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The two Drosophila cytochrome C proteins can function in both respiration and caspase activation

2005; Springer Nature; Volume: 25; Issue: 1 Linguagem: Inglês

10.1038/sj.emboj.7600920

1460-2075

Eli Arama, Maya Bader, Mayank Srivastava, Andreas Bergmann, Hermann Steller,

Plant Genetic and Mutation Studies

Article15 December 2005free access The two Drosophila cytochrome C proteins can function in both respiration and caspase activation Eli Arama Eli Arama Strang Laboratory of Cancer Research, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Maya Bader Maya Bader Strang Laboratory of Cancer Research, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Mayank Srivastava Mayank Srivastava Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Andreas Bergmann Andreas Bergmann Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Hermann Steller Corresponding Author Hermann Steller Strang Laboratory of Cancer Research, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Eli Arama Eli Arama Strang Laboratory of Cancer Research, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Maya Bader Maya Bader Strang Laboratory of Cancer Research, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Mayank Srivastava Mayank Srivastava Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Andreas Bergmann Andreas Bergmann Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Hermann Steller Corresponding Author Hermann Steller Strang Laboratory of Cancer Research, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Author Information Eli Arama1, Maya Bader1, Mayank Srivastava2, Andreas Bergmann2 and Hermann Steller 1 1Strang Laboratory of Cancer Research, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA 2Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA *Corresponding author. Strang Laboratory of Cancer Research, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. Tel.: +1 212 327 7075; Fax: +1 212 327 7076; E-mail: [email protected] The EMBO Journal (2006)25:232-243https://doi.org/10.1038/sj.emboj.7600920 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cytochrome C has two apparently separable cellular functions: respiration and caspase activation during apoptosis. While a role of the mitochondