hydroxide elution 
 buffer ( 0.5 m nh4oh , 0.5 mm edta ) . samples were lyophilized 
 in a speedvac and solubilized in 45 l 2 sds sample buffer 
 ( 6 buffer diluted to 2 with 0.35 m tris - hcl/0.28% sds , 
 ph 6.8 buffer [ 6 buffer : [ 0.35 m tris - hcl/0.28% sds , ph 6.8 
 buffer ] , and 5% glycerol , 10% sds , 100 mm dtt , 0.002% bromophenol 
 blue ] ) with 10% -mercaptoethanol ( bme ) at room temperature 
 for 30 min . the sds - solubilized proteins were resolved on nupage novex 
 412% bis - tris gels ( np0335box , invitrogen ) at 100 v for 20 
 min , followed by 150 v until the dye front reached the bottom of the 
 gel . gels were fixed for 30 min in a 5% acetic acid / methanol solution 
 and rinsed twice with double distilled water ( ddh20 ) for 
 2 min each . to minimize background , gels were washed in ddh20 overnight with shaking at 4 c , sensitized for 2 min in a 
 0.02% sodium thiosulfate solution , and rinsed twice for 30 s each 
 with ddh20 . fresh silver nitrate ( 0.1% ) solution was incubated 
 for 30 min , followed by two 30-s washes in ddh20 . gels 
 were developed with two changes of freshly prepared 0.01% formaldehyde 
 in 2% sodium carbonate solution , rinsed with 1% acetic acid , and stored 
 in the same solution at 4 c . briefly , all surfaces in contact 
 with the gel were wiped with 70% ethanol and precautions were taken 
 to minimize contamination of gel and gel pieces with keratin and dust 
 in air . protein bands were excised on a clean glass plate with a # 11 
 scalpel blade ( blade was wiped with 70% ethanol before use , and changed 
 between each band ) . gel bands were cut into smaller pieces approximately 
 3 mm , transferred to 0.6 ml eppendorf tubes , and washed 
 by vortexing in 50 l solution a ( 50 mm ammonium bicarbonate 
 [ emd # 00113164 ] ) . gel pieces were partially dehydrated in 50 l solution b ( 50% 
 acetonitrile [ acn ; j.t.baker # 982903 ] , 25 mm ammonium bicarbonate ) 
 for 15 min at room temperature , centrifuged briefly , and the liquid 
 was discarded . samples were dried in a speedvac for 5 min without 
 heating , followed by an incubation in 30 l solution c ( 50 mm 
 ammonium bicarbonate , 10 mm dtt ) for 15 min at 56 c to rehydrate 
 the gel pieces and reduce disulfide bonds . tubes were cooled at room 
 temperature for 15 min , centrifuged briefly , and the liquid discarded . 
 to inhibit reformation of disulfide bonds , thiols were quenched by 
 adding 30 l solution d ( 100 mm iodoacetamide [ sigma # i1149 ] 
 in 50 mm ammonium bicarbonate ) in a 15 min incubation in the dark . samples were centrifuged , washed in solution a for 15 min , partially 
 dehydrated in solution b for 15 min , centrifuged briefly , and dried 
 in a speedvac for 5 min without heating . trypsin enzyme solution was 
 prepared by adding 100 l resuspension buffer to 20 g 
 porcine trypsin ( promega , # v5111 ) and mixing the resuspended enzyme 
 with 310 l of solution a ( sufficient for 10 samples ) . the enzyme 
 solution was added to each sample at 28 l / tube , incubated on 
 ice for 10 min , excess solution removed and 10 l of solution 
 e ( 25 mm ammonium bicarbonate ) added to each tube incubated at 37 
 c overnight . fifty microliters of solution e was 
 added to the gel pieces and incubated at room temperature for 20 min 
 with occasional vortexing . analytes were pooled with the other 10 
 l of solution e from the previous step . peptides were extracted 
 by incubating each sample in 50 l of solution f ( 5% formic 
 acid [ j.t . baker # 983203 ] and 50% acn ) for 20 min . the tubes 
 were centrifuged and the peptide solution was pooled into the solution 
 e washes . ms / ms was performed by dissolving the peptide samples 
 in 5% formic acid and loading into a 200 m  5 cm precolumn 
 packed in - house with 5 m ymc ods - a c18 beads ( waters , ma , # ymc086529 ) 
 using a micro agilent 1100 hplc system ( agilent technologies ) . peptides 
 were desalted online with 95% water ( j.t . baker # 983103 ) , 
 5% acn , and 0.1% formic acid ( v / v ) for 10 min at 10 l / min . the flow rate was split before the precolumn to produce approximately 
 200 l / minutes flow at the column . following elution , peptides 
 were directed to a 75 m  5 cm analytical column packed 
 with 5 m ymc ods - a c18 beads and eluted using a 1 h gradient 
 ( 15 to 80% acn with 0.1% formic acid ) into a ltq linear ion - trap mass 
 spectrometer ( thermo - electron , usa ) . ms / ms spectra were acquired in 
 a data - dependent acquisition mode automatically selecting and fragmenting 
 the five most intense peaks from each spectrum generated . peak lists 
 were generated from the ms / ms .raw file using mascot distiller 2.0.0.0 
 ( matrix science ) to produce .mgf files under default parameters with 
 the exception that for each ms / ms peak , lists were generated assuming 
 a 2 + and a 3 + charge . all .mgf files were analyzed and matched to 
 the ipi database using the mascot 2.1.04 database search engine ( matrix 
 science ) with trypsin as digestion enzyme , carbamidomethyl of cysteine 
 as a fixed modification , and methionine oxidation as a variable modification . peptide and ms / ms mass tolerances were set at 2 da and 0.8 
 da , respectively , with 1 miss - cleavage tolerance and the significance 
 threshold set to 0.01 ( p < 0.01 ) . finally , an 
 ion score cutoff of 30 was chosen to produce a false - positive rate 
 of less than 1% in the ms data . the most logical assignment of the 
 peptide in the database was reported , and when peptides matched to 
 more than one database entry , only the highest scoring protein was 
 considered . the network of proteins associating with cx32 was arranged 
 according to subcellular localization and visualized in an interaction 
 network generated using the osprey network visualization system ( osprey 
 version 1.2.0 ; samuel lunenfeld research institute ) . molecular functions 
 and protein networks were analyzed using the database for annotation , 
 visualization and integrated discovery ( david ) v6.7 and ingenuity systems pathway analysis software ( ipa version 
 8.8 , ingenuity systems inc . ) . protein interaction 
 by co - ip was performed as described above with the following minor 
 modifications : protein g beads were coupled with monoclonal cx32 or 
 polyclonal antibodies ( table 1 ) directed against 
 proteins identified by htms analysis . livers were placed in borosilicate 
 glass culture tubes ( vwr , # 47729570 ) in 2 ml ice - cold ripa 
 buffer with fresh protease inhibitors . tissue was homogenized using 
 a variable speed tissue tearor ( biospec products # 985370395 ) 
 and passed through a 26-gauge needle 810 times to break up 
 nuclei . samples were incubated on ice for 20 min , and centrifuged 
 at 16000 rcf for 30 min . postnuclear supernatants were adjusted to 
 5 mg / ml protein with fresh ripa buffer , and 1 mg of protein was used 
 in each co - ip . protein g agarose bead volumes were scaled down to 
 50 l ( antibody : bead ratio was maintained ) . in figure 2c , 50 g of input protein and 10 ul of protein 
 g agarose beads were used for the pm and mitochondrial ips . no lysate 
 ( nl ) controls were performed by substituting an equal volume of ripa 
 buffer for protein lysate . in supplemental figure 2 , supporting information , the rigg isotype control was performed 
 in parallel to the wt , ko , and nl sfxn ips , using 3.0 g rigg 
 antibody/50 l bead slurry , and 1 mg of wt protein lysate . eluted , 
 dried , and resuspended ip product was run on 12.5% tris - hcl polyacrylamide 
 gels ( prepared in - house ) and transferred to immobilon psq pvdf membranes 
 ( millipore , ma , # ipvh00010 ) for 60 min at 100 v or overnight at 22 
 v. abbreviations : 
 atp5a1 , atp synthase , 
 h+ transporting , mitochondrial f1 complex , alpha subunit 1 ; bip , heat 
 shock 70 kda protein 5 ( glucose - regulated protein , 78 kda ) ; cav-1 , 
 caveolin-1 ; cx32 , connexin32 ; cx26 , connexin26 ; coxiv , cytochrome 
 c oxidase ( complex iv ) ; flot-1 , flotillin-1 ; nakatpase , sodium  potassium adenosine triphosphatase ; sfxn-1 , 
 sideroflexin-1 ; vdac1 , voltage - dependent anion - selective channel protein 
 1 ; ip , immunoprecipitation ; icc , immunocytochemistry . bead slurry refers to a 1:1 ratio 
 of protein g agarose beads and pbs . liver isolated from wt and ko mice was 
 homogenized in ripa buffer containing protease inhibitors ( 1 mm naf , 
 50 g / ml aprotinin , 1 mm sodium orthovanadate , 1 mg / ml pmsf , 
 10 mm pbs ) . samples processed for western analysis were diluted 
 to a 1 final concentration using 6 sds sample buffer ( 0.35 
 m tris - hcl/0.28% sds , ph 6.8 , and 5% glycerol , 10% sds , 100 mm dtt , 
 0.002% bromophenol blue ) with 10% bme and solubilized at room temperature 
 for 30 min . samples subjected to ip and lyophilization were resuspended 
 in 2 final sds sample buffer . proteins were resolved under reducing / denaturing 
 conditions on 10% or 12.5% tris - hcl polyacrylamide gels ( prepared 
 in - house ) and transferred to immobilon psq pvdf membrane at 100 v 
 for 60 min . membranes were blocked in 5% ( w / v ) skim milk powder - pbs 
 ( 10 mm phosphate , 154 mm nacl ) with 0.1% tween-20 ( blocking buffer ) 
 for 1 h and incubated in primary antibody diluted in the same buffer 
 overnight at 4 c ( table 1 ) . membranes 
 were rinsed twice in 0.1% pbst ( 10 mm phosphate , 154 mm nacl with 
 0.1% tween-20 ) and twice in blocking buffer for 10 min prior to a 
 1 h incubation in hrp - conjugated antimouse or antirabbit ( jackson 
 immunoresearch laboratories , inc . , pa ; [ antimouse # 115 - 035 - 146 ; 1:2000 ] 
 and [ antirabbit # 711 - 096 - 152 ; 1:5000 ] ) secondary antibody diluted 
 in blocking buffer . signal was detected on x - ray film using supersignal 
 west pico chemiluminescent substrate ( pierce , # 34808 ) . data were expressed 
 as the percentage of sfxn-1 normalized to nak - atpase localizing to either plasma membrane or mitochondrial membranes 
 in ko samples relative to wt . t cells were maintained in dulbecco s 
 modified eagle 
 medium / f-12 ( dmem / f-12 ; invitrogen # 12400 - 024 ) with 10% fetal bovine 
 serum ( invitrogen , # 12483 - 020 ) and 1% penicillin / streptomycin antibiotic 
 cocktail . the full cdna coding sequence of the human cx32 gene ( accession 
 # : nc_000023 ) was cloned into the pcdna6/v5-his b backbone ( invitrogen , 
 # v220 - 01 ) , and the empty vector was used as a negative control . t 
 cells were plated in 6-well tissue culture plates on # 1.5 glass coverslips 
 ( zeiss # 474030 - 9000 - 000 ) . four micrograms of plasmid dna ( human cx32 
 or empty vector ) and 10 l of lipofectamine 2000 reagent ( invitrogen , 
 # 11668 - 019 ) were each diluted in 250 l warmed opti - mem reduced 
 serum media ( invitrogen , # 31985 - 088 ) and incubated for 5 min . the 
 diluted plasmid and lipofectamine solutions were gently mixed together 
 and incubated for 20 min . the medium in each well was replaced with 
 1.5 ml warmed dmem with serum ( no antibiotic ) , and 500 l of 
 dna : lipofectamine complexes . cells were incubated with transfection 
 reagents overnight , and protein expression was allowed to proceed 
 for 24 h. cells were washed 
 with 10 mm cold 
 tissue culture pbs , fixed with 3.7% formaldehyde / pbs for 10 min , and 
 washed again in pbs . fixed cells were incubated with monoclonal anti - cx32 
 ( 1 g / ml , # 138200 ) and polyclonal anti - sfxn-1 ( 2.5 g / ml ) 
 antibodies diluted in antibody buffer ( 10 mm pbs [ 10 mm phosphate , 
 154 mm nacl ] , 3% bsa , 0.3% triton - x , ph 7.2 ) overnight at 4 c . 
 cells were washed 3 in 10 mm pbs for 5 min at room temperature , 
 and incubated with cy3 antirabbit igg secondary antibody ( 1:800 , jackson 
 immunoresearch laboratories , inc . # 711 - 166 - 152 ) , and atto488 antimouse 
 igg secondary antibody ( 1:1000 , alexis biochem , # 211 - 205td - c100 ) in 
 antibody buffer for 1 h at room temperature . coverslips were washed 
 3 in 10 mm pbs for 5 min at room temperature , and mounted onto 
 glass microscope slides using prolong gold antifade reagent ( invitrogen , 
 # p36930 ) . colocalization was assessed by confocal laser scanning microscopy 
 on a leica tcs sp5 ( leica microsystems ) in z - stacks captured with 
 leica las af software ( version 2.41 ) using an hcx pl apo cs 63 
 1.4 oil objective with a pinhole size of 1 airy unit ( or equal optical 
 slices for multiple fluorophores ) . to enrich for membrane - integrated cx32 microdomains , 
 we used a modified lipid raft fractionation protocol to float triton - insoluble 
 membranes in a sucrose gradient ( figure 1a ) . given connexin antibody cross - reactivity , ko fractionations were performed in parallel to ensure specificity 
 ( supplemental figure 1 , supporting information ) . in wt lysates , cx32 was detected in fractions 4 , 5 , and 6 ( figure 1a , b ) . no labeling was observed in any of the ko 
 fractions using the same monoclonal cx32 antibody ( supplemental figure 
 1 , table 1 , supporting information ) . fractions 
 4 , 5 , and 6 were located at the interface of 5% and 30% sucrose layers 
 and in the lightest of the 30% sucrose gradients ( figure 1a ) . fractions 46 were enriched for lipid 
 raft markers caveolin-1 ( cav-1 ) and flotillin-1 ( flot-1 ) ( figure 1b , table 1 ) . fractions 4 
 and 5 were also positive for inner mitochondrial membrane markers 
 ( cytochrome c oxidase subunit iv [ coxiv ] ) ( figure 1b and table 1 ) . the endoplasmic reticulum 
 marker ( heat shock 70 kda protein 5 ( glucose - regulated protein , 78 
 kda ) [ bip ] ) was detected at low levels in fractions 5 and 6 ( figure 1b and table 1 ) . fractions 
 710 were devoid of cx32 and enriched for bip ( figure 1b and table 1 ) . ( b ) cx32 was detected in fractions 4 , 5 , and 6 
 using monoclonal anti - cx32 ( table 1 ) . sucrose fractions 46 were pooled and incubated 
 with monoclonal 
 anti - cx32 chemically coupled to protein g agarose beads ( table 1 ) . wt cx32 ip produced several bands that were visually 
 absent in the ko purification in silver - stained gels , notably in the 
 lower molecular weight range ( figure 2a , boxes , 
 wt vs ko ) . a lysate - free reaction was also prepared by incubating 
 cx32-coupled beads in ripa buffer only ( figure 2a , nl ) . immunoblotting liver lysates or 10% of the cx32 wt and ko 
 ip input with polyclonal anti - cx32 antibody ( table 1 ) demonstrated strong , specific enrichment of cx32 in pooled 
 immunopurified fractions ( figure 2a , 
 bottom panel compare wt f46 to wt ip f46 and ko , nl 
 controls ) . following excision of the unique silver - stained bands in 
 wt and corresponding regions in ko lanes ( figure 2a , boxes ) , proteins were 
 eluted from the gel pieces , digested with trypsin , and analyzed by 
 lc table 2 presents mascot scores , 
 number of peptides , and the percent coverage for each protein identified 
 from 5 mg of input protein ( pooled liver fractions 46 ) . only 
 proteins ( a ) with mascot scores of 100 or higher in one or both paired 
 analyses ( b ) for which at least two peptides were identified that 
 were ( c ) absent from ko ip - htms analyses or ( d ) for which the same 
 peptide was identified in ips of both 1 mg and 5 mg wt protein fractions 
 absent from ko met criteria to be included in the reported network 
 ( table 2 ) . the complete list of proteins detected 
 is provided as supplemental table 1 , supporting 
 information . identification of novel cx32 interactors by ip - htms of 
 cx32-enriched 
 liver membrane fractions and validation by co - ip of tissue lysates . ( a ) silver stain of proteins found in sucrose gradient fractions 46 
 ( figure 1b ) following ip with monoclonal anti - cx32 
 ( table 1 ) . negative controls included ip reactions 
 from ko fractions 46 ( supplemental figure 1 , supporting information ) and a no lysate ( nl ) mock ip reaction . ( a ) western blot of cx32-enriched fractions 46 ( f46 , 
 5 g ) and 1 l ( 10% ) of the corresponding ip products 
 from these same fractions analyzed in ( a ) . analysis confirmed presence 
 of bait ( cx32 ) in wt samples and lack of cross - reactivity in ko samples . cx32-m indicates use of monoclonal anti - cx32 ; cx32-p indicates use 
 of polyclonal anti - cx32 antibody ( table 1 ) . ( b ) ip - htms identified one known ( cx26 ) and 17 novel cx32 interacting 
 partners in replicate screens of 1 mg and 5 mg input protein . interaction 
 network generated by osprey version 1.2.0 of proteins present in wt 
 but not ko ip - htms . proteins identified in both the 1 mg and 5 mg 
 screens are represented with larger icons and thicker interaction 
 lines than proteins identified only in the 5 mg screen . ( c ) reciprocal 
 co - ips of liver lysates for cx32 and cx26 . ( d ) reciprocal co - ips for 
 cx32 and sfxn-1 . specificity was also verified in independent ips 
 using an isotype igg control ( supplemental figure 2 , supporting information ) . ( e ) ip for cx32 and blotting for 
 atp5a1 did not validate a cx32-atp5a1 interaction . peptides detected 
 in wt and not 
 ko in two independent screens of 1 mg input and 5 mg input . mascot 
 scores and peptide coverage are reported only for both the 1 mg and 
 5 mg input screen separated by /. eighteen 
 interactors were identified ( figure 2b ) , including 
 the known cx32 binding partner , cx26 . in total , 72% of the proteins identified by endogenous 
 cx32 ip - htms were intrinsic membrane proteins ; 50% were mitochondrial 
 resident proteins ( figure 2b ) . we validated 
 by co - ip one plasma membrane ( cx26 ) and two inner mitochondrial membrane 
 proteins , one novel ( sfxn-1 ) and one predicted to be an artifact of 
 detergent - insoluble sucrose fractionation ( atp5a1 ) ( figure 2c  e ) . ko controls validated specificity of the interaction , 
 which was further confirmed by immunoprecipitating wt protein using 
 an isotype control rabbit igg antibody ( chemicon ( millipore ) , # pp64 ) . 
 only the sfxn-1 antibody specifically immunoprecipitated cx32 ( supplemental 
 figure 2 , supporting information ) . protein 
 interaction between cx32 and atp5a1 ( figure 2e ) despite detection of cx32 in the crude liver homogenates ( pre - ip ) , 
 effective immunoprecipitation ( ip ) , and clearing of target protein 
 from lysate post - ip ( post - ip ) . atp5a1 was detectable in both pre and 
 post cx32 ip as well as in the ko control ( figure 2e ) . in addition to confirming antibody specificity and 
 screening for 
 nonspecific interactions , we also used cx32 null mutant liver lysates 
 to assess the effect of complex formation on the stability of validated 
 cx32 binding partners . loss of cx32 effectively reduced total cx26 
 but not sfxn-1 protein levels in liver . where immunoblotting for cx26 
 produced a band at approximately 24 kda in wt liver lysates , cx26 
 was barely detected in cx32 ko lysates ( figure 2c , top panel , lysate , supplemental figure 3 , supporting information ) . loss of cx32 had no effect on overall 
 levels of sfxn-1 , detected as a single band at approximately 35 kda 
 ( figure 2d , top panel , lysate ) . to explore the cx32-sfxn-1 interaction , 
 we sought to localize the cx32-sfxn-1 complex with increasingly rigorous 
 subcellular membrane fractionation techniques . our initial ip - htms 
 screen was performed by targeting cx32 primarily integrated into plasma 
 membrane lipid rafts . in this separation , however , lipid rafts partially 
 cofractionate with mitochondrial membranes ( figure 1a ) . to separate plasma from mitochondrial membranes , we performed 
 detergent - free fractionation of liver tissue using a discontinuous 
 op gradient ( figure 3a ) . in both wt and ko 
 samples , plasma membrane markers ( nak - atpase ) were enriched at the 210% interface ; the vdac1/porin marker 
 for outer mitochondrial membranes was detected at the 17.525% 
 interface ( figure 3b , wt m , ko m ) . in two separate 
 gradients , mitochondrial membrane contamination was never detected 
 in plasma membrane fractions ; low levels of nak - atpase , however , were consistently detected in mitochondrial fractions 
 ( figure 3b , compare nak - atpase with porin - positive fractions ) . sfxn-1 was enriched in mitochondrial 
 ( m ) but was also detected at lower levels in plasma membrane fractions 
 ( pm ) ( figure 3b ) . this finding was further 
 supported by the distribution of sfxn-1 across fractions 46 
 of our original detergent - resistant membrane sucrose gradient representing 
 both plasma membrane and mitochondrial membranes ( supplemental figure 
 4 , supporting information ) . cx32 interacts with sfxn-1 
 at plasma membrane . loss of cx32 reduces 
 sfxn-1 levels at the plasma membrane . ( a ) schematic of the optiprep 
 ( op ) gradient used to generate plasma membrane ( pm ) and mitochondrial 
 membrane ( m ) fractions from wt and ko postnuclear supernatants cleared 
 of cytosolic proteins . the plasma membrane fraction floated at the 
 interface between the 2.5% and 10% op gradient , while the mitochondrial 
 fraction was found at the interface between the 17.5% and 25% op gradient . ( b ) sfxn-1 localizes primarily to the porin - positive mitochondrial 
 fractions with some sfxn-1 detected in nakatpase - positive plasma membrane fractions . a replicate purification is presented 
 in the right panel with less mitochondrial protein loaded to highlight 
 the observation that , in ko liver tissue , sfxn-1 is reduced only in 
 the pm and not from the mitochondrial fraction . ( c ) cx32 interaction with sfxn-1 is primarily detected 
 at the pm but is also present in mitochondrial membranes following 
 biochemical fractionation . ( d ) confocal colocalization of ectopically 
 expressed cx32 ( green ) and endogenous sfxn-1 ( red ) in hek cells transfected 
 with human cx32 suggests that in situ interaction 
 is only detected at the plasma membrane at both junctional ( insets 
 and orthogonal views , arrow ) and non junctional ( arrow ) membranes . ( e ) to further purify mitochondrial membranes from plasma membranes 
 and subfractionate inner ( i m ) and outer ( om ) mitochondrial membranes , 
 sucrose gradient flotation was performed on crude mitochondria purified 
 under detergent - free conditions . fractions were assayed for expression 
 of cx32 and cellular fractionation markers ( supplemental figure 5 , supporting information ) . ( f ) in purified mitochondrial 
 preparations , cx32 localizes to i m ; sfxn-1 localizes to both i m and 
 om . next , pooled plasma membrane ( pm)-specific 
 or mitochondrial ( m)-specific 
 fractions isolated from the op gradient ( figure 3a ) were immunoblotted for cx32 ( figure 3c , 
 left panel ) . as expected , cx32 was enriched in plasma membrane fractions 
 of wt samples ( figure 3c , left panel , wt pm ) . low levels of cx32 , however , were also present in mitochondrial fractions 
 ( figure 3c , left panel , wt m ) . finally , we 
 immunoprecipitated sfxn-1 from both plasma membrane and mitochondrial 
 membrane fractions , and immunoblotted for cx32 ( figure 3c , right panel ) . the interaction between these two proteins 
 was strongest at the plasma membrane , but was also detectable in mitochondrial 
 membranes ( figure 3c , right panel ) . ko controls 
 confirmed specificity of cx32 detection in both compartments ( figure 3c , left and right panels ) . to rule out the 
 possibility that this mitochondrial pool of cx32 
 was not carry over from contaminating plasma membrane present in mitochondrial 
 fractions , we purified crude mitochondrial preparations under detergent - free 
 conditions , isolated contaminating plasma membranes from mitochondria 
 by sucrose gradient flotation ( figure 3e , supplemental 
 figure 5 , supporting information ) , and 
 separated the inner and outer mitochondrial membranes prior to western 
 blotting . as shown in supplemental figure 5 , contaminating plasma membrane in our mitochondrial preparations 
 was found in fractions 815 overlapping with fractions 1316 
 enriched for coxiv ( supplemental figure 5 ) . fractions 1723 represented pure mitochondria devoid of 
 plasma membrane contamination in both wt and ko flotations ( supplemental figure 5 ) . consistent with the op 
 gradients ( figure 3a - c ) , cx32 was enriched 
 at plasma membrane but was also clearly detectable in the mitochondria - only 
 fractions ( supplemental figure 5 ) . a biologically 
 relevant interaction between cx32 and sfxn-1 would require that both 
 interacting partners be present in the same mitochondrial membrane 
 compartments . outer mitochondrial membranes ( om ) were further isolated 
 from pooled fractions 1723 using a 0.6% digitonin solubilization 
 protocol . inner mitochondrial membranes ( i m ) in remaining mitoplasts 
 were osmotically disrupted in a 150 mm kcl solution and collected 
 by centrifugation . the om sample was enriched in vdac1/porin and devoid 
 of coxiv ( figure 3f ) . the i m sample was enriched 
 in coxiv and devoid of vdac1/porin ( figure 3f ) . sfxn-1 was 
 found equally distributed across both om and i m compartments ( figure 3f ) . while these data clearly show that cx32 
 and sfxn-1 are detected 
 at both pm and i m , biochemical fractionation can not rule out an exchange 
 of interacting proteins from different compartments postlysis . to 
 address this , we localized in situ cx32-sfxn-1 interactions 
 by confocal microscopy in human cx32-transfected hek cells ( figure 3d ) . cx32 ( green ) puncta overlap with the sfxn-1 
 ( red ) signal at both junctional membranes ( arrows ) , and nonjunctional 
 membranes ( arrows ) . this staining pattern is consistent 
 with the hypothesis that cx32 in gap junctional plaques at plasma 
 membrane can interact with sfxn-1 at the plasma membrane and/or between 
 apposed plasma and outer mitochondrial membranes , but not with the 
 hypothesis that the interactions can occur at the inner mitochondrial 
 membrane ( figure 3d ) . to explore consequences 
 of this pm - om interaction we 
 compared sfxn-1 levels in both mitochondrial and plasma membrane compartments 
 of wt and ko liver . we found that localization of sfxn-1 at the plasma 
 membrane , but not at inner and outer mitochondrial membranes , was 
 reduced by 54  17% in ko liver as determined by densitometry 
 of replicate independent fractionations compared to wt ( 100% ) ( figure 3b ) . this change represented a shift in subcellular 
 localization ( figure 3b ) without altering total 
 sfxn-1 levels ( figure 2d ) suggesting that plasma 
 membrane cx32 likely interacts with sfxn-1 resident in outer mitochondrial 
 membrane and that detection of sfxn-1 at plasma membrane may represent 
 a subpool of sfxn-1 removed from apposing mitochondrial membranes 
 during co - ip without affecting total cellular levels . taken together , the interaction of cx32-sfxn-1 
 at the plasma membrane , and reciprocal localization at the inner mitochondrial 
 membrane suggested the presence of overlapping organelle - specific 
 cx32 protein networks in liver . to explore the nature of these networks , 
 we performed functional annotation clustering using david and protein interaction network analysis using 
 ingenuity systems pathway analysis software ( figure 4 ) . cx32 interactors clustered into two main functional classifications : 
 ( 1 ) drug metabolism , lipid metabolism , molecular transport and ( 2 ) 
 endocrine system development and function , molecular transport , cellular 
 development . pathway analysis identified three overlapping pathways 
 implicated in ( 1 ) liver metabolic response to adipose - derived leptin , 
 ( 2 ) liver response to insulin / insulin receptor signaling , and ( 3 ) 
 interferon gamma responsiveness / iron metabolism impaired in hepatic 
 carcinoma ( figure 4a ) . dysfunction in leptin 
 and insulin pathways , potentially mediated through disruptions in 
 cx32 interactions with slc27a5 , slc25a10 , ass1 , abcd3 , slc25a5 , and 
 gp6 ( figure 4a ) is consistent with previous 
 reports that c57bl/6x129/sv cx32 null mutant mice exhibit a small 
 reduction ( 17% ) in body weight also observed in our colony backbred to a pure c57bl/6 lineage ( data 
 not shown ) . proposed regulation of interferon gamma responsiveness / iron 
 metabolism by a cx32 interactome with sfxn-1 , pmse1 , pmse2 , and pebp-1 
 ( figure 4a ) is , however , novel . to test directly 
 whether cx32 ko mice show any dysfunction in these pathways , we measured 
 circulating ferritin ( figure 4b ) , transferrin 
 ( figure 4c ) , iron ( figure 4d ) , total iron binding capacity ( figure 4e ) , and percent transferrin saturation ( figure 4f ) . cx32-null mutant mice exhibited a mild , significant hyperferritinemia 
 ( figure 4b ) without any indication of systemic 
 iron overload ( figure 4c  e ) or changes 
 in transferrin saturation ( figure 4f ) . taken 
 together , this phenotype is consistent with hyperferritinemia observed 
 following liver injury and as a result of systemic inflammatory conditions . ( a ) 
 proteins ( listed by gene name ) are represented as nodes ( shapes indicate 
 functional annotation ) . a documented biological relationship between 
 two nodes is represented as a gray or orange edge ( line ) . gray edges 
 represent interactions within a single network ; orange edges cross - link 
 nodes from multiple interacting networks . proteins identified in our 
 screen are represented as gray nodes with black edges ( lines ) . white 
 nodes represent gene products that participate in these networks , 
 but were not identified in this study . the direct cx26cx32 
 interaction validated in our screen is a known interaction and thus 
 appears as a gray edge . all gray and orange edges are supported by 
 references maintained by the ingenuity pathways knowledge base . solid 
 gray edges represent direct interactions ; indirect interactions are 
 represented by dashed lines . circular lines describe a feed - back loop 
 wherein a node has been shown to self - regulate . ( b ) serum analysis 
 of blood collected from wt and ko animals reveals that ko animals 
 exhibit mild hyperferritinemia , without concurrent changes in serum 
 transferrin ( c ) iron ( d ) , total iron binding capacity ( e ) , or % transferrin 
 saturation ( f ) . here , 
 we report a novel endogenous cx32-interacting network in 
 mouse liver identified by immunoaffinity purification of cx32-enriched 
 subcellular membrane microdomains . using unbiased ip - htms , we mapped 
 18 interacting partners . fifty percent were resident mitochondrial 
 proteins , 22% were cytoplasmic proteins , 17% localize to the endoplasmic 
 reticulum , and 11% were predicted to be resident in plasma membrane . 
 by reciprocal co - ip , subcellular fractionation , and confocal microscopy , 
 we found that cx32 forms protein  protein complexes at the plasma 
 membrane ( i.e. , with cx26 ) and either sequesters sfxn-1 at the plasma 
 membrane or forms protein  protein complexes between cx32 and 
 sfxn-1 resident in apposing plasma membranes and outer mitochondrial 
 membranes . bioinformatic analysis places these interactions into three 
 overlapping networks : ( 1 ) liver metabolic response to adipose - derived 
 leptin , ( 2 ) liver response to insulin / insulin receptor signaling , 
 and ( 3 ) interferon gamma responsiveness / iron metabolism . the observation 
 that cx32 , integrated into plasma membrane , can interact with resident 
 outer mitochondrial membrane proteins further suggests a novel role 
 for connexins in possibly transiently tethering mitochondria to connexin - enriched 
 plasma membrane domains ( figure 5 ) . together , 
 these data highlight a novel plasma membrane / mitochondrial signaling 
 nexus mediated by the cx32 interactome . cx32 ( orange ) forms heterotypic and heteromeric gap junction 
 channels with cx26 ( blue ) . sfxn-1 ( red ) is present on both outer and inner mitochondrial 
 membranes , and possibly at plasma membrane . sites of cx32-sfxn-1 interaction 
 include ( 1 ) plasma membrane gap junctional plaques either as ( i ) transient 
 interactions , ( ii ) novel plasma membrane resident proteins , ( iii ) 
 part of the docking mechanism tethering subplasmalemmal mitochondria 
 to the plasma membrane , or ( iv ) to promote the transport of small 
 molecules into mitochondria , and ( 2 ) the inner mitochondrial membrane 
 ( unknown role ) . this study represents 
 the first endogenous connexin network mapped 
 by unbiased ms approaches . the majority of reported connexin interactors 
 have been identified using ectopically expressed tagged proteins , 
 or exogenous peptides as  bait  . these data have provided essential 
 insight into channel - independent connexin function , notably for cx43 , however , tissue - specific network 
 analyses are lacking . to our knowledge , only two ms - based proteomic 
 screens have been performed , both using a c - terminal fragment of cx43 
 as bait . the primary obstacle has been the challenge 
 associated with affinity purifying endogenous membrane channel complexes 
 in sufficient amounts for ms detection . to address this , we enriched 
 for cx32-positive membrane microdomains by subcellular fractionation 
 prior to ip - htms . success of this strategy was 
 validated by the fact that our top  hit was the known 
 cx32 binding partner , cx26 . cx26 and 
 cx32 have been shown to colocalize in heterotypic ( and presumably 
 heteromeric ) channels in liver hepatocytes . moreover , we also find that cx26 protein levels are reduced in liver 
 of cx32 ko mice , consistent with earlier reports . this reduction is likely due to a loss of cx26 protein 
 stability in absence of cx32/cx26 complexes , as steady - state cx26 
 mrna levels in wt and ko liver are comparable . certainly , further improvements can be made to increase 
 identification 
 of connexin - interacting proteins by ip - htms . we have recently developed 
 a gel - free centrifugal proteomic reactor to analyze membrane proteins and have begun to adapt this technology to analyze 
 fractionated membrane microdomains enriched in cx32 . our centrifugal 
 proteomic reactor is a simplified , user - friendly , adaptation of gel - free 
 digestion . we exploit the fact that most proteins are positively charged 
 at ph <3 and thus a protein / trypsin mixture will bind tightly to 
 a small volume of strong cation exchange ( scx ) beads while nonionic 
 detergents and contaminants are readily washed away . we then use dtt and iodoacetamide for protein reduction 
 and alkylation , activate the trypsin by increasing ph to 8 , and elute 
 the resulting peptides in alkaline buffer for lc  ms / ms analysis . using this gel - free methodology , we have been 
 able to detect 945 plasma membrane proteins and 955 microsomal membrane 
 proteins in hepatic cells . more than 
 800 out of these 945 and 955 proteins were not identified by the conventional 
 in - gel digestion approach . we anticipate 
 that these improvements will further expand upon this study s 
 identification of a mitochondrial protein - connexin interactome . application 
 will require further optimization as we have found that the phospholipid - cx32 
 interactions in our fractionated microdomains 
 complicate the required ph - dependent binding to cationic beads . despite 
 clear presence of cx32 in ip samples , detection of bait connexins 
 by htms we are currently exploring the efficacy of mild 
 cross - linking followed by delipidation to circumvent this problem 
 and increase the number of connexin binding proteins we can identify . despite the fact that we may be able to identify more interacting 
 partners in future , we report here that the majority of novel cx32 
 interactors detected were resident mitochondrial proteins . in previous 
 targeted approaches , , cx32 has been shown to 
 colocalize with the tight junction proteins occludin and claudin-1 , 
 and the scaffolding protein zona occludens-1 . in the brain , oligodendrocytic cx32 colocalizes with zo-1 and the 
 zo-1-associated nucleic acid - binding protein . in lipid rafts , cx32 interacts with caveolin-1 , the ubiquitously 
 expressed calcium binding protein , calmodulin , and the membrane - associated 
 guanylate kinase family member , discs large homolog-1 ( dlgh-1 ) . cx32 presumably sequesters dlgh-1 at the plasma membrane as acute 
 removal enables its translocation to the nucleus . here , we show that the cx32-sfxn-1 interaction may have 
 a similar effect with cx32 sequestering a subpool of the resident 
 mitochondrial protein sfxn-1 to the plasma membrane . comparison of 
 our quantitative biochemical analyses and confocal localization studies , 
 however , suggests that the primary function of the cx32-sfxn-1 interaction 
 is likely not to regulate localization of binding partners . first , in situ interaction with sfxn-1 at the plasma membrane as 
 assessed by confocal microscopy is not ubiquitous in cx32-overexpressing 
 heks . second , unlike the effect of cx32 null - mutation on cx26 stability , 
 overall sfxn-1 protein levels are not significantly altered , nor is 
 mitochondrial subcellular localization determined by biochemical assessment 
 in cx32 ko liver lysates . rather , by confocal assessment in heks ectopically 
 expressing human cx32 , small , regionally distinct pools of sfxn-1 , 
 apparently at the outer mitochondrial membrane , colocalize with plasma 
 membrane - resident cx32 . in null mutant liver lysates , sfxn-1 levels 
 at plasma membrane are quantitatively reduced compared to wt , as determined 
 by direct biochemical assay and mass spectrometry . taken together , these observations provide tantalizing support for the emerging hypothesis 
 that connexins may transiently tether mitochondria to the plasma membrane 
 when binding partners come into apposition , further supported by evidence that analagous complex associations 
 with cx43 are transient , yet strong enough to be coimmunoprecipitated . despite this evidence , we were mindful 
 that mitochondrial contaminants 
 routinely copurify with detergent - resistant lipid raft preparations 
 and are often misinterpreted as bona fide interactors . spurious interactions with f(1)/f(0 ) atpase 
 subunits and voltage - dependent anion selective channels are common . here , we used co - ip and bioinformatic analyses 
 to rule out cx32 interactions with the atpase subunit atp5a1 and the 
 unidentified protein similar to atp synthase , h+ transporting , mitochondrial 
 f1 complex , o subunit identified in our ip - htms screen . moreover , f(1)/f(0 ) atpase subunits 
 are not predicted to participate in the three main pathways identified 
 by ingenuity pathway analysis . our validation , however , of sfxn-1-cx32 
 complexes at both plasma and mitochondrial membranes is consistent 
 with a previous report that cx32 is present in isolated liver mitochondria . indeed , cx43 is certainly capable of translocating 
 to the inner mitochondrial membrane of cardiomyocytes , where cx43 channels protect cells from excitotoxic 
 insult . it is , however , important to note that in this study , while we have 
 biochemical evidence indicating that cx32 and sfxn-1 are both present 
 in inner mitochondrial membranes , we do not have evidence to suggest 
 that an interaction occurs at this location . in situ imaging indicates that the primary interaction between these two 
 proteins occurs between plasma membrane - localized cx32 and outer mitochondrial 
 membrane sfxn-1 . thus , while our data is the first demonstration that 
 cx32 is present at inner mitochondrial membranes , we must conclude 
 that the low level cx32-sfxn-1 interaction detected by ip in isolated 
 mitochondrial membranes is likely due to contamination of these fractions 
 with trace amounts of plasma membranes as shown in our data . given 
 that intracellular interactions between cx32 and sfxn-1 were rarely 
 detected in our hek cell transfectants , we favor the hypothesis that 
 the interaction seen in tissue lysates is due to transient interactions 
 of outer mitochondrial membrane sfxn-1 with cx32-containing gap junction 
 plaques . sfxn-1 is a five - membrane spanning integral membrane 
 protein predicted to be either a channel or carrier protein . mutations 
 in sfxn-1 are the genetic determinant of transient embryonic and neonatal 
 anemia presented in the flexed - tail ( f / f ) mouse model , wherein defects in iron metabolism lead to a 
 transient iron overload and intramitochondrial iron deposits in erythrocytes . interestingly , presentation of iron defects 
 coincides with the onset of hepatic erythropoiesis , and persists through 
 postnatal day 7 subsiding between the second and third week postnatal . this time course corresponds closely with the 
 developmental increase in hepatic cx32 levels that do not reach adult 
 levels until postnatal day 21 . iron overload 
 has been shown to reduce the presentation of cx32 at plasma membrane 
 in favor of intracellular distribution . thus , iron deficits in sfxn-1-deficient mice may promote the mitochondrial 
 localization of cx32 , although this hypothesis remains to be tested . 
 here , we asked the reverse question : what effect does cx32 null mutation 
 have on ferritin status and iron metabolism ? based on the predicted 
 involvement of cx32 protein  protein interactions with the interferon 
 gamma response in hepatocytes , we highlight a novel regulation of 
 isolated hyperferritinemia observed in the absence of iron overload , 
 that is characteristic of dysregulated chronic inflammatory responses . ferritin elevations are seen in a number of 
 liver disorders including alcoholic and nonalchoholic steatohepatitis , 
 viral hepatitis , and acute liver injury . moreover , elevated ferritin levels are associated with insulin resistance 
 and purported to be the hepatic manifestation of metabolic syndrome . our data implicate the connexin - interacting 
 nexus identified here in leptin , insulin , and interferon gamma signaling 
 pathways . interestingly , these pathways also link the cx32 interactome 
 identified in this study to the regulation of liver tumorigenesis . for example , the interactions between cx32 and the mitochondrial 
 solute carrier proteins slc25a10 and slc25a5 as well as one of rate - limiting 
 synthases in the arginine biosynthesis pathway ( ass1 ) are predicted 
 to underlie the metabolic response to leptin in hepatocytes , notably 
 in the exchange of citrate for malate between cytosol and mitochondria 
 required for fatty acid synthesis , lipogenesis , and the regulation 
 of protein catabolism , ammonia levels , and nitric oxide generation . cx32 interaction may act to stabilize membrane presentation , as 
 shown here and by others for cx26 , given 
 that both cx32 and slc25a5 are downregulated in human heptatocellular 
 carcinomas . certainly , mutations in ass1 
 that impair protein stability are known causes of citrullinemia type 
 i leading to liver failure , as well ethanol - induced liver injury . this pathway analysis is consistent with previous reports that cx32 
 null mutant mice are mildly underweight compared to controls . further , given that sfxn-1 likely facilitates 
 transport of components , 
 notably pyridoxine , required for iron utilization into and out of 
 the mitochondria , its function may depend 
 upon a network of channel transporters moving required components 
 through plasma membrane to mitochondria . the interaction of cx32 at 
 the plasma membrane and sfxn at the outer mitochondrial membrane could 
 serve to tether a pool of subplasmalemmal mitochondria to the plasma 
 membrane , and potentially facilitate transport of pyridoxine into 
 the mitochondria . in support of this hypothesis , another cx32 interactor 
 identified in this study , ant-2 , has been shown to function as both 
 a mitochondrial and plasma membrane translocase . moreover , the known cx32 interactor 
 caveolin-1 also binds to the abcd transporters , and our identification of a potential cx32-abcd3 interaction , 
 possibly including caveolin-1 in complex , provides further support 
 for the idea that cx32 participates in the tethering of mitochondria 
 to lipid raft domains . as cx32 localizes to the basolateral membrane 
 domains of hepatocytes wherein the majority of transporters are located , these interactions would presumably enable transient 
 mitochondrial docking at site of metabolic import ( figure 5 ) . similar functional interactions have been described 
 in developing muscle cells between the l - type calcium channel 2/1 
 subunit and the atp5b subunit of the mitochondrial atp synthase complex . in summary , this work describes the identification 
 of a network 
 of liver - specific cx32 interacting proteins enriched in mitochondrial 
 targets . these interactions were validated by co - ip and detergent - free 
 subcellular fractionation , and provide evidence for a novel signaling 
 nexus made possible by cx32 interactions with resident proteins at 
 plasma and mitochondrial membranes that are implicated in liver function 
 and hepatocyte growth , differentiation , and tumor progression .
connexins 
 are the structural subunits of gap junctions and act 
 as protein platforms for signaling complexes . 
 little is known about 
 tissue - specific connexin signaling nexuses , given significant challenges 
 associated with affinity - purifying endogenous channel complexes to 
 the level required for interaction analyses . here , we used multiple 
 subcellular fractionation techniques to isolate connexin32-enriched 
 membrane microdomains from murine liver . 
 we show , for the first time , 
 that connexin32 localizes to both the plasma membrane and inner mitochondrial 
 membrane of hepatocytes . using a combination of immunoprecipitation - high 
 throughput mass spectrometry , reciprocal co - ip , and subcellular fractionation 
 methodologies , 
 we report a novel interactome validated using null 
 mutant controls . 
 eighteen connexin32 interacting proteins were identified . 
 the majority represent resident mitochondrial proteins , a minority 
 represent plasma membrane , endoplasmic reticulum , or cytoplasmic partners . 
 in particular , connexin32 interacts with connexin26 and the mitochondrial 
 protein , sideroflexin-1 , at the plasma membrane . 
 connexin32 interaction 
 enhances connexin26 stability . converging bioinformatic , biochemical , 
 and confocal analyses 
 support a role for connexin32 in transiently 
 tethering mitochondria to connexin32-enriched plasma membrane microdomains 
 through interaction with proteins in the outer mitochondrial membrane , 
 including sideroflexin-1 . 
 complex formation increases the pool of 
 sideroflexin-1 that is present at the plasma membrane . 
 together , these 
 data identify a novel plasma membrane / mitochondrial signaling nexus 
 in the connexin32 interactome .