C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1

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1 C. elegans mitohonrial fator WAH-1 promotes phosphatiylserine externalization in apoptoti ells through phospholipi sramlase SCRM-1 Xiaohen Wang 1,, Jin Wang, Keiko Gengyo-Ano 3, Lihuan Gu 4, Chun-Ling Sun 1, Chonglin Yang 1, Yong Shi 1, Tetsuo Koayashi 3, Yigong Shi 4, Shohei Mitani 3, Xiao-Song Xie an Ding Xue 1, Externalization of phosphatiylserine, whih is normally restrite to the inner leaflet of plasma memrane, is a hallmark of mammalian apoptosis 14. It is not known what ativates an meiates the phosphatiylserine externalization proess in apoptoti ells. Here, we report the evelopment of an annexin V-ase phosphatiylserine laelling metho an show that a majority of apoptoti germ ells in Caenorhaitis elegans have surfae-expose phosphatiylserine, iniating that phosphatiylserine externalization is a onserve apoptoti event in worms. Importantly, inativation of the gene enoing either the C. elegans apoptosis-inuing fator (AIF) homologue (WAH-1), a mitohonrial apoptogeni fator, or the C. elegans phospholipi sramlase 1 (SCRM-1), a plasma memrane protein, reues phosphatiylserine exposure on the surfae of apoptoti germ ells an ompromises ell-orpse engulfment. WAH-1 assoiates with SCRM-1 an ativates its phospholipi sramling ativity in vitro. Thus WAH-1, after its release from mitohonria uring apoptosis, promotes plasma memrane phosphatiylserine externalization through its ownstream effetor, SCRM-1. Although phosphatiylserine externalization is a wiesprea phenomenon in mammalian apoptosis 14, it is unlear whether it ours uring apoptosis in other organisms. To examine whether phosphatiylserine is externalize in apoptoti ells in C. elegans, we evelope a gentle surgial proeure to expose C. elegans gonas an then use fluoresene-onjugate annexin V, a highly speifi phosphatiylserine-ining protein, to stain apoptoti germ ells that are on the surfae of gonas. When expose gonas from e-1(e17) animals (whih are efetive in ellorpse engulfment an thus allow assay of more germ-ell orpses 7 ) were staine, % of germ-ell orpses were laelle with annexin V, resulting in right ring-shape staining (Fig. 1a, ). We onfirme that the ells laelle with annexin V orrespone to apoptoti germ ells y their raise is-like morphology uner Nomarski optis an onense Hoehst 3334 hromosomal DNA staining pattern, harateristi of apoptoti ells. These phosphatiylserine-positive ells were not staine with propiium ioie an thus were not neroti or amage germ ells (ata not shown). In aition, no annexin V staining was oserve in expose gonas of e-1(e17); e-3(n717) animals (Fig. 1), in whih all germ-ell eaths were loke y a e-3(n717) loss-of-funtion mutation, proviing further onfirmation that annexin V only stains apoptoti ells. A higher perentage of phosphatiylserine staining (84%) was oserve in germ-ell orpses in animals (Fig. 1), whih are also efetive in ell-orpse engulfment 7. In aition, animals of ifferent ault ages ha similar perentages of germ-ell orpses staine with annexin V (see Supplementary Information, Tale S1). γ-irraiation was also use to inue apoptosis in the germline of wil-type animals an resulte in a similar perentage of germ-ell orpses (%) with surfae-expose phosphatiylserine (Fig. 1). These results iniate that phosphatiylserine is expose on the surfae of a signifiant portion of germ-ell orpses in C. elegans, an that phosphatiylserine externalization is a onserve apoptoti event in C. elegans. This phosphatiylserine staining protool was then use to searh for regulatory fators an enzymes involve in promoting phosphatiylserine externalization uring C. elegans apoptosis. It has een reporte previously that miroinjetion or overexpression of human AIF in ulture ells an inue surfae phosphatiylserine exposure 8,9. However, it is unlear whether AIF has a iret ausal effet on phosphatiylserine externalization. This possiility was examine y using RNA interferene (RNAi) to reue C. elegans wah-1 expression an then staining for phosphatiylserine in e-1(e17); wah-1(rnai) animals. Compare with % of germ-ell orpses staine in e-1(e17); ontrol(rnai) animals, only % of germ-ell orpses in e-1(e17); wah-1(rnai) animals were laelle with annexin V (Fig. 1). Similarly, 1 Department of Moleular, Cellular, an Developmental Biology, University of Colorao, Bouler, CO 839, USA. MDermott Center for Human Growth an Development, University of Texas Southwestern Meial Center, Dallas, TX 739, USA. 3 Department of Physiology, Tokyo Women s Meial University, Shool of Meiine, an CREST, JST, Tokyo, 1-8, Japan. 4 Department of Moleular Biology, Prineton University, Prineton, NJ 844, USA. Current aress: National Institute of Biologial Sienes, Beijing,, China. Corresponene shoul e aresse to D.X. ( ing.xue@olorao.eu) Reeive 13 Otoer ; aepte 8 Marh 7; pulishe online 1 April 7; DOI:.38/n74 NATURE CELL BIOLOGY VOLUME 9 NUMBER MAY 7 41

2 a e-1(e17) e-1(e17); e-3(n717) Nomarski Annexin-V Hohest3334 Annexin/Hohest None wah-1-n RNAi wah-1-c RNAi e-1(e17) N γ-irraiation srm-1(tm98) e Pie-1 Pie-1 Pie-1 GFP GFP GFP WAH WAH-1 147K44E Array None e-1(e17) Figure 1 wah-1 promotes phosphatiylserine exposure on the surfae of apoptoti germ ells. (a, ) The expose gona of a e-1(e17) (a) hermaphroite animal or a e-1(e17); e-3(n717) () animal was staine with annexin V an Hoehst Germ-ell orpses were ientifie y their raise-utton-like morphology uner the Nomarski optis an the onense Hoehst 3334 staining pattern. Nomarski, annexin V, Hoehst 3334 an merge images of annexin V an Hoehst 3334 staining of the gonas are shown. Germ-ell orpses staine with oth Hoehst an annexin V are iniate y white arrows. Those that were staine y Hoehst ut not y annexin V are iniate with re arrows. () reues germ-ell orpse phosphatiylserine staining in e-1(e17) an e- (n1994) animals. experiments were performe using three ifferent RNAi onstruts. The onstrut ontains full-length wah-1 DNA sequene. wah-1-n RNAi an wah-1-c RNAi ontain 1 ase pair an 3 ase pair DNA sequenes erive from the amino an aroxyterminal regions of wah-1, respetively. The perentage of phosphatiylserine signifiantly reue germ-ell orpse phosphatiylserine staining in animals. The perentages of germ-ell orpses laelle with annexin V were similar in e-1(e17); wah-1(rnai) animals of ifferent ault ages (see Supplementary Information, Tale S1) an in animals treate with two aitional onstruts (Fig. 1), wah-1-n RNAi an wah-1-c RNAi, whih ontain targeting sequenes erive from the amino-terminal an aroxy-terminal regions of wah- 1, respetively, suggesting that the effet of on phosphatiylserine exposure is speifi an inepenent of age. These results iniate that the ativity of wah-1 is important for phosphatiylserine externalization in apoptoti germ ells. staining (y axis) was alulate y iviing the numer of germ-ell orpses with phosphatiylserine staining y the numer of total germ-ell orpses sore 48 h after the L4 to ault moult. () Phosphatiylserine exposure in apoptoti germ ells inue y γ-irraiation. Wil-type or srm-1(tm98) animals were age 4 h after L4 to ault moult efore irraiation at 1 Gy. Annexin V staining of apoptoti germ ells was performe an sore 3 h after irraiation. Data shown in an are mean ± s.e.m. from three inepenent experiments. In eah experiment, germ-ell orpses from 1 gona arms were sore. (e) Overexpression of wah-1 in C. elegans germline promotes phosphatiylserine exposure in apoptoti germ ells. The GFP-fusion onstruts shown on the left were injete into the e-1(e17) animals to reate omplex DNA arrays that failitate gene expression in the germline. F an F3 generations of transgeni animals were use for annexin V staining. Eah numere array represents an inepenent transgeni line an at least germ-ell orpses from 1 gona arms were sore for eah line. The sale ar in a represents. µm. We then examine whether wah-1 an promote phosphatiylserine externalization in C. elegans germ ells y overexpressing the ative form of WAH-1 (WAH ) uner the ontrol of the pie-1 promoter in an in-frame fusion with GFP (P pie-1 GFP::WAH ; refs, ). Overexpression of WAH in germ ells i not inrease the numer of germ-ell eaths in e-1(e17) animals (see Supplementary Information, Fig. S1), whih is onsistent with our previous oservation that overexpression of WAH alone oes not ause etopi somati ell eath in C. elegans. However, overexpression of WAH in germ ells signifiantly inrease the perentage of germ-ell orpses staine with annexin V 77% of germ-ell orpses from the 4 NATURE CELL BIOLOGY VOLUME 9 NUMBER MAY 7

3 a e e-1(n199) e-1(e17) e-7(n9) e-7(n94) Ault age (germ ells) f 4 4 n = n = 3 n = n = g h i j Comma L Comma L1 n = 4 e-7(n94) 4 n = n = n = 3 n = n = 4 e-1(e17) n = 4 n = n = 3 n = Comma L1 Comma L e-1(n199) e-7(n9) e-7(n1994) n = n = Comma Developmental stages (somati ells) n = Figure affets the learane of apoptoti ells. (aj) L1 or L larvae from the following animals were treate with wah-1 RNAi (fille ars) or ontrol RNAi (empty ars) as previously esrie : e-1(n199) (a, f), e-1(e17) (, g), e-7(n9) (, h), e- 7(n94) (, i) an (e, j). The L4 larval stage progeny of the treate animals were transferre to fresh RNAi plates an their progeny were sore for ell orpses. In ae, the numers of germ-ell orpses were sore every 1 h after the L4 to ault moult from one gona arm of the animals. The y axis iniates the average numer of germ-ell orpses. The error ars represent s.e.m. Fifteen animals were sore for eah time point in ae. In fj, the numers of somati-ell orpses were sore at the following emryoni or larval stages: ean or omma (omma), 1.-fol (1.), twofol (),.-fol (.), threefol (3), fourfol (4) an early L1 larvae (L1). The y axis iniates the average numer of ell orpses ounte in the hea region of emryos or larvae. The error ars represent s.e.m. Fifteen animals were sore for eah evelopmental stage in fj unless otherwise iniate. In all panels, ata erive from or ontrol RNAi treatment at multiple time points or evelopmental stages were ompare y two-way analysis of variane. Post ho omparisons were one y Fisher s protete least squares ifferene (PLSD). Single asterisks iniate P <. an oule asterisks iniate P <.1. All other points ha P values >.. NATURE CELL BIOLOGY VOLUME 9 NUMBER MAY 7 43

4 a N tm98 tm8 SCRM-1 CstF N srm-1(tm98) srm-1(tm8) N (33 ± 3 min) tm98 ( ± 3 min) tm8 (7 ± 3 min) Comma 1.-fol -fol.-fol Developmental stages 3-fol 4-fol Duration of ell orpses (min) Comma N srm-4(tm4) srm-(tm); srm-3(tm31) 1.-fol -fol.-fol Developmental Stages 3-fol 4-fol e N (33 ± 3 min) srm-4 srm-; srm-3 (34 ± min) Duration of ell orpses (min) f Anti-SCRM-1 (FITC) FITCDAPI h Anti-SCRM-1 (FITC) FITCDAPI g i Figure 3 SCRM-1 is a plasma memrane protein important for ell-orpse engulfment. (a) Western lot analysis of the srm-1 eletion mutants. C. elegans lysates were mae from the iniate mix-stage animals an western lot analysis was performe using affinity-purifie anti-scrm- 1 antioy. C. elegans spliing fator CstF-4 was use as a loaing ontrol. (e) Time-ourse analysis of emryoni-ell orpses in the srm- 1 mutants () or in the srm-4 srm-; srm-3 triple mutant (). Cell orpses were sore at six emryoni stages an analyse statistially as esrie in Fig.. Fifteen animals were sore at eah stage. The error ars iniate s.e.m. Single asterisks iniate P <. an oule asterisks iniate P <.1. All other points ha P values >.. Fourimensional mirosopy analysis of ell-orpse urations in the srm-1 mutants () or the srm-4 srm-; srm-3 triple mutant (e) are also shown. The urations of 31 ell orpses eah from N emryos (n = 4, open ars), srm-1(tm98) emryos (n =, lak ars), srm-1(tm8) emryos (n =, grey ar) an srm triple-mutant emryos (n =, lak ars in e) were measure. The numers in parentheses iniate the average urations of ell orpses (± s.e.m.) from eah genotype. The y axis iniates the numer of ell orpses within a speifi uration range (shown on the x axis). (fi) SCRM-1 loalizes to plasma memrane. Images of FITC (SCRM- 1 antioy staining) an FITCDAPI C. elegans emryos are shown: a 1- ell stage wil-type (f) or srm-1(tm8) (g) emryo an a wil-type (h) or srm-1(tm8) (i) emryo at approximately -ell stage. The sale ars iniate 1 µm. 44 NATURE CELL BIOLOGY VOLUME 9 NUMBER MAY 7

5 e-1(e17) animals transgeni for the P pie-1 GFP::WAH onstrut were staine (Fig. 1e). In omparison, less than 3% of germ-ell orpses were staine in e-1(e17) animals arrying either a ontrol transgene (P pie-1 GFP) or a transgene expressing a mutant WAH-1 protein that fails to ativate its effetor (P pie-1 GFP::WAH-1 147(K44E) ; see elow, Fig. 1e an Supplementary Information, Fig. Sa). As overexpression of WAH-1 inreases phosphatiylserine externalization, whereas wah-1 RNAi reues phosphatiylserine exposure in apoptoti germ ells, wah- 1 may iretly ativate the phosphatiylserine externalization proess in apoptoti ells. Given that surfae-expose phosphatiylserine an at as an eat-me signal to inue phagoytosis 11, we examine whether wah-1 affets ell-orpse engulfment in C. elegans. has een shown to elay normal progression of apoptosis, ompromise apoptoti DNA egraation an inhiit ell killing in sensitize geneti akgrouns. However, no ovious ell-orpse engulfment efet was etete in wah- 1(RNAi) animals. To examine this more arefully, animals that were partially efetive in ell-orpse engulfment were treate with wah-1 RNAi an we examine whether enhane the engulfment efet in these mutants. Geneti analyses in C. elegans have ientifie several genes that funtion in two separate pathways to meiate orpse removal in oth germ ells an somati ells, with e-1, e- an e-7 funtioning in one pathway an e-, e-, e- an e- 1 in the other 1. Doule mutants of genes ating in the same pathway have weaker engulfment efets than oule mutants of genes ating in ifferent pathways 13. Interestingly, strongly enhane the engulfment efet in e-1(n199) animals, whih ontain a partial loss-of-funtion allele of e-1 (ref. 14). In e-1(n199); wah-1(rnai) animals, the numers of persistent germ-ell orpses were signifiantly higher than those of the e-1(n199); ontrol(rnai) animals in four ault stages examine (Fig. a). The enhanement of the persistent germ-ell orpse phenotype was weaker in e-1(e17); wah-1(rnai) animals (Fig. ), whih ontain a putative null allele of e-1 (ref. 14). similarly enhane the germ-ell orpse engulfment efet assoiate with oth weak (n9) an strong (n94) alleles of e-7 (Fig., ), ut not that onferre y either weak or strong mutations in the e-, e-, e- an e-1 genes (Fig. e an ata not shown), whih funtion in a ifferent engulfment pathway. ause a similar efet in removal of somati apoptoti ells: it enhane the somati ell-orpse engulfment efets assoiate with oth weak an strong e-1 an e-7 mutations (Fig. fi), ut not those of the e-, e-, e- an e-1 mutants (Fig. j an ata not shown). Altogether, these oule-mutant analyses learly iniate that wah-1 funtions in the engulfment pathway meiate y e-, e-, e- an e-1 in phagoytes 1 to affet ell orpse learane, proaly through promoting phosphatiylserine externalization in ying ells. Consistent with this moel, i not enhane the engulfment efet of the psr-1(tm49) mutant (see Supplementary Information, Fig. S3a), whih is efetive in PSR-1 a putative phosphatiylserine-reognizing reeptor that funtions in the e-, e-, e- an e-1 engulfment pathway 1. As WAH-1 oes not ontain any omain or iohemial property that is suggestive of its iret involvement in transilayer lipi movement, it may funtion through a lipi-transloating enzyme to promote phosphatiylserine externalization in apoptoti ells. Phospholipi sramlases (PLSCRs), whih have een impliate in promoting ATP-inepenent, iiretional lipi sramling 1719, oul e potential targets. A BLAST searh using the sequenes of four human phospholipi sramlases ientifie eight sramlase homologues in C. elegans (see Supplementary Information, Fig. S4a an Supplementary Methos), all of whih ontain a onserve Ca ining motif an a putative transmemrane omain 17,. They were name srm genes for sramlases. Of the eight SCRM proteins, SCRM-1 is most homologous to four human PLSCRs an thus was haraterize first (see Supplementary Information, Fig. S4). To investigate the funtions of srm-1, two eletion mutations were generate in the srm-1 lous (see Supplementary Information, Fig. S4), srm-1(tm98) an srm-1(tm8). Both eletions aolishe SCRM-1 protein expression in mutant animals ase on the western lot analysis using anti-scrm-1 antioies (Fig. 3a), an thus are strong loss-offuntion or null mutations. In a time-ourse analysis of emryoni ell orpses, more ell orpses were oserve in srm-1(tm98) an srm- 1(tm8) emryos than in wil-type emryos (Fig. 3). The inrease in ell orpses in all emryoni stages in the srm-1 mutants is likely to e ause y a efet in ell-orpse engulfment, as four-imensional mirosopy analyses of the uration of emryoni ell orpses iniate that ell orpses in oth srm-1 mutants on average persiste longer than those in wil-type emryos. Speifially, the average ell-orpse uration in srm-1(tm98) an srm-1(tm8) emryos was an 7 min, respetively, ompare with 33 min in wil-type emryos (Fig. 3). These results iniate that the ell-orpse engulfment proess is ompromise in the srm-1 mutants. We also examine whether srm-1 affets phosphatiylserine externalization in apoptoti ells an foun that srm-1 eletions reue phosphatiylserine exposure on the surfae of germ-ell orpses: for example, 84% of germ-ell orpses were laelle with annexin V in e- (n1994) animals (Fig. 4a), whereas in srm-1(tm98); an srm-1(tm8); oule mutants, only 9 an 7% of germ-ell orpses were staine, isplaying % an 17% of reutions in staining, respetively. Similar reutions in phosphatiylserine staining were oserve in germ-ell orpses in srm-1(tm98); e-(n181) an srm-1(tm8); e-(n181) animals (Fig. 4a), as well as in germ-ell orpses of γ-irraiate srm-1(tm98) animals (Fig. 1), onfirming that the srm-1 mutations reue phosphatiylserine externalization on the surfae of apoptoti germ ells. As oth srm-1 eletions mutations ause signifiant, ut not strong, efets in ell-orpse engulfment an phosphatiylserine externalization, we examine whether other C. elegans SCRM proteins ontriute to these two proesses. Three eletion mutations (tm, tm31 an tm4) were isolate in three losely relate SCRM-1 paralogues (srm-, srm-3 an srm-4; see Supplementary Information, Fig. S4 an Supplementary Methos) an these mutations alone i not ause any efet in ell-orpse engulfment (see Supplementary Information, Fig. S3) or phosphatiylserine exposure in apoptoti ells (Fig. 4). Moreover, ell-orpses engulfment seeme normal in a triple mutant of these paralogues, srm-4(tm4) srm-(tm); srm-3(tm31), as its emryoni ell-orpse numers an average uration were inistinguishale from wil-type animals (Fig. 3, e). Phosphatiylserine staining in the srm triple mutant was only milly reue (Fig. 4). Moreover, treatment of srm-4(tm4) srm-(tm); e-(n181); srm-3(tm31) animals with srm-1 RNAi, whih fully phenoopies srm-1 eletions in reuing phosphatiylserine exposure NATURE CELL BIOLOGY VOLUME 9 NUMBER MAY 7 4

6 a srm-1(tm98); srm-1(tm8); e-(n181) n = 4 srm-1(tm98); e-(n181) srm-1(tm8); e-(n181) e-(n181) srm-1(tm98); e-(n181) srm-(tm); e-(n181) n = n = e-(n181); srm-3(tm31) srm-4(tm4); e-(n181) n = srm-4(tm4) srm-(tm); e-(n181); srm-3(tm31) None 9 srm-1 RNAi e-(n181) srm-4(tm4) srm-(tm); e-(n181); srm-3(tm31) n = srm-1(tm98); srm-1(tm8); e-(n181) srm-1(tm98); e-(n181) srm-1(tm8); e-(n181) Figure 4 srm-1 an wah-1 funtion in the same pathway to promote phosphatiylserine exposure in apoptoti ells. (a) srm-1 ut not srm-, srm-3 or srm-4 affets phosphatiylserine exposure in apoptoti germ ells. () srm-1 an wah-1 funtion in the same pathway to promote phosphatiylserine externalization. Phosphatiylserinestaining assays were arrie out in various srm; e- an srm; e- mutants, or srm; e- an srm; e- mutants treate with srm-1 in apoptoti ells, i not result in further reution of phosphatiylserine staining (Fig. 4), suggesting that srm-, srm-3 an srm-4 o not ontriute signifiantly to phosphatiylserine externalization uring apoptosis. Iniviual RNAi treatment of the four remaining srm genes (srm-, srm-, srm-7 an srm-8) i not proue an ovious efet in phosphatiylserine exposure (see Supplementary Information, Fig. S3). Thus, of the four worm sramlases that were genetially analyze, srm- 1 was the only one that signifiantly affete phosphatiylserine externalization in apoptoti ells an the ell-orpse engulfment proess. To examine the expression pattern an suellular loalization of SCRM-1 in C. elegans, rait polylonal antioies against SCRM-1 were generate. In western lot analyses, affinity-purifie SCRM-1 antioies speifially reognize a protein an with the preite RNAi or an sore as esrie in Fig. 1. The genotypes of the animals staine y annexin V are iniate. For eah set of experiments, multiple ifferent strains in tripliates were sore linly for phosphatiylserine staining 48 h after the L4 to ault moult. The error ars iniate s.e.m. Data shown represent three inepenent experiments unless iniate otherwise. In eah experiment at least germ-ell orpses from 1 gonaal arms were sore. relative moleular mass of SCRM-1 (3,) in wil-type worm lysate; this an was asent in lysates from oth srm-1 eletion mutants (Fig. 3a). Wil-type C. elegans emryos staine with anti- SCRM-1 antioies isplaye a plasma-memrane staining pattern that was oserve in all ells throughout emryogenesis (Fig. 3f, h an ata not shown), whereas no speifi staining was oserve in srm-1(tm8) mutant emryos (Fig. 3g, i). Similar plasmamemrane loalization of SCRM-1 was oserve in germ ells of wil-type animals, ut not in those of srm-1(tm8) animals (see Supplementary Information, Fig. S, e). This plasma-memrane loalization pattern of SCRM-1 is onsistent with its oserve role in promoting phosphatiylserine reistriution in the plasma memrane uring apoptosis. 4 NATURE CELL BIOLOGY VOLUME 9 NUMBER MAY 7

7 a Input LUC SCRM-1 SCRM-1(M) M r (K) 7 37 Luiferase SCRM-1 SCRM-1(M) MBP MBPWAH-1 MBPWAH-1 K44E WAH-1 17 Interation with SCRM-1 WAH WAH WAH WAH WAH-1 19 WAH-1 19 WAH-1 38 K38 K38 H393 K491 R31 K44 4 EGTA. mm Ca 1 mm Ca e Perentage NBDPS protetion 3 Perentage NBDPS protetion 3 Buffer SCRM-1 WAH-1 SCRM-1 SCRM-1 SCRM-1(M) WAH-1 WAH-1 K44E WAH Reation time (h) Figure WAH-1 interats speifially with SCRM-1 to ativate its phospholipi-sramling ativity. (a) WAH-1 assoiates with SCRM-1 in vitro. Purifie MBP, MBPWAH-1 or MBPWAH-1 K44E protein (. µg eah) immoilize on the amylose resin was inuate with S-methioninelaelle (iniate y asterisk) luiferase (Lu), SCRM-1 or SCRM-1 mutant (M). The oun proteins were resolve on a 1% SDSPAGE an etete using a phosphoimager. Lanes 13: % of the laelle proteins use in the ining reations. () WAH-1 38 is suffiient to meiate ining to SCRM- 1. Purifie GST or GSTSCRM-1 (. µg eah) immoilize on glutathione Sepharose eas was inuate with similar amounts of the full-length or trunate WAH-1 proteins with a six histine tag. The oun proteins were resolve on SDSPAGE gels an visualize y western lot analysis using an antioy against the His tag. Interation etween WAH-1 an SCRM-1 is iniate. () Strutural moelling of WAH-1. The human AIF struture is use to iniate the surfae loations of the mutate resiues in WAH-1. Resiues in human AIF that orrespon to the mutate resiues in WAH-1 are highlighte in yellow (K38, K388, H393, K491 an R31) an re (K44). Eah resiue was sustitute with glutamate. Lys 44, ut not the other five resiues, is ritial for the ining of WAH-1 to SCRM-1. () WAH-1 ativates the phospholipi-sramling ativity of SCRM- 1. Purifie SCRM-1 ( µg) an WAH (1. µg), either alone or together, were reonstitute with µg liposomes, to whih NBDPS (.% in moles) was ae after the proteoliposomes were seale. SCRM- 1(M) WAH-1, WAH-1 K44E SCRM-1 or a uffer-only ontrol without any protein were similarly reonstitute with liposomes. The proteoliposomes prepare as suh were equally ivie into six aliquots, supplemente with either 4 mm EGTA or CaCl at the iniate onentration, an inuate at 37 C in the ark for h. The sramlase ativity was measure an presente as perentage NBDPS protetion as esrie in etail in Methos. (e) Time-ourse analysis of SCRM-1 phospholipi-sramling ativity. The sramlase ativity assays were arrie out as esrie in, exept that aliquots were remove an fluoresene was measure at the iniate times. Data shown in an e represent three inepenent experiments, eah in upliates. The error ars iniate s.. NATURE CELL BIOLOGY VOLUME 9 NUMBER MAY 7 47

8 We next examine whether wah-1 an srm-1 at aitively to promote phosphatiylserine exposure in apoptoti ells. treatment of srm-1(tm98); or srm-1(tm8); animals i not result in further reution of phosphatiylserine staining in apoptoti germ ells ompare with ; wah-1(rnai) animals (Fig. 4). Similar results were oserve with treatment of e- an srm-1; e- animals (Fig. 4). These results suggest that wah-1 an srm-1 are likely to funtion in the same pathway to promote phosphatiylserine externalization. We then examine whether WAH-1 interats with SCRM-1 in vitro using a maltose ining protein (MBP)-fusion protein pullown assay. In vitro-synthesize S-methionine-laelle SCRM-1 was pulle own speifially y MBPWAH-1, ut not y MBP alone or y a mutant MBPWAH-1 K44E protein (Fig. a an see elow). In aition, a SCRM-1 mutant protein in whih all ysteine resiues were replae with alanine i not in to any of the MBPWAH-1 or MBP proteins. Moreover, MBPWAH-1 faile to in SCRM-, SCRM-3 or SCRM-4 (see Supplementary Information, Fig. Sa, ), whih is onsistent with the oservations that these three sramlases o not affet phosphatiylserine externalization in worms. Taken together, these results strongly suggest that WAH-1 interats speifially with SCRM-1 in vitro. To ientify the region of WAH-1 that is important for ining to SCRM- 1, the ining of SCRM-1 to various WAH-1 trunations was examine an a 17-resiue region of WAH-1 (amino ai 38) was shown to e suffiient for ining of WAH-1 to SCRM-1 (Fig. ). The WAH-1 struture was then moelle on the human AIF protein struture 1 an six potential surfae resiues were ientifie in this region that may affet the ining of WAH-1 to SCRM-1 (Fig. ). Sustitution of the K44 resiue, whih loates at a ifferent surfae region of WAH-1 from the other five resiues (Fig. ), speifially aolishe the ining of WAH-1 to SCRM-1 (Fig. a), ut i not affet the ining of WAH-1 to DNA (see Supplementary Information, Fig. S). When WAH-1 K44E was overexpresse in the C. elegans germline, it faile to enhane phosphatiylserine externalization in apoptoti germ ells as the wil-type WAH-1 protein i (Fig. 1e an see Supplementary Information, Fig. Sa), proviing further in vivo eviene that the WAH-1SCRM-1 interation is important for WAH-1 to promote phosphatiylserine exposure in apoptoti ells. Finally, we examine whether SCRM-1 possesses a phospholipisramling ativity, an whether WAH-1 affets the ativity of SCRM-1 using an in vitro phospholipi-sramling assay. Briefly, purifie reominant SCRM-1, WAH-1 or oth were inorporate into liposomes an fluoresently laelle NBDPS was use as a proe to measure the phospholipi-sramling ativity of SCRM-1. As shown in Fig., SCRM-1 y itself ha arely etetale phospholipi-sramling ativity, whih was not ativate y Ca at various onentrations teste (ata not shown). WAH-1 y itself ha no phospholipi-sramling ativity, ut oul ativate the phosphatiylserine-sramling ativity of SCRM-1 y tenfol. In ontrast, the SCRM-1 mutant that i not interat with WAH-1 showe no response to WAH-1 ativation an the WAH-1 K44E mutant that i not in SCRM-1 faile to ativate the phosphatiylserine-sramling ativity of SCRM-1 (Fig. ), iniating that the aility of WAH-1 to in SCRM-1 is ruial for its ativation of SCRM-1. The ativation of SCRM-1 y WAH-1 was strongest at a molar ratio of :1 (SCRM-1:WAH-1), at a ph of approximately.7 an at a salt onentration of approximately mm NaCl (see Supplementary Information, Fig. S, e an ata not shown). A time ourse analysis reveale that, uner these onitions, the phosphatiylserine-sramling ativity of the SCRM-1WAH-1 omplex was etete early in the reation (1% NBDPS protetion after 3 min), was approximately linear for the first h an reahe a plateau at 3% of NBDPS protetion after 4 h (Fig. e) whih was estimate to e lose to the equilirium for NBDPS istriution etween the two leaflets of the proteoliposomes (see Methos). The results from these in vitro an in vivo assays provie strong eviene that WAH-1 an speifially interat with SCRM-1 an ativate its phospholipi sramling ativity in a Ca -inepenent fashion. How phospholipi asymmetry on the lipi ilayer is generate, maintaine an altere (in some iologial events suh as apoptosis) is a funamental ut poorly unerstoo issue in ell iology 3. We have evelope an in vivo phosphatiylserine-staining tehnique to etet surfae-expose phosphatiylserine in apoptoti ells in C. elegans an have use this metho, in omination with the geneti an reverse-geneti tools availale in C. elegans, to isset the moleular pathway that meiates the phosphatiylserine-externalization proess uring apoptosis. These stuies have le to the ientifiation of a C. elegans phospholipi sramlase (SCRM-1) as the lipi-transloating enzyme that atalyzes the phosphatiylserine-externalization proess an WAH-1, an apoptogeni fator release from mitohonria uring apoptosis, as the iret ativator of the SCRM-1 lipi sramlase, thus estalishing a new mitohonria to plasma memrane signalling pathway that promotes ell-surfae hanges in apoptoti ells. Similar approahes an now e applie to isset moleular pathways that ontrol the generation an maintenane of phospholipi asymmetry in C. elegans an in other organisms. METHODS Annexin V staining. The C. elegans gona is a large ytoplasmi synytium ontaining many germ-ell nulei. The ying germ ells quikly ellularize an pinh off the synitium very early in the ell-eath proess 4. Thus, all apoptoti germ ells are visile on the surfae of gonas an are aessile to annexin V staining one gonas are issete out of the animals. For annexin V staining, gravi hermaphroite animals were arefully ut open with a surgial lae, at the hea or the tail region, on a epression slie in gona issetion uffer ( mm NaCl, 3 mm KCl, 3 mm Na HPO 4, mm MgCl, mm HEPES, μg ml 1 peniillin, μg ml 1 streptomyin, μg ml 1 neomyin, mm Gluose, 33% FCS an mm CaCl ) to expose their anterior or posterior gonas from the animal oies. The issete animals were then transferre y a mouth pipette to another epression slie with the issetion uffer ontaining 4 μm Hoehst 3334 an 1 μl Alexa Fluro 488-onjugate annexin V (Moleular Proes, Eugene, OR) for 4 min at C efore they were transferre to a thir epression slie ontaining fresh issetion uffer. The staine animals were then mounte on a slie with a % agar pa an examine using a Nomarski mirosope equippe with an epifluoresene etetor. To sore the perentage of germ ell orpses staine y annexin V, the germ-ell orpses were first ientifie y their raise-utton like morphology using Nomarski optis an then the onense Hoehst 3334 staining using the DAPI filter, efore they were examine for annexin V staining. Overexpression of WAH-1 in C. elegans germ ells. To express wah-1 in the germline, a mixture of the following DNA was injete into the e-1(e17) animals to reate omplex DNA arrays that failitate gene expression in the germline : the WAH-1 expression onstrut linearize with SaII (1 μg ml 1 ), the prf4 (1 μg ml 1 ) onstrut linearize with EoRI as a ominant oinjetion marker an wil-type C. elegans genomi DNA igeste with SaI ( μg ml 1 ). F an F3 generations of Roller transgeni animals were examine for the expression of GFP efore they were staine with annexin V. Quantifiation of ell orpses an extra ells. The numer of somati-ell orpses in the hea region of living emryos or L1 larave, an the numer of germ-ell orpses in one gona arm from animals at various ault ages were sore using Nomarski optis, as previously esrie,4. 48 NATURE CELL BIOLOGY VOLUME 9 NUMBER MAY 7

9 Four-imensional mirosopy. Four-imensional mirosopy analysis of the uration of ell orpses was performe as esrie previously 1. For Fig. 3 an e, the urations of four ell ivisions in the MS ell lineage from the MS ell to MS.aaaa ell were also followe to ensure that the emryos assaye ha similar rates of evelopment. The average uration of four ell ivisions in N emryos is 8 ± 3 min, 8 ±. min for srm-1(tm98) emryos, 87 ±. min for srm- 1(tm8) emryos an 9 ± 3.9 min for the srm triple mutant. Preparation of liposomes. Lipis issolve in hloroform were mixe at a esignate omposition (phosphatilyholine, phosphatilyethanolamine, phosphatiylserine, phosphatiylinositol, sphingomyelin an holesterol at a weight ratio of 4:18.:::1.:1) an rie uner a stream of nitrogen. The lipis were further rie uner vauum for 1 h followe y suspension in a lipi uffer (1% rerystallize soium holate, 1 mm ithiothreitol (DTT) an mm TrisMES at ph 7.) to yiel a final lipi onentration of mg ml 1. The lipi mixture was soniate in a ath-type soniator for three -min yles uner nitrogen until the lipi preparation eame lear. Lipi preparations are then reay for reonstitution with proteins, or an e store at 4 C for up to a week. When ialyse or ilute in an assay uffer y twentyfol to reue the etergent onentration to elow.