2021 Development 126, 2021-2031 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 DEV3958 C. elegans MAC-1, an essential member of the AAA family of ATPases, can bind CED-4 and prevent cell death Dayang Wu1, Pei-Jiun Chen2, Shu Chen1, Yuanming Hu1, Gabriel Nuñez1,* and Ronald E. Ellis2,*,§ 1Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, and 2Department of Biology, The University of Michigan, Ann Arbor, Michigan 48109, USA *Joint senior authors §Author for correspondence (e-mail: [email protected]) Accepted 9 February; published on WWW 6 April 1999 SUMMARY In the nematode Caenorhabditis elegans, CED-4 plays a central role in the regulation of programmed cell death. To identify proteins with essential or pleiotropic activities that might also regulate cell death, we used the yeast twohybrid system to screen for CED-4-binding proteins. We identified MAC-1, a member of the AAA family of ATPases that is similar to Smallminded of Drosophila. Immunoprecipitation studies confirm that MAC-1 interacts with CED-4, and also with Apaf-1, the mammalian homologue of CED-4. Furthermore, MAC-1 can form a multi-protein complex that also includes CED-3 or CED-9. A MAC-1 transgene under the control of a heat shock promoter prevents some natural cell deaths in C. elegans, and this protection is enhanced in a ced-9(n1950sd)/+ genetic background. We observe a similar effect in mammalian cells, where expression of MAC-1 can prevent CED-4 and CED-3 from inducing apoptosis. Finally, mac1 is an essential gene, since inactivation by RNA-mediated interference causes worms to arrest early in larval development. This arrest is similar to that observed in Smallminded mutants, but is not related to the ability of MAC-1 to bind CED-4, since it still occurs in ced-3 or ced4 null mutants. These results suggest that MAC-1 identifies a new class of proteins that are essential for development, and which might regulate cell death in specific circumstances. INTRODUCTION proteins, which also promote survival (Hengartner et al., 1992; Hengartner and Horvitz, 1994). Genetic experiments indicate that ced-9 prevents cells from undergoing programmed deaths by regulating the activities of ced-3 and ced-4 (Hengartner et al., 1992; Shaham and Horvitz, 1996). In addition, ectopic expression of these genes in the touch neurons of nematodes suggests that ced-4 works by activating ced-3, and that ced-9 somehow blocks this activation (Shaham and Horvitz, 1996). What factors determine which cells will be protected by ced9, and which will die? Recent results indicate that CED-9 is inactivated by the product of the egl-1 gene (Conradt and Horvitz, 1998). EGL-1 binds CED-9, and this binding apparently allows CED-4 and CED-3 to kill cells. EGL-1 contains a BH3 domain, like some related proteins that induce cell death; these include Bik, Bid, Harakiri and Bad. The expression of egl-1 is probably controlled by proteins that regulate the deaths of specific cells. These regulatory genes might include ces-1 and ces-2, which control specific neuronal deaths in the pharynx (Ellis and Horvitz, 1991; Metzstein et al., 1996). The discovery of physical interactions between CED-3, CED-4 and CED-9 helped elucidate the molecular basis of cell death. When expressed in mammalian or yeast cells, wild-type Programmed cell death is a conserved mechanism for killing cells, both during development and for tissue homeostasis (Ellis et al., 1991). The nematode C. elegans is a leading model for genetic and molecular analysis of cell death. In C. elegans hermaphrodites, 131 of the 1090 somatic cells produced during development undergo programmed deaths (Sulston and Horvitz, 1977; Sulston et al., 1983). Loss-of-function mutations in either ced-3 or ced-4 cause all of these cells to survive (Ellis and Horvitz, 1986), showing that both genes are essential for cell death to occur. These deaths are likely to be ‘suicides’, since genetic mosaic analysis indicates that ced-3 and ced-4 act within dying cells (Yuan and Horvitz, 1990). CED-3 belongs to a conserved family of intracellular cysteine proteases, termed caspases, that are homologous to mammalian interleukin-1β-converting enzyme (Miura et al., 1993; Yuan et al., 1993). In both nematodes and mammals, CED-3 and related caspases are thought to kill cells by cleaving specific targets (Cohen, 1997). The nematode gene ced-9 is required to protect cells that normally survive from undergoing programmed deaths; its product is homologous to the mammalian Bcl-2 and Bcl-XL Key words: Apoptosis, Programmed cell death, CED-4, Smallminded, AAA proteins 2022 D. Wu and others CED-9 interacts with CED-4 and prevents cell death, whereas mutant CED-9 neither binds CED-4 nor prevents death (Chinnaiyan et al., 1997; James et al., 1997; Spector et al., 1997; Wu et al., 1997a,b). Thus, CED-9 might function by binding and inactivating CED-4. When expressed in mammalian cells, CED-3 also binds CED-4, forming a multiprotein complex that includes CED-9 (Chinnaiyan et al., 1997; Wu et al., 1997a). Thus, CED-4 regulates cell death by bringing together molecules that promote apoptosis with those that inhibit it. CED-4 is homologous to the mammalian protein Apaf-1, which also promotes cell death (Zou et al., 1997). This similarity is restricted to their amino-termini, which each contain a caspase recruitment domain similar to that found in the prodomains of CED-3 and mammalian caspases like caspase-9 (Hofmann et al., 1997). Thus, homophilic interactions between caspase recruitment domains might allow Apaf-1 to interact with caspase-9, and CED-4 with CED-3 (Li et al., 1997; Hu et al., 1998; Pan et al., 1998). Once bound, Apaf-1 promotes the processing and activation of caspase-9, just as CED-4 promotes the activation of CED-3 (Li et al., 1997; Hu et al., 1998). In C. elegans, the genes of the cell death pathway were each identified by viable mutations (Trent et al., 1983; Ellis and Horvitz, 1986; Hengartner et al., 1992). Because these genetic screens might have missed essential genes that act in the cell death process, or which induce cell death in specific circumstances, we used the yeast two-hybrid system to find proteins that bind CED-4. We identified MAC-1, which is a member of a family of ATPases associated with a variety of cellular activities, known as AAA ATPases (Confalonieri and Duguet, 1995). In the presence of CED-4, MAC-1 can form a multi-protein complex with CED-3 or CED-9. Furthermore, expression of MAC-1 prevents cell deaths in nematodes and in mammalian cells. Finally, we show that inactivation of mac-1 blocks larval development in C. elegans, a phenotype similar to that of the AAA family member Smallminded of Drosophila. MATERIALS AND METHODS Reagents and cell lines The 293T cell line was described by Wu et al. (1997b). Mouse monoclonal antibodies against Flag epitope (M2; Kodak/IBI), HA epitope (12CA5; Boehringer Mannheim, Indianapolis IN) and Myc epitope (9E10; Santa Cruz Biotech) were obtained from the indicated sources. Rabbit antibodies against Flag, HA and Myc were from Santa Cruz Biotech. The enhanced chemiluminescence (ECL) system was from Amersham. Protein A- or Protein G-Sepharose 4B were from Zymed Laboratories. Other chemicals were from Sigma, unless indicated otherwise. Identification and cloning of the MAC-1 cDNA A mixed stage C. elegans cDNA library fused to the GAL4 DNAactivation domain of pACT vector (a gift from Robert Barstead) was screened with a CED-4S bait using the yeast HF7c strain as described by Wu et al. (1997b). Positive library clones were identified by βgalactosidase staining (Wu et al., 1997b). Plasmids were recovered by transformation into bacteria and growth in M9 medium lacking leucine. The cDNA inserts were characterized by restriction enzyme mapping and nucleotide sequence analysis on an automated DNA sequencer (Applied Biosystems). False-positive clones were eliminated by testing the interaction with empty vector and irrelevant ‘bait’. A positive clone cDNA corresponding to MAC-1 was further characterized. Plasmids that express GAL-4-CED-3, GAL4-CED-9 in pACT and GAL-4 in pGBT8 have been described (Wu et al., 1997b). Two hybrid yeast growth assays were performed as described by Wu et al. (1997b). Transfection, immunoprecipitation and western blot analysis Either 3-5×106 in 100 mm or 1-2×106 in 60 mm of human embryonic kidney 293T cells in tissue culture dishes were transfected with the indicated amount (see figure legends) of plasmid DNA by the calcium phosphate method. pcDNA3-ced-4-Myc, -ced-3-HA and -HA-ced-9 constructs were described by Wu et al. (1997a). For immunoprecipitations, cells from each dish were lysed in 0.5-1 ml of isotonic lysis buffer containing 0.2% Nonidet P-40 and proteinase inhibitors at 18-22 hours after transfection, and the soluble lysates were incubated with different antibodies overnight at 4°C with 5% (v/v) of protein A- or Protein G-Sepharose 4B beads. Complexes were centrifuged, washed with lysis buffer at least four times, separated by 10-15% SDS-PAGE and immunoblotted. Signals were detected by enhanced chemiluminescence system (Amersham). About 5% of the total lysate was used for immunoblotting to assess the expression of the transfected constructs. Apoptosis assay 1×105 293T cells in each well of 12-well plates were transiently cotransfected in triplicate with 0.2 µg of a reporter plasmid pcDNA3-βgal with or without 0.2 µg pcDNA3-ced-3-HA, 0.05 µg pcDNA3-ced4-Myc, 1 µg pcDNA3-HA-ced-9, 1 µg pcDNA3-Flag-MAC-1, 1 µg pcDNA3-Flag-MAC-1 (1-239) and 1 µg pcDNA3-Flag-MAC-1 (234813) by the calcium phosphate method. The total amount of transfected plasmid DNA was always 2 µg per 3 wells and was adjusted by adding vector pcDNA3 plasmid. The percentage of apoptotic cells was determined 18 hours after transfection in triplicate cultures by analysis of at least 300 cells expressing β-galactosidase (Wu et al., 1997a). Construction and analysis of transgenic nematodes Nematodes were raised at 20°C as described by Brenner (1974). We generated transgenic animals by co-injection of 50 ng/µl of the plasmid pRF4, which carries the rol-6(su1006sd) mutation, with 100 ng/µl of an hsp16-2::mac-1 plasmid, an hsp16-41::mac-1 plasmid, or an empty hsp16-2 vector (pPD49.78). In each case, the injected DNA formed an extra chromosomal array, which we followed by scoring the Rol-6 phenotype (Mello et al., 1991). Individual unc-29(e1072) fog-3(q504); vEx8[hsp16-2::mac-1 rol-6(su1006sd)] females were prepared by standard crosses, and mated with ced-9(n1950gf); him5(e1490) males. We transferred the fertilized animals to fresh plates, and subjected them to a 1-hour heat shock at 31°C. The plates were returned to 20°C for an additional 90 minutes, after which the parents were removed. Once the embryos had matured, we scored the pharynges of Rol larvae and young adults for alterations in the normal pattern of programmed cell death (Ellis and Horvitz, 1991; Hengartner et al., 1992). Similar crosses were carried out in parallel to study two other transgenes, vEx9[hsp16-41::mac-1 rol-6(su1006sd)] and vEx10[hsp16-2 vector pPD49.78 rol-6(su1006sd)]. We used heat shocks of a 1-hour duration at 31°C because higher temperatures or longer exposures led to significant lethality in both the experimental and the control animals. In these experiments, we usually scored 14 cells in the anterior pharynx of each animal (Ellis and Horvitz, 1991). However, when we examined the effects of an empty vector in wildtype animals, the dorsal pharynx from one of the nine animals could not be scored, so three cells from this region were not included in the data set. The hsp16-41::mac-1 transgene also prevented programmed deaths, but its effect was weaker than that of the hsp16-2::mac-1 transgene (data not shown). MAC-1 is essential and can interact with CED-4 2023 RNA-mediated interference We generated RNA complementary to the mac-1 gene by using in vitro transcription from T7 promoters. The RNAs were precipitated and resuspended in 1× injection buffer (Fire, 1986), allowed to anneal, and their concentration estimated by ethidium staining. The injections were carried out as described by Guo and Kemphues (1995) and Fire et al. (1998). The double-stranded RNA for the unc-4 control corresponded to the entire predicted unc-4 transcript. RT-PCR and northern blot analysis Nematodes for RNA preparation were grown in liquid culture, as described by Sulston and Brenner (1974). RNA from mixed stage worms was used as a template for RT-PCR analysis. The RT-PCR reactions were performed using a cDNA cycle kit (Invitrogen). The products of reverse transcription were amplified with either the SL1 primer (GGTTTAATTACCCAAGTTTGAG) or SL2 primer (GGTTTTAACCCAGTTACTCAAG), and primers complementary to the 5′ region of the MAC-1 coding sequence. The products were purified and sequenced using an automated DNA sequencer (Applied Biosystems model 373A). We used four strains for northern analysis: N2 XX hermaphrodites, which were harvested as a mixed population, him-5(e1490) XX and XO animals (Hodgkin et al., 1979), which were harvested as a mixed population containing 30% males and 70% hermaphrodites, fem-3(q96gf,ts) XX animals, which were harvested as adults after shifting the cultures to the restrictive temperature of 25°C, and fem-1(hc17ts) XX animals, which were also harvested as adults after shifting the cultures to 25°C. The fem-3(q96gf,ts) mutants produce only sperm (Barton et al., 1987) whereas the fem-1(hc17ts) animals produce only oocytes (Kimble et al., 1984). For northern analysis, mac-1 antisense RNA fragment B (Fig. 9) was radio-labeled by incorporation of [32P]UTP and hybridized to blots containing nematode RNAs. SL2 primer in combination with any of several primers from the 5′ region of mac-1. The sequence of these PCR products overlaps with that of the original cDNA, and defines the 5′-end of the message. A BLAST search identified two partial EST sequences from C. elegans that correspond to the mac-1 message (Kohara, 1996). Our composite cDNA contains an ORF encoding a protein of 813 amino acids, with a predicted molecular mass of 89 kDa (Fig. 2A). A search of protein databases revealed that MAC-1 contains two conserved ATPase domains (CADs); each domain includes Walker A and B signature sequences and an AAA minimal consensus motif (Fig. 3A; Swaffield and Purugganan, 1997; Patel and Latterich, 1998). Thus, we named this protein MAC-1, for member of the AAA family that binds CED-4. Although MAC-1 shares amino acid and structural similarity with many AAA proteins, it most resembles mouse VCP (Egerton et al., 1992), yeast CDC48 (Fröhlich et al., 1991) and Smallminded, a Drosophila protein required for larval development (Long et al., 1998). The percentage identity between these proteins is particularly high within two regions of MAC-1, amino acids 204-455 and 507-757; these regions correspond to the conserved AAA modules characteristic of this family of ATPases (Fig. 3A,C). MAC-1 interacts with CED-4 in mammalian cells To verify that MAC-1 associates with CED-4, 293T human RESULTS Identification of MAC-1 To search for proteins that bind CED-4, we screened a C. elegans cDNA library using GAL4-CED-4 as ‘bait’ in the yeast two-hybrid assay. From a screen of 2× 106 clones, four cDNAs were found whose products interact with GAL4-CED-4, but not with control baits (Wu et al., 1997b). The products of two of these four cDNAs bind CED-4 in other assays. One contains the ced-9 coding region, fused to the GAL4 transcriptional activation domain (Wu et al., 1997b). Here we describe the other, whose product we call MAC-1 (see below). In the twohybrid system, CED-4 interacts with MAC-1, CED-3 and CED-9 but not with the control vector, as expected from previous results (Fig. 1A). Using GAL4-MAC-1 as bait, MAC1 associates with CED-4 but not with CED-3 or CED-9, which shows the specificity of the CED-4-MAC-1 interaction (Fig. 1B). MAC-1 is a novel member of the AAA family of ATPases Sequence analysis revealed that our cDNA contained an insert of approx. 2.4 kb fused to the GAL4-DNA-transcription activation domain. Because it was unclear if the cDNA contained the entire mac-1 coding region, we used the reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify mixed stage C. elegans RNA, using forward primers corresponding to either the SL1 or SL2 trans-spliced leaders (Krause and Hirsh, 1987) and reverse primers from the 5′ region of our cDNA. We could amplify fragments using the Fig. 1. Interaction of MAC-1, CED-3, CED-4 and CED-9 in yeast cells. (A) A plasmid expressing CED-4S fused to the GAL4 DNA binding domain was co-transfected with indicated plasmids expressing MAC-1, CED-3 or CED-9 fused to the GAL4 DNAactivation domain. Growth of yeast cells in the absence of leucine (Leu−), tryptophan (Trp−) and histidine (His−) is indicative of protein-protein interaction. Growth in the absence of leucine and tryptophan is shown as control. (B) A plasmid expressing MAC-1 fused to the GAL4 DNA transcription activation domain was cotransfected with indicated plasmids expressing CED-3, CED-4 or CED-9 fused to the GAL4 DNA-binding domain. 2024 D. Wu and others A MPGGMGFPSDPALLPRVQAHIRKFPGTKYFKPELVAYDLQQEHPEYQRKNHKVFMGMV REALERIQLVAKEENDEKMEEKEAMDDVQEIPIVKALETRKRKAPAAGRKSTGQAAAA KEVVLSDDSEDERAARQLEKQIESLKTNRANKTVLNLYTKKSAPSTPVSTPKNQATKK PPGASAAPPALPRGLGAVSDTISPRESHVKFEHIGGADRQFLEVCRLAMHLKRPKTFA TLGVDPPRGFIVHGPPGCGKTMFAQAVAGELAIPMLQLAATELVSGVSGETEEKIRRL FDTAKQNSPCILILDDIDAIAPRRETAQREMERRVVSQLCSSLDELVLPPREKPLKDQ LTFGDDGSVAIIGDSPTAAGAGVLVIGTTSRPDAVDGRLRRAGRFENEISLGIPDETA REKILEKICKVNLAGDVTLKQIAKLTPGYVGADLQALIREAAKVAIDRVFDTIVVKNE GHKNLTVEQIKEELDRVLAWLQGDDDPSALSELNGGLQISFEDFERALSTIQPAAKRE GFATVPDVSWDDIGALVEVRKQLEWSILYPIKRADDFAALGIDCRPQGILLCGPPGCG KTLLAKAVANETGMNFFSVKGPELLNMYVGESERAVRTVFQRARDSQPCVIFFDEIDA LVPKRSHGESSGGARLVNQLLTEMDGVEGRQKVFLIGATNRPDIVDAAILRPGRLDKI LFVDFPSVEDRVDILRKSTKNGTRPMLGEDIDFHEIAQLPELAGFTGADLAALIHESS LLALQARVLENDESVKGVGMRHFREAASRIRPSVTEADRKKYEHMKKIYGLKQATPPS V Fig. 2. Deduced amino acid sequence and structure of MAC1. (A) Amino acid sequence (single letter amino acid code) predicted from the MAC-1 cDNA. (B) Schematic structure of MAC-1. Conserved ATPase Domains (CADs) are indicated by gray boxes. 58 116 174 232 290 348 406 464 522 580 638 696 754 812 813 B 1 204 455 507 757 813 MAC-1 kidney cells were transiently co-transfected with expression plasmids producing Flag-tagged MAC-1 and Myc-epitopetagged CED-4. We prepared immunoprecipitates using a monoclonal antibody to Flag, and separated them by SDS polyacrylamide gel electrophoresis. Immunoblotting with a rabbit antibody to Myc showed that CED-4 coimmunoprecipitates with MAC-1 (Fig. 4A). The MAC-1CED-4 interaction is specific, because it requires coexpression of both MAC-1 and CED-4, and was not detected in precipitates formed using a control antibody (Fig. 4A). We also measured the ability of MAC-1 to associate with three mutant CED-4 proteins, each of which causes a loss-offunction in C. elegans. The mutation n1948 causes an amino acid change (Ile to Asn) at position 258, the mutation n1894 creates a stop codon at position W401 (Yuan and Horvitz, 1992), and the mutation G328 creates a stop codon at amino acid 328 (Wu et al., 1997a). MAC-1 co-immunoprecipitates CED-4 I258N and CED-4 W401, but not CED-4 G328 (Fig. 4A). Thus, amino acids 328 to 401 of CED-4 are required for association with MAC-1. We engineered two mutants, MAC-1-N (amino acids 1-239) and MAC-1-C (amino acids 234-813) to determine which portions of MAC-1 were required for its interaction with CED4 (Fig. 4B). We then co-transfected 293T cells with expression plasmids for both Myc-tagged CED-4 and either wild-type or mutant Flag-tagged MAC-1. Our results show that the Nterminal and C-terminal regions of MAC-1 can interact independently with CED-4, although the binding ability of each mutant protein is significantly lower than that of wild-type MAC-1 (Fig. 4C). To confirm these results, we performed reciprocal experiments in which CED-4 was immunoprecipitated using anti-Myc antibodies. Immunoblotting with anti-Flag antibodies confirmed that CED-4 can associate with the wild-type and both mutant MAC1 proteins (Fig. 4D). MAC-1 associates with CED-3, CED-9 and Apaf-1 in mammalian cells Because CED-4 interacts with both CED-3 and CED-9 in mammalian cells (Chinnaiyan et al., 1997; Wu et al., 1997a), we assessed the ability of MAC-1 to associate with CED-3 and CED-9. A plasmid producing Flag-tagged MAC-1 was cotransfected with plasmids expressing HA-tagged CED-3 or CED-9, in the presence or absence of CED-4. Immunoprecipitation of MAC-1 complexes with anti-Flag antibodies revealed that CED-3 and CED-9 each coimmunoprecipitate with MAC-1, even in the absence of CED4 (Fig. 5A). However, expression of CED-4 significantly increased the amounts of CED-3 and CED-9 that coimmunoprecipitate with MAC-1 (Fig. 5A). We verified these results in reciprocal experiments, in which CED-3 or CED-9 were immunoprecipitated using anti-HA antibodies. In these experiments, either CED-3 or CED-9 could coimmunoprecipitate MAC-1 (Fig. 5B). Because MAC-1 does not interact with CED-3 or CED-9 in the yeast two-hybrid system (Fig. 1), this co-immunoprecipitation might involve an adapter protein present in 293T cells. One candidate is Apaf1, a homologue of CED-4 that is expressed in 293T cells (Li et al., 1997). To see if MAC-1 can bind Apaf-1, we coexpressed Myc-tagged Apaf-1 and Flag-tagged MAC-1, and immunoprecipitated MAC-1 with anti-Flag antibodies. Immunoblotting revealed that Apaf-1 co-immunoprecipitates with MAC-1 (Fig. 5C). Genomic organization and expression of mac-1 in C. elegans A BLAST search using the cDNA sequence showed that mac1 is located on the YAC clones Y48C3 and Y81G3, which are being sequenced by the C. elegans Genome Sequencing Consortium (Sulston et al., 1992). This result places mac-1 on the right arm of chromosome II, between the genes rtw-3 and rsn-2 (Fig. 6A,B). Comparison of the genomic and cDNA sequences shows that mac-1 contains seven exons, and is trans-spliced to the SL2 leader sequence (Fig. 6C). We used northern analysis to measure the size of the mac-1 transcript, and study its expression pattern. RNA from a mixed population of males and hermaphrodites of all ages contains a single mac-1 transcript of approx. 2.4 kb; this size matches the length of our cDNA. The mac-1 mRNA is found throughout larval development (data not shown), but is expressed at highest levels in hermaphrodites that are producing oocytes (Fig. 6D). MAC-1 is essential and can interact with CED-4 2025 Elevated expression of MAC-1 in C. elegans prevents programmed cell deaths To determine if MAC-1 could regulate programmed cell deaths in C. elegans, we created a transgenic strain of animals in which expression of mac-1 could be driven by the heat shock promoter hsp16-2 (Candido et al., 1989). Initial observation of 13 transgenic animals subjected to a heat shock during early embryogenesis revealed that 10 pharyngeal cells (out of a possible 182) had failed to undergo programmed deaths, whereas only 1 cell failed to die (out of a possible 123) in control animals that had also been heat shocked. Because this effect was small, we next studied the expression of transgenic mac-1 in cells that were genetically predisposed towards survival. In ced-9(n1950gf)/+ progeny of wild-type mothers, one third of the cells that would normally die instead survive (Hengartner et al., 1992). We observed this same frequency following heat shock of ced-9(n1950gf)/+ transgenic animals that carried an empty hsp16-2 vector (Fig. 7). By contrast, heat shock of similar animals carrying the hsp16-2::mac-1 transgene doubles the number of surviving cells (Fig. 7). The extra cells we observe in these animals are in the same positions, and share the same morphologies, we have observed for surviving cells in ced-3 or ced-4 mutants. Thus, expression of MAC-1 can prevent programmed cell deaths in C. elegans. MAC-1 prevents CED-3 and CED-4 from causing apoptosis in mammalian cells To determine if MAC-1 could prevent cell death in mammalian cells, we co-transfected 293T cells with expression constructs producing either wild-type or mutant MAC-1, and plasmids expressing CED-3 and CED-4, and then assayed the cells for apoptosis. Expression of either CED-3, or of both CED-3 and CED-4, induces apoptosis in 293T cells (Wu et al., 1997a). This effect can be blocked by co-expression of CED-9 (Wu et al., 1997a). Significantly, the expression of either wild-type MAC-1, or of the MAC-1-C (amino acids 234-813) mutant, can prevent the induction of cell death by either CED-3, or by CED-3 plus CED-4 (Fig. 8A). However, the MAC-1-N (amino acids 1-239) mutant had no effect on cell death. Further experiments showed that apoptosis induced by CED-3, or by A MAC-1 mVCP CDC48 smallminded MP GG- MGFP S D P A L L P RV QA HI RK FP GTK Y FK P E L V A Y DL QQE H P E Y QRK NHK V F MGMV RE A L E R I QL V A K E E NDE K ME E K E A MDDV QE I P I V K A L E T RK RK A P A A GR K S T GQA A A A K E V V L S - - DDS E D E RA A RQL E K M- - - - - - - - - A S GA DS K G- - - - DD- L S TA I L K QK NRP NRL I V DE A I NE DNS V V S L S QP K MDE L QL F RGD TV L L K GK K RRE A V CI - - - - - V L S DD T CS D E K I R MNRV V R NNL - RV RL GDV I S I QP CP DV K Y GK RI HV L P I D MGE E HK P L L DA S GV DP RE - - - - E DK TA TA I L RR K K K DN ML L V DD A I NDDNS V I A I NS N T MDK L E L F RGD TV L V K GK K RK D TV L I - - - - - V L I DDE L E D GA CRI NRV V R NNL - RI RL GDL V T I HP CP DI K Y A T RI S V L P I A MK K A - K P L L HD HL I T I RV K K Y L E E HI GE TY L DV K QMT RE L MQK Y P E Y S RRK F GP F RQL V HQA FS I I S E S Y NL DK V S S S E E DCV S E DS E P P P T NS V MNN MMNS L Y S QP R K P L A P K P I S E P I DI S S GDE NE D DS N TK T T NGD 136 120 130 139 MAC-1 mVCP CDC48 smallminded - - - - - - - - - - - - - - - QI E S L K T NRA NK TV L NL Y TK K S A P S TP V S TP K NQA TK K P P GA S A A P P A L P - - - - - - - - - - R GL GA V S D T I S P RE S HV K - - - - - - - - - - - - - - - - FE HI GGA DRQ F L E V CRL A - MH L K RP K T FA T L D TV E GI T GNL F E V Y L K P Y F L E A Y RP I RK GDI F L V RGGMRA V E FK V V E T DP S P Y C I V A P D TV I HCE GE P I K RE DE E E S L NE V - - - - - - - - - - - - - - - - - - - - - - - - - - - GY DDV GGCRK Q L A QI K E MV E L P L RHP A L FK A I D T I E GI T GNL F DV F L K P Y FV E A Y RP V RK GDH FV V RGGMRQV E FK V V DV E P E E Y A V V A QD T I I HWE GE P I NRE DE E N N MNE V - - - - - - - - - - - - - - - - - - - - - - - - - - - GY DDI GGCRK Q MA QI RE MV E L P L RHP QL FK A I GV A A A A A P P P P TP A V QGS A L K R L ME E V P E I A V A A K K A K P N T I HV S S S E A I QK L H QV V GNRA K NL S E DA V P RS K DHR NV P GL Y QQL HQNQS RDRL RK FK RDL E V QHP TE S F RDI GGMDS T L K E L CE ML - I H I K S P E FY F QL 234 233 243 278 MAC-1 mVCP CDC48 smallminded GV DP P RGF I V H GP P GCGK T MFA QA V A GE L A I P ML QL A A TE L V S GV S GE TE E K I R RL F D TA K QNS P CI L I L DDI DA I GV K P P RGI L L Y GP P GT GK T L I A RA V A NE T GA F F F L I NGP E I MS K L A GE S E S NL R K A FE E A E K NA P A I I F I DE L DA I GI K P P RGV L MY GP P GT GK T L MA RA V A NE T GA F F F L I NGP E V MS K MA GE S E S NL R K A FE E A E K NA P A I I F I DE I DS I GL L P S RGL L L H GP P GCGK T F L A RA I S GQL K MP L ME I P A TE L I GGI S GE S E E RI R E V F DQA I GY S P CV L F I DE I DA I GDS P TA A GA GV L V I - - - - - - - - A HV I V M - - - - - - - - S NV V V I - - - - - - - - - SVVVI 374 344 354 392 MAC-1 mVCP CDC48 smallminded GT TS RP DA V DGRL RRA GR FE NE I S L GI A A T NRP NS I DP A L RR F GR F DRE V DI GI A A T NRP NS I DP A L RR F GR F DRE V DI GI A A T T RP DV L DP GL RRI GR F DHE I A I HI P DE TA R E K I L E K I CK - V NL A GDV T L K QI A K L TP GY V GA DL QA L I RE A A K V A I DRV - - - - - - - - - - - - - - - F D T I V V - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K NE GHK P DA T GR L E I L QI H TK N MK L A DDV DL E QV A NE T HGHV GA DL A A L CS E A A L QA I RK K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P DA T GR L E V L RI H TK N MK L A DDV DL E A L A A E T HGY V GA DI A S L CS E A A MQQI RE K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P S RK E R RE I L RI QCE GL S V DP K L NY DK I A E L TP GY V GA DL MA L V S RA A S V A V K RRS MK K F RE L HA A S E K N MT TV T L DDDE P S E DA GE TP V P D S K GE E TA K DA E A E QK V DGDK E 467 426 436 532 MAC-1 mVCP CDC48 smallminded - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - TS A K DK S E GDS P NI - - - - - - - - - - - - - - - - - - - - - - NL TV E QI K E E L DRV L A WL QGDDD- - P S A L S E L NGGL Q- - - - - - - - - - - - I S FE D FE RA L S T I QP A A K RE G FA TV P DV S WDD I GA L V E V RK Q - - - - - - - - - - - - - - - - - - MDL I DL E DE T I DA E V MNS L A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V T MDD F RWA L S QS NP S A L RE T V V E V P QV T WE D I GGL E DV K RE - - - - - - - - - - - - - - - - - - MDL I DL DE D E I DA E V L DS L G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V T MDN F R FA L GNS NP S A L RE T V V E S V NV T WDD V GGL DE I K E E K S P QK T P K K S A E K P T DA A MDV DNV A P E E P K K A V E QE V D S S S S NDE Y Y E P T L A E L T N F L DN P P E E FA DP N F C L T L I D FV DA I K V MQP S A K RE G F I TV P D T T WDD I GA L E K I RE E 544 488 498 672 MAC-1 mVCP CDC48 smallminded L E WS I L Y P I K R A DD FA A L GI DC RP QGI L L CGP P GCGK T L L A K A V A NE T GMN F FS V K GP E L L N MY V GE S E RA V R TV F QRA RDS QP CV I L QE L V QY P V E H P DK F L K F GMT - P S K GV L FY GP P GCGK T L L A K A I A NE CQA N F I S I K GP E L L T MWF GE S E A NV RE I F DK A RQA A P CV L L K E TV E Y P V L H P DQY TK F GL S - P S K GV L FY GP P GT GK T L L A K A V A TE V S A N F I S V K GP E L L S MWY GE S E S NI RDI F DK A RA A A P TV V L K L A V L A P V K Y P E ML E RL GL TA - P S GV L L CGP P GCGK T L L A K A I A NE A GI N F I S V K GP E L MN MY V GE S E RA V RA C F QRA RNS A P CV I MAC-1 mVCP CDC48 smallminded I V DA A I I I DP A I QI DP A I I I DP A I MAC-1 mVCP CDC48 smallminded DRK K Y E H MK K - - - - - - - - - - - - I Y GL - K QA TP P S - - - - - - - - - - - - - - - - - - - - - - - - - - - - V DI RK Y E MFA QT L QQS RG- F GS F R FP S GNQGGA GP - - - - S QGS GG GT GGS V Y TE - - DNDDDL Y G E L RRY E A Y S QQ MK A S RGQFS N F N F NDA P L GT TA T DNA NS NNS A P S GA GA A F GS N A E E DDDL Y S DRK I Y DK L RL - - - - - - - - - - - - K Y A A P RV P T L N D- - - - - - - - - - - - - - - - - - - - - - - - - - - - K ************ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E T P K KA T N GNSS I A P RRE TA QRE ME RRV V S QL CS S L DE L V L P P RE K P L K DQL T F GD DGS V A I A P K RE K T HGE V E RRI V S QL L T L MDGL - - - - K QR- - - - - - - - - - - - - - - A P K RDK T NGE V E RRV V S QL L T L MDGM- - - - K A R- - - - - - - - - - - - - - - GGNRQWA S K D ME RRI V S QL I S S L DNL - - - - - - - - - - K A NE F GQ - - - - - - I - ●●●●●● L RP GR L DK I L FV D FP S V E DRV DI L RP GR L DQL I Y I P L P D E K S RV A I L RP GR L DQL I Y V P L P D E NA RL S I L RP GR L D T I L Y V GFP E QS E R TE I F F DE I DA L V P K RS HG- E - - S S GGA RL V NQL L T E MDGV E GRQK V F L I GA T NRP D F F DE L DS I A K A RGGNI GDGGGA A DRV I NQI L T E MDGMS TK K NV F I I GA T NRP D F L DE L DS I A K A RGGS L GDA GGA S DRV V NQL L T E MDGMNA K K NV FV I GA T NRP D F F DE F DS L CP K RS DG- GDGNN S GT RI V NQL L T E MDGV E E RK GV Y I L A A T NRP D ●●●●●● ************ L RK S TK NGT RP ML GE DI D F HE I A QL P E L A GF T GA DL A A L I HE S S L L A L QA RV L - - - - - - - E NDE S V K GV GM- - - - - - - - - - - - - - - - - - - - L K A N L R- - - K S P V A K DV DL E F L A K MT N- - GFS GA DL TE I CQRA CK L A I RE S I E S E I RR E RE RQT NP S A ME V E - - - - - - - - - - - - E DDP V P E I L NA Q L R- - - K TP L E P GL E L TA I A K A T Q- - GFS GA DL L Y I V QRA A K Y A I K DS I E A HRQH E A E K E V K V E GE DV E MT DE GA K A E QE P E V DP V P Y I L K A T TK NGK RP V L A D DV DL DE I A A Q TE - - GY T GA DL A GL V K QA S MFS L RQS L N- - - - - - - NGD T NL DDL CV - - - - - - - - - - - - - - - - - - - - - R- - H F RE A A S RI RP S V TE A RRDH FE E A MR FA RRS V S DN TK E H FA E A MK TA K RS V S DA RS QH F QE A L QQL RP S V NE Q 681 627 637 810 791 750 772 920 813 806 835 943 C B Percent Identity 1 1 2 3 4 2 23.2 3 4 21.1 42.3 69.4 20.7 20.3 1 1 2 3 4 MAC-1 mVCP 757 813 MAC-1 1 12.3% 201 43.9% 427 451 47.6% 698 806 1 8.2% 212 41.7% 436 461 44.3% 708 835 1 18.5% 232 49.1% 474 635 62.7% 884 943 CDC48 Smallminded AAAAAA AAAAAA AAAAAA AAAAAA AAAAAA AAAAAA AAAAAA 455 507 204 mVCP CDC48 Smallminded Fig. 3. MAC-1 is a member of the AAA family of ATPases. (A) Alignment of MAC-1 with mouse VCP (accession number 400712), yeast CDC48 (accession number 1705679) and Drosophila Smallminded (accession number 1770214). Positions in which a majority of family members have identical residues are shown by black boxes. Two conserved ATPase domains or CADs are boxed. A set of Walker A (P-loop) and B signature sequences, and the AAA minimal consensus motif (Swaffield and Purugganan, 1997; Patel and Latterich, 1998) within each CAD are indicated by underlined stars, dots and solid lines, respectively. (B) Percentage amino acid identity between MAC-1, Smallminded, VCP and CDC48. (C) Percentage amino acid identity by regions of the proteins. CADs are indicated by gray boxes. The percentage amino acid identity in the aligned regions is indicated. The number above boxes refers to amino acid residue number. 2026 D. Wu and others Fig. 4. Interaction of MAC-1 with CED-4 in mammalian cells. (A) Interaction of MAC-1 with wt (wild-type) and mutant CED-4. Human embryonic kidney 293T cells were transiently co-transfected with 5 µg of plasmids expressing the indicated proteins. In the case of transfection with one plasmid, cells were co-transfected with empty plasmid so that the total amount of transfected plasmid DNA was always 10 µg. Cellular lysates were immunoprecipitated with mAb anti-Flag or isotype-matched control (indicated by cont). Immunoprecipitates were immunoblotted with rabbit anti-Myc or anti-Flag antibody. Expression of wt and mutant CED-4 protein in total lysates is shown in lower panels. (B) Schematic structure of wt and mutant MAC-1 proteins. Grey areas denote ATPase domains. (C,D) Interaction of CED-4 with wt MAC-1 (WT), MAC-1-N (N) and MAC-1-C (C). Transfection and immunoprecipitation analysis were performed as indicated in A. Expression of CED-4 in total lysates is shown in lower part of C. CED-3 plus CED-4, was inhibited by MAC-1 in a dosedependent manner (Fig. 8B). Finally, co-expression of MAC1 and CED-9 inhibits apoptosis of 293T cells more effectively than expression of either CED-9 or MAC-1 alone (Fig. 8B). MAC-1 is essential for larval development in C. elegans To study the effect of reducing MAC-1 activity in C. elegans, we used RNA-mediated interference (Guo and Kemphues, 1995; Fire et al., 1998). This procedure involves injecting RNA corresponding to a target gene into the germ line of an adult hermaphrodite, causing the inactivation of that gene in its progeny. For many targets, this method reproduces the phenotype of known loss-of-function mutations (Guo and Kemphues, 1995; Lin et al., 1995; Hunter and Kenyon, 1996; Rocheleau et al., 1997). Because double-stranded RNA is far more effective than antisense RNA (Fire et al., 1998), we used double-stranded RNA in most of our experiments. We refer to the progeny of animals injected with double-stranded RNA complementary to mac-1 as mac-1(dsRNAi) animals. We first observed mac-1(dsRNAi) animals produced using construct A (Fig. 9A). These animals complete embryogenesis and hatch, but most stop growing early in the second stage of larval development, and a few arrest later, in the L3 stage; all of these animals continue to move normally (Fig. 9B). Most of the mac-1(dsRNAi) animals remain arrested indefinitely, but some slowly continue development and eventually reach adulthood. In addition to this arrest, we observed other defects in mac-1(dsRNAi) animals. Even in the presence of food, the pharynges of young individuals did not pump normally. After several days, the intestines of most animals had large numbers of vacuoles, and some sections of the intestine were almost completely degraded (Fig. 9C). Finally, among the mac1(dsRNAi) animals that eventually reached adulthood, we observed defects in vulval and gonadal morphogenesis. These defects included the formation of a protruding vulva, and aberrant uterine structure. To confirm that these results were caused by inactivation of mac-1, and not of another nematode gene containing the CAD domain characteristic of AAA proteins, we repeated these injections using constructs B and C, each of which corresponds to a region of the transcript unique to mac-1. The mac1(dsRNAi) animals produced using constructs B and C resemble those produced using construct A, although the severity of the effect was slightly weaker (Fig. 9B). To determine if this phenotype required injection of double- MAC-1 is essential and can interact with CED-4 2027 stranded RNA, we also used antisense RNA from construct B in a series of control experiments; these mac-1(RNAi) animals showed the same phenotype of larval arrest (data not shown). Finally, we used a construct corresponding to the unc-4 transcript in a series of control injections. Among the unc-4(dsRNAi) animals, 51% were unable to move backwards, and 39% backed poorly, but none showed a developmental arrest (n=57). This inability to move backwards is characteristic of unc-4(lf) mutants (Miller et al., 1992; White et al., 1992), so our results support the conclusion of Fire et al. (1998) that injection of double-stranded RNA can specifically reproduce the phenotype caused by null mutations in a gene. The larval arrest of mac-1(dsRNAi) animals is not suppressed by ced-3 or ced-4 To determine if this larval arrest was caused by inappropriate activation of the programmed cell death pathway, we carried out a series of injections using ced-3(n718) and ced-4(n1416) mutants. Because all of our dsRNA constructs resulted in the same phenotype, we adopted fragment D for these experiments, since it is co-extensive with B and C, and does not include sequences homologous to other nematode AAA messages. We found that inactivation of mac-1 caused the ced3 and ced-4 animals to arrest at roughly the same point in development as the wild-type. At 4 days after fertilization, 90% of the N2 mac-1(dsRNAi) animals were still L2 or L3 larvae (n=73), whereas untreated N2 animals were mature adults. Similarly, 24% of the ced-3; mac-1(dsRNAi) animals were L1 larvae, and 73% were L2 or L3 animals (n=143). Finally, 29% of the ced-4; mac-1(dsRNAi) individuals were L1 larvae, and 68% were L2 or L3 animals (n=66). That some of the ced-3 and ced-4 individuals arrest at an earlier age than their wildtype counterparts is probably due to the fact that untreated ced3 and ced-4 animals occasionally arrest early in development (Ellis and Horvitz, 1986; Hengartner et al., 1992). DISCUSSION We identified MAC-1 through a yeast two-hybrid screen for Fig. 5. MAC-1 associates with CED-3, CED-9 and Apaf-1 in mammalian cells. (A,B) 293T cells were transiently co-transfected with 5 µg of plasmids expressing the indicated proteins. Cellular lysates were immunoprecipitated with mAb anti-Flag (A), anti-HA (B) or isotype-matched control mAb (indicated by C). Immunoprecipitates were immunoblotted with rabbit anti-HA, anti-Myc or anti-Flag antibody (A) or anti-Flag (B). Expression of MAC-1, CED-3, CED-4 and CED-9 in total lysates is shown in the lower part of the panels. (C) MAC-1 associates with Apaf-1. 293T cells were transiently co-transfected with 5 µg of plasmids expressing Flag-tagged MAC-1 and Myc-tagged Apaf-1. Cellular lysates were immunoprecipitated with mAb anti-Flag and immunoblotted with rabbit anti-Myc or anti-Flag antibodies. Expression of Apaf-1 in total lysates is shown in the lower part of the panel. Non-specific bands are indicated by a star. 2028 D. Wu and others CED-4-interacting proteins. We used this approach because we suspected that genes with lethal or pleiotropic phenotypes might not have been identified in genetic screens for cell death mutants. We have shown that MAC-1 is essential for larval development in C. elegans, can bind to CED-4 in both yeast and mammalian cells, and can regulate programmed cell death in nematodes and mammalian cells. 1997). Instead, MAC-1 most resembles the Smallminded protein of Drosophila (Figs. 3B,C; Long et al., 1998). This similarity is particularly significant in the N-terminal 200 amino acids, since this domain differs greatly in most AAA family members. Strikingly, smallminded mutant flies arrest development as second instar larvae, and mac-1(dsRNAi) worms arrest as L2 larvae. This result suggests that mac-1 and smallminded identify a new subfamily of AAA proteins that regulate growth or development in young larvae. MAC-1, a novel member of the AAA family of ATPases What is the function of MAC-1 in larval The predicted amino acid sequence of MAC-1 suggests that it development? is a member of the AAA family of ATPases. To date, more than 100 members of the AAA family have been identified in Two experiments shed light on the normal function of mac-1 eukaryotes, eubacteria and archaebacteria (Swaffield and in nematodes. First, elevated (or ectopic) expression of mac-1 Purugganan, 1997). Members of this family have one or two can prevent programmed cell deaths, which suggests that copies of a highly conserved region of 230-250 amino acids, MAC-1 might help regulate cell death. Second, inactivation of termed the conserved ATPase domain or CAD; this domain mac-1 by RNA-mediated interference causes animals to arrest includes the AAA minimal consensus motif and a set of Walker as L2 larvae. Although most worms arrest permanently, some A and B signature sequences characteristic of ATPases eventually complete development, perhaps because the double(Swaffield and Purugganan, 1997; Patel and Latterich, 1998). stranded RNA that inactivates mac-1 is degraded. These mature MAC-1 contains two such CAD domains. Members of the AAA family participate in a wide variety of cellular functions, including control of the cell cycle, vesicular transport during protein secretion, biogenesis of organelles, formation of protein complexes, regulation of transcription, mitochondrial membrane-bound proteolysis, and proteosomal regulation (Confalonieri and Duguet, 1995; Swaffield and Purugganan, 1997; Patel and Latterich, 1998). The precise biochemical functions of these ATPases are not understood, but it has been proposed that they modulate the folding, transfer, assembly or degradation of protein complexes in an ATP-dependent manner (Confalonieri and Duguet, 1995). Mammalian VCP and its yeast and Xenopus orthologs, CDC48 and p97, three AAA family members that share a high level of amino acid and structural similarity with MAC1, participate in organelle membrane-fusion events (Confalonieri and Duguet, 1995). However, it is unlikely that MAC-1 is the C. elegans Fig. 6. Genomic organization and expression of mac-1 in C. elegans. (A) Genetic map position of the ortholog of VCP/CDC48/p97, mac-1 locus. Genes shown in gray have been positioned only by their location on the physical map, and since two nematode AAA not by genetic recombination. (B) The position of the mac-1 locus on the YACs Y48C3 and Y81G3 is members, still uncharacterized, shown, based on preliminary data from the Genomic Sequencing Project (Sulston et al., 1992). (C) The exhibit even greater homology structure of the mac-1 locus. (D) Expression of mac-1 in wild-type and mutant C. elegans. The femwith this subfamily (Beyer, 3(gf) animals produce only sperm, and the fem-1(lf) animals produce only oocytes. vector 4 8 4 2 0 2 6 10 12 Number of Animals hsp16-2::mac-1 4 2 0 2 4 6 8 10 12 Extra cells in anterior pharynx 14 Fig. 7. MAC-1 promotes cell survival in C. elegans. Histograms show the number of cells in the anterior pharynx that failed to undergo programmed deaths. This value is 0 in wild-type animals, 5.3 in ced-9(n1950)/+ controls, and 10.0 in ced-9(n1950)/+; hsp162::mac-1 animals. The bars indicate the mean, ± one standard deviation. animals show additional defects, such as the formation of vacuoles in the intestine, and abnormal vulval and gonadal development. We do not know if these problems reflect a widespread requirement for mac-1 in larval development, or are a side effect of slowed development. However, animals that fail to develop because of starvation have not been seen to exhibit these problems (L. Avery, personal communication; our unpublished observations). Our results show that the requirement for mac-1 during larval development is unlikely to involve the regulation of programmed cell death, since mutations in ced-3 and ced-4 do not suppress the arrest of mac-1(dsRNAi) animals. Since the screen that identified mac-1 was capable of finding proteins that are essential, or which have pleiotropic effects on development, it should not be surprising that MAC-1 appears to have more than one function, and that its major function might be unrelated to programmed cell death. In this respect, several members of the AAA family of ATPases, including mammalian VCP, are known to be involved in diverse cellular functions, including cycle regulation (Madeo et al., 1997b), endocytosis (Pleasure et al., 1993), membrane transport (Rabouille et al., 1995) and proteasome regulation (Dai et al., 1998). What is the function of MAC-1 in programmed cell death? Although it remains possible that MAC-1 does not bind and regulate CED-4 during normal development, our results show that it is capable of controlling programmed cell death in nematodes and mammalian cells. Four models can account for these observations. First, mac-1 might help protect all cells that should survive from undergoing programmed deaths. The fact that increased expression of MAC-1 prevents programmed cell deaths from occurring, both in mammalian cells and in transgenic nematodes, suggests that mac-1 could protect cells from inappropriate activation of CED-4 and CED-3. However, mac-1(dsRNAi) animals do not resemble ced-9(lf) mutants, which die early in development from ectopic cell deaths (Hengartner et al., 1992). Furthermore, mutations in either ced3 or ced-4 suppress the lethality caused by mutations in ced-9 (Hengartner et al., 1992), but not that caused by inactivation of mac-1. This result shows that mac-1(dsRNAi) worms do not die because of inappropriate activation of the cell death pathway. Thus, if MAC-1 plays a general role in preventing programmed cell deaths, it is likely to act as an accessory to CED-9, rather than being essential in its own right. The fact that expression of MAC-1 shows a greater protective effect if CED-9 activity has also been increased is consistent with this hypothesis. Genetic studies suggest that the ced-8 gene might also be a non-essential partner in the cell death process (reviewed by Hengartner, 1997). A second possibility is that mac-1 regulates the survival or A 70 60 % Apoptotic Cells hsp16-2 50 40 30 20 10 0 + - Vector CED-3 CED-4 CED-9 MAC-1 B % Apoptotic Cells Number of Animals MAC-1 is essential and can interact with CED-4 2029 + - + - - - WT N C + - - + + - + - WT - + + - - N C + + - + + + - + + - - + + + + - - WT N C 80 60 40 20 0 CED-3 CED-4 CED-9 MAC-1 + + - + + + + + + + + - - - 0.1 0.2 0.5 1.0 + + + + + + + + + + + + 0.1 0.2 0.5 0.1 0.1 0.1 0.1 0.2 0.5 - - - Fig. 8. MAC-1 inhibits apoptosis induced by CED-3 and CED-4 in 293T cells. (A) 293T cells were transiently transfected with plasmids producing the indicated proteins (see Materials and methods), and a reporter plasmid expressing β-gal (Wu et al., 1997a). 16-20 hours after transfection, cells were stained with X-gal and examined by light microscopy. The results represent the percentage of blue cells that exhibit morphologic features of apoptosis (Wu et al., 1997a) and are given as the mean ± s.d. of triplicate cultures. WT, wild-type Mac-1; N, Mac-1-N; C, Mac-1-C (see Fig. 4). (B) MAC-1 enhances the protective ability of CED-9 against apoptosis. 2030 D. Wu and others induce chromatin condensation and cell death, even without co-expression of CED-3 (James et al., 1997). Similarly, when CED-4 is expressed in the touch neurons of C. elegans ced-3 mutants, some of these cells die (Shaham and Horvitz, 1996), which is consistent with this model. If CED-4 is able to induce some deaths in the absence of CED-3, perhaps MAC-1 regulates this activity. Finally, mac-1 might regulate the survival of cells in response to conditions not found in the laboratory. In mammals, programmed cell death plays an important role in the elimination of cells that have been infected with certain viruses (Tschopp et al., 1998), or that have become damaged or cancerous (Ellis et al., 1991). We do not know if these stimuli induce programmed deaths in nematodes, but if so, mac-1 might regulate this response. Furthermore, since MAC1 is an essential protein, one exciting possibility is that MAC1 induces death if normal cell growth or differentiation have been seriously perturbed. One observation supports this possibility – a missense mutation of the gene CDC48, a homologue of mac-1 that is required for cell division in yeast, can induce some features typically found during programmed cell death, such as membrane staining with annexin V, DNA fragmentation, and chromatin condensation (Madeo et al., 1997a). Perhaps MAC-1 regulates CED-4 activity to allow complete induction of the cell death program in analogous situations during C. elegans development. Because these results have broad implications for how cell death is controlled during normal development, and how essential genes might influence this process, we have initiated a search for mutations that inactivate mac-1. Such mutations would allow more detailed study of its function and behavior through genetic mosaic analysis or study of mac-1 transgenes. Fig. 9. RNA-mediated inactivation of mac-1 arrests development of C. elegans. (A) The location of the three mac-1 fragments used to produce double stranded or antisense RNAs. Exon boundaries are shown as black lines, and the regions encoding the CAD domains in gray. (B) The frequency of larval arrest among mac-1(RNAi) and unc-4(RNAi) animals. Animals scored as arrested were still young larvae 6 days after fertilization. mac-1 A, n=85, mac-1 B, n=37, mac1 C, n=42, unc-4, n=43. (C) Nomarski photomicrograph of the posterior half of a mac-1(dsRNAi) individual, age 9 days. The arrows indicate vacuoles that have formed in part of the intestine. Anterior is to the left, dorsal is up. death of a small number of cells during development. Because of the defects mac-1(dsRNAi) animals show in feeding, we examined the anterior pharynges of these animals, but did not observe any abnormal deaths, or cells that had failed to die (data not shown). We also examined the heads and ventral cords of ced-1(e1735); mac-1(dsRNAi) larvae to determine if extra cell deaths or inappropriate survivals had occurred in these areas, but observed no differences from the ced-1 controls (data not shown). However, we could not accurately study cell deaths that normally occur after the developmental arrest in mac-1(dsRNAi) worms, so it remains possible that MAC-1 regulates cell death during late larval development or adulthood. A third possibility is that MAC-1 regulates the ability of CED-4 to kill cells by a mechanism that does not require CED3. 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