( a ) c16:0 to c18:0 elongase activity is significantly blunted in delovl6 mutants , whereas elongase activities on other fatty acids measured are not affected . microsomal preparations from control or delovl6 mutant animals were incubated with radioactive malonyl - coa and the indicated fatty - acyl - coa . elongation was quantified by incorporation of the aqueous metabolite malonyl - coa into lipid soluble fatty acids , as described in . ( b ) gas chromatography flame ionization detector ( gc - fid ) analysis reveals that delovl6 mutant larvae have reduced levels of c18:0 , the product of elovl6 elongase activity . ( c ) lethality of delovl6 mutants is fully rescued to expected mendelian ratios by ubiquitous expression ( with actin - gal4 ) of delovl6 from a uas transgene ( chi square test 1.149<3.841= where p=0.05 , n=141 , * * * * p<0.0001 ) . ( d ) human elovl6 and drosophila elovl6 are functionally equivalent , since the lethality of delovl6 mutant flies is fully rescued to expected mendelian ratios by ubiquitous expression ( with actin - gal4 ) of human elovl6 from a uas transgene ( chi square test 2.38<3.841= where p=0.05 , n = 76 , * * p<0.01 ) ( e ) the lethality of delovl6 mutant flies is most strongly rescued by c18:0 , the product of elovl6 . synchronized 1 instar larvae of indicated genotypes were grown on standard food supplemented with indicated fatty acids ( 5% ) . ( f ) 
 delovl6 mutants are not hypersensitivite to drugs such as g418 ( protein biosynthesis inhibitor ) or etoposide ( topoisomerase inhibitor ) . 30 synchronized l1 larvae were grown in vials with food supplemented with either g418 ( 50g / ml ) or etoposite ( 25m ) . oxygen consumption was measured in the presence of only tmpd as substrate , which can be directly oxidized by complex iv . the values were corrected for non - mitochondrial oxygen consumption ( oxygen consumption in the presence of complex iv inhibitor kcn ) and normalized to tissue weight . ( n=3 ) ( h , i ) overexpression of pgc1 ( fly spargel ) in delovl6 mutant female prewandering larvae leads to increased mitochondrial abundance , assessed by porin levels ( h , representative of 6 biological replicates ) and citrate synthase activity ( i , n=4 ) in pre - wandering larvae . see supplementary information figure 12 for image of the uncropped full western blot . ( j ) delovl6 ( either n or c terminally tagged ) localizes to the mitochondrial outer membrane . s2 cell lysates (  total  ) were successively fractionated to yield crude mitochondria ( which include mitochondrial - associated membranes , mams ) , pure mitochondria ( lacking mams ) , and mitochondrial outer membranes ( om ) , inner membranes ( i m ) and inter - membrane space ( ims ) . endogenous porin and atpsyn- were used as positive controls for om and i m respectively . ( k ) lipidomic analysis of standard fly food reveals low levels of c18:0 in the food . ( a , c , d , e , f , g , i ) . p0.05 , * p<0.05 , * * p<0.01 , of two tailed t - test . ( a ) lipidomic ( gc - fid ) profiles of purified mitochondria from delovl6 mutant 3 instar larvae do not show major differences compared to control animals . ( b - c ) 
 delovl6 mutant larvae do not have reduced amounts of mitochondria , quantified via levels of porin ( b , representative of 3 biological replicates ) or citrate synthase activity ( c , n=3 ) . ( d ) 
 delovl6 mutant larvae have fragmented mitochondria , which is rescued by dietary c18:0 supplementation . mitochondrial morphology from fat bodies of control or elovl6 mutant female larvae , fed control or c18:0 ( 10% ) supplemented food , visualized with mito - gfp . images are representative of 8 areas of 4 larvae from each genotype and food conditions . ( e - e ) only c18:0 , and not shorter , longer , or desaturated fatty acids , restores mitochondrial fragmentation to control levels in hela cells grown in medium containing delipidated serum . mitochondria were visualized with mitotracker ( red ) ( e ) and mitochondrial fragmentation was quantified by normalizing the number of mitochondrial particles to total mitochondrial area ( e ) . ( n=15 ) ( f - f  ) reduced lipoic acid ( la ) levels do not lead to mitochondrial fragmentation . lipoic acid synthase ( lias ) was knocked down by rnai in hela cells , leading to significantly reduced la levels , assayed by immunoblotting of total cell lysates with antibody detecting la ( f  ) . unlike removal of c18:0 , this does not lead to mitochondrial fragmentation . representative images in ( f ) , quantified in ( f ) ( n=6 ) . ( g , h ) hela cells growing in medium containing delipidated serum do not display reduced levels of protein lipoylation ( g ) or reduced levels of lipoylated proteins ( h ) . hela cells were grown in medium containing delipidated serum for either 24 hours ( the same timepoint used for all other experiments in this manuscript where mitochondrial fragmentation is assessed ) ( g - h ) , or for an extended period of time  4 days ( h ) . lipoic acid levels were assayed by immunobloting total cell lysates with an anti - lipoic acid antibody ( g ) , and levels of lipoylated proteins were assessed with specific antibodies ( h ) . p0.05 , * p<0.05 , * * p<0.01 , of two tailed t - test , error bars represent s.d . all scale bars : 10m . ( a ) 
 dmfn ( official gene symbol marf ) knockout larvae ( 1 instar ) have fragmented mitochondria , visualized with mito - gfp . ( b ) endogenous mfn runs as a main band plus a laddering of apparently increasing molecular weights on an sds - page gel . specificity is controlled by blotting lysates from control and mfn knockout female larvae with anti - mfn antibody . mfn knockout larvae survive for multiple days as small l1/l2 larvae and eventually die . synchronised 1 instar larvae were grown on standard fly food and examined every 24 hours for developmental stage and percent survival ( n=30 ) . oxygen consumption of inverted , digitonin permeabilized , female larval tissues was measured with an oroboros oxygraph chamber and normalized to tissue weight . oxygen consumption was measured in the presence of the following substrates : gmn ( glutamate and malate ) , gmd ( glutamate , malate and adp ) , gmcd ( glutamate , malate , cytochrome c and adp ) , gmscd ( glutamate , malate , succinate , cytochrome c and adp ) , ets ( glutamate , malate , cytochrome c , adp and uncoupling reagent ) , and sc(rot)u ( glutamate , malate , cytochrome c , adp and rotenone ) . ( n=5 ) ( e ) endogenous dmfn is post - translationally modified in a c18:0-dependent manner in drosophila . mfn2 from delovl6 female mutants migrates in an sds - page gel differently , compared to mfn2 from control animals . all indicated bands are dmfn , since they disappear in lysates from dmfn knock animals  see extended data figure 3b . ( f ) endogenous mfn2 is post - translationally modified in a c18:0-dependent manner in human hela cells . mfn2 immunoprecipitated from hela cells treated 24 hours with medium containing standard or delipidated serum , and then 2 hours in the absence or presence of c18:0 ( 100m ) , lysed in 8 m urea  see methods . mfn2 is more heavily ubiquitinated in cells treated with delipidated serum than in control cells and this is reversed by supplementing the medium with c18 . hela cells were cotransfected with tagged versions of mfn2 ( myc ) and ubiquitin ( ha ) . quantification of ubiquitination , normalized to myc - mfn2 in the ip is shown below the lane . a cyclohexamide ( chx ) chase experiment was performed to block de novo synthesis of mfn2 , thereby looking at turnover of existing mfn2 protein in vivo . hela cells treated with medium containing delipidated serum plus / minus c18:0 were treated with 100m chx and then lysed at indicated time points to compare mfn2 protein levels . dmfn transcript levels in 24-hour female prewandering larvae were determined by quantitative rt - pcr , normalized to rp49 ( in triplicates ) . p0.05 , * p<0.05 , * * p<0.01 , * * * p<0.001 of two tailed t - test , error bars represent s.d . ( a ) sirna depletion of other ubiquitin ligases targeting mfn ( besides huwe1 , shown in main figure 3 ) does not rescue the mitochondrial fragmentation induced by removal of c18:0 ( a ) , quantified in ( a ) . ( n=15 ) ( b ) sirna depletion of park2 in hek293 cells , as in hela cells ( a ) , does not rescue the mitochondrial fragmentation induced by removal of c18:0 ( b ) , quantified in ( b ) . ( n=15 ) ( c ) huwe1 knock down efficiency controlled by detecting huwe1 protein levels . ( d ) survival to pupation of elovl6 mutants is fully rescued by ubiquitous expression ( daughterless - gal4 ) of rnai targeting dhuwe1 . elovl6 mutants expressing huwe1 rnai survive to pupation at expected mendelian frequencies ( chi square test 0.86<3.841= where p=0.05 ) . p0.05 , * p<0.05 , * * p<0.01 , * * * p<0.001 of two tailed t - test , error bars represent s.d . ( a ) the mitochondrial associated membranes ( mam ) band is strongly reduced or absent in percoll gradients of crude mitochondrial fractions from delovl6 mutant animals , compared to controls . ( b ) purity control of mitochondrial preps show that pure mitochondrial fractions are lacking markers of other subcellular organelles such as calnexin ( er ) and lamin ( nuclei ) . see supplementary information figure 17 for image of the uncropped full western blot . ( b ) quantification of ( b ) shows that levels of the er marker calnexin are reduced in crude mitochondrial fractions from delovl6 mutants , compared to controls , in agreement with reduced mams in delovl6 mutants . values show densitometry ratios of calnexin levels in crude mitochondrial fractions , normalized to total lysate calnexin . ( c - c  ) electron microscopy of drosophila s2 cell mitochondria ( c ) reveals cristae abnormalities in delovl6 depleted cells , quantified in ( c ) ( n=200 ) . significance of the difference was calculated with a mann - whitney test ( * p<0.05 ) . ( n=200 , * * * * p<0.0001 of two tailed t - test ) ( c and c  ) error bars : s.d . ( a ) c18:0 removal for 24 hours does not induce expression of upr target genes , quantified by quantitative rt - pcr , normalized to hrpl19 . bip is a readout for ire1 activation , chop is a readout for atf6 activation , and perk is a readout of its own activation due to a positive transcriptional feedback loop . y - axis is displayed in logarithmic scale to fit all datapoints on one graph . ( b ) p - eif2 , a upr marker , does not increase upon removal of c18:0 whereas it is induced by tunicamycin , a positive control . ( c - c ) knocking down mediators of the upr response does not inhibit mitochondrial fragmentation upon c18:0 removal . hela cells were transfected with either control sirnas or sirnas targeting upr mediators as indicated . ( c ) shows the mitochondrial fragmentation index and ( c ) shows representative images . ( n=15 ) ( d - d ) inhibiting er stress by means of a chemical chaperone , tudca , does not rescue mitochondrial fragmentation upon c18:0 removal . ( d ) shows mitochondrial fragmentation index ( n=15 ) and ( d ) shows representative images . ( a , c , d ) * p<0.05 , * * p<0.01 of two tailed t - test , error bars : s.d . ( a ) c17:0-azide is a functional analog of c18:0 in that it induces mitochondrial fusion in hela cells , whereas other c18:0 derivatives are not . ( b - b ) transferrin receptor ( tfr1 ) is the most enriched protein in a c17:0-azide pull - down , and it regulates mitochondrial morphology . hela cells were treated with c17:0-azide for 2 hours , and covalently bound proteins were precipitated by lysing cells under denaturing conditions ( 8 m urea ) , and linking the c17:0-azide to an alkyne - labelled resin via click chemistry ( b ) . precipitated proteins were identified by mass spec , and peptide counts were normalized to peptide counts in a negative control pull - down from cells not treated with c17:0-azide ( n=3 ) ( b , column 2 ) . indicated proteins were also tested by sirna - mediated knockdown for effects on mitochondrial morphology ( column 3 ) . ( c ) tfr1 is covalently bound to the c18:0 derivative c17:0-azide in hela cells in a zdhhc6-dependent manner . helas were treated with c17:0-azide for 2 hours , and subsequently lysed in denaturing conditions ( 8 m urea ) . similar to panel ( b ) , the c17:0-azide was  clicked  onto a biotinylated alkyne , and the labelled proteins were pulled - down with streptavidin beads . after washing , immunoprecipitated proteins were eluted off beads in laemmli buffer containing biotin , and analyzed by immunoblotting . the palmitic acid analog c15:0-azide was used as a positive control since tfr1 is known to also be palmitoylated . c17:0-azide pulls down more tfr1 than equal amounts of c15:0-azide , indicating that tfr1 palmitoylation can not account for the c17:0 signal . hela cells were transfected with either control or tfr1 targeting sirnas prior to treatement with medium containing delipidated serum + / c18:0 . representative images are shown here and quantification of mitochondrial fragmentation is shown in the main figure 3h . ( n=15 ) ( a , a ) hela cells can not grow in the presence of dfo , an iron chelator ( a ) whereas they grow in delipidated serum lacking c18:0 at a comparable rate to cells in control medium ( a ) . ( n=3 ) ( b , b ) treatment of hela cells with medium containing delipidated serum ( lacking c18:0 ) for 24h does not lead to transcriptional activation of iron deficiency response genes ( b ) , which are activated by dfo - mediated iron chelation ( 24h ) as a positive control ( b ) . ( n=3 ) ( c ) treatment of hela cells with medium containing delipidated serum for 24 hours or 4 days does not lead to a drop in levels of the succinate dehydrogenase protein sdhb , which contains an fe - s cluster . ( d - f  ) treatment of hela cells with medium containing delipidated serum for 24 hours or 4 days does not lead to a drop in activities of enzymes containing lipoylated subunits ( pdh and ogdh ) ( d - e ) or fe - s cluster containing subunits ( sdh ) ( f - f  ) . dfo treatment to chelate iron from the medium , or sirna - mediated depletion of the enzymes were used as positive controls ( d , e , f  ) . ( g , g ) treatment of hela cells with medium containing delipidated serum ( 24h ) does not cause a reduction in transferrin uptake . cells were treated with 25 g / ml alexa 488 coupled transferrin for 30 minutes . representative images ( g ) and quantification of the amount of transferrin per cell in ( g ) ( n=5 ) . ( h ) treatment of hela cells with medium containing delipidated serum ( 24h ) does not reduce association of transferrin containing vesicles with mitochondria . crude mitochondria were fractionated from cells growing in medium containing or lacking c18:0 , and the amount of transferrin that copurifies with mitochondria was analyzed and quantified by immunoblotting . ( a , a , b , b , d , d , e , e , f , f , f p0.05 , * p<0.05 , * * * p<0.001 , * * * * p<0.0001 of two tailed t - test , error bars : s.d . ( a ) treatment of hela cells with 1m gambogic acid does not induce apoptosis . 10 m , gambogic acid was used as a positive control for apoptosis induction , assessed by cleaved caspase-3 levels . 1m gambogic acid neither induces caspase cleavage ( shown here ) nor causes cells to die ( not shown ) . cells were treated with 10m gambogic acid for 1 hour , or for all other concentrations for 3 hours . ( b ) activation of tfr1 by treating cells with 1m gambogic acid leads to mitochondrial fragmentation which is reversed by 1 hour c18:0 pretreatment . representative images are shown here and quantification of mitochondrial fragmentation is shown in main figure 3i ( n=15 ) . ( c ) treatment of hela cells with c18:0 to inhibit tfr1 causes reduced jnk signaling activity , assayed by p - jun kinase levels on an immunoblot . ( d , d ) removal of c18:0 , as well as treatment with gambogic acid , induces shuttling of phosphorylated jun kinase into the nucleus . cells were stained with phosho jun kinase antibody ( d ) and relative levels of nuclear to cytosolic phospho jun kinase signal was quantified ( d ) ( n=37 cells ) . * * * p<0.001 of two tailed t - test . hela cells were treated with the jnk inhibitor sp600125 30 minutes prior to gambogic acid treatment to activate tfr1 . representative images are shown here and quantification of mitochondrial fragmentation is shown in main figure 3j ( n=15 ) . ( a , b ) dietary c18:0 supplementation ( 10% ) significantly increases lifespan of male parkin ( a ) and pink1 ( b ) mutant flies . ( c ) dietary c18:0 supplementation rescues atp levels of 1-week old male pink1 mutant adult flies . ( d - d ) dietary c18:0 supplementation significantly improves locomotor defects of 2-week old male pink1 mutant flies . locomotion quantified as animals climbing up past a threshold in given amount of time ( technical duplicates , biological quadruplicates , 10 animals per assay ) . ( e ) 
 parkin loss - of - function in flies leads to mitochondrial fragmentation which is rescued by dietary supplementation with c18:0 . guts from 14-day old female control or park mutant adult flies expressing mitogfp and grown on food supplemented + / c18:0 ( 10% ) were dissected and mitochondria were imaged . ( 3 animals per condition , 6 optical areas per animal ) for panels ( b - d ) control flies are the revertant line pink1 . ( a , b , c , d , d , e ) error bars show s.d . , * p<0.05 , n.s .
summarymitochondria are involved in a variety of cellular functions including atp production , amino acid and lipid biogenesis and breakdown , signaling and apoptosis1 - 3 . 
 mitochondrial dysfunction has been linked to neurodegenerative diseases , cancer , and aging4 . 
 although transcriptional mechanisms regulating mitochondrial abundance are known5 , comparatively little is known about how mitochondrial function is regulated . 
 we identify here the metabolite stearic acid ( c18:0 ) and transferrin receptor ( tfr1 ) as mitochondrial regulators . 
 we elucidate a signaling pathway whereby c18:0 stearoylates tfr1 , thereby inhibiting its activation of jnk signaling . 
 this leads to reduced ubiquitination of mitofusin via huwe1 , thereby promoting mitochondrial fusion and function . 
 we find that animal cells are poised to respond to both increases and decreases in c18:0 levels , with increased c18:0 dietary intake boosting mitochondrial fusion in vivo . 
 intriguingly , dietary c18:0 supplementation can counteract the mitochondrial dysfunction caused by genetic defects such as loss of the parkinsons genes pink or parkin . 
 this work identifies the metabolite c18:0 as a signaling molecule regulating mitochondrial function in response to diet .