%, the lipi preparation eomes tightly seale small unilamellar liposomes. Reonstitution of SCRM-1 an WAH-1 into proteoliposomes. Designate amounts of SCRM-1 an WAH-1, alone or together, were mixe in a volume of μl with DTT at a final onentration of 1 mm. Five mirolitres of the lipi preparation ( μg) esrie aove was then ae to the protein, or the protein mixture, an mixe thoroughly y vortexing in the presene of 1 mm MgCl. The mixture was inuate at room temperature for 1 h an then 4 C overnight. The reonstitution mixture was then ialyze in a ialysis is (Millipore, Billeria, MA; pore size =. μm) against 3. ml assay uffer for 4 min. The efault assay uffer ontains mm soium triine at ph.7, mm KCl, mm NaCl an. mm EGTA. The inorporation of SCRM-1, mutant SCRM- 1, WAH-1, or WAH-1 K44E into liposomes, either alone or together, was examine y % SDSPAGE an Coomassie lue staining an foun to e omparale in all experiments (ata not shown). Phospholipi sramlase assay. The ialyze proteoliposomes, as esrie aove, were ilute in.9 ml assay uffer, followe y aition of NBDPS (. mol%) an mixe y vortexing. The proteoliposomes were then equally ivie into six aliquots: μl eah with the aition of either 4 mm EGTA or CaCl at a esignate onentration, an inuate at 37 C in the ark. In the assays to examine the ativation of SCRM-1 y WAH-1 (Fig. ), soium ithionite was ae to five of the six aliquots to a final onentration of mm to quenh the fluoresene of unprotete NBDPS 11, an the resiual fluoresene was measure (R) in a FLUOstar OPTIMA fluoresene reaer. The aliquot without aition of ithionite was measure, whih provie a reaing of total fluoresene (T). One aliquot serve as the akgroun (B), to whih 1% NP4 was ae in aition to mm ithionite to quenh NBDPS in oth memrane leaflets. The phospholipi-sramlase ativity was presente as perentage NBDPS protetion, whih equals to (RB)/T. For the time-ourse analysis of the SCRM-1 ativity (Fig. e), at eah esignate time point, mm soium ithionite was ae into the reation mixture to quenh the fluoresene of unprotete NBDPS, an the resiual fluoresene was measure immeiately in a FLUOstar OPTIMA fluoresene reaer. This proeure for preparing proteoliposomes shoul yiel small unilamellar vesiles (SUV) with an average iameter of nm, of whih the surfae area of the inner leaflet an e alulate to oupy aout 31% of the total surfae area of oth leaflets. The 3% NBDPS protetion oserve after 4 h was estimate to e lose to, if not at, the equilirium for NBDPS istriution etween the inner an outer leaflets of the proteoliposomes. Of note, this in vitro phosphatiylserine-sramling assay may e far from optimal ompare with the in vivo situation eause of ifferenes in the atual lipi ompositions an the potential presene of other protein fators. Note: Supplementary Information is availale on the Nature Cell Biology wesite. ACKNOWLEDGEMENTS We thank T. Blumenthal for anti-cstf-4 antioy an Geraline Seyoux for the pte onstrut. This researh was supporte y a Burroughs Wellome Fun Career Awar an a Human Frontier Siene Program (HFSP) grant (RGP1/-C) to D.X., a grant from the Ministry of Euation, Culture, Sports, Siene an Tehnology of Japan to S.M., an grants from the National Institutes of Health (NIH) to D.X., X.S.X. an Y.S. AUTHOR CONTRIBUTIONS X.C.W. performe most of the experiments. X.C.W. an D.X. esigne an interprete most of the experiments. J.W. an X.S.W. performe the in vitro proteoliposome assays an relate ata analysis. K.G.A., T.K. an S.M. isolate srm eletion alleles. L.G., Y.G.S., C.L.S., Y.S. an C.L.Y. ontriute to the experiments. X.C.W., X.S.X. an D.X. wrote the paper an others ommente on the manusript. COMPETING FINANCIAL INTERESTS The authors elare that they have no ompeting finanial interests. Pulishe online at Reprints an permissions information is availale online at reprintsanpermissions/ 1. Faok, V. A. et al. Exposure of phosphatiylserine on the surfae of apoptoti lymphoytes triggers speifi reognition an removal y marophages. J. Immunol. 148, 71 (199).. Ashman, R. F., Pekham, D., Alhasan, S. & Stunz, L. L. Memrane unpaking an the rapi isposal of apoptoti ells. Immunol. Lett. 48, 91 (199). 3. Martin, S. J. et al. Early reistriution of plasma memrane phosphatiylserine is a general feature of apoptosis regarless of the initiating stimulus: inhiition y overexpression of Bl- an Al. J. Exp. Me. 18, 4 (199). 4. 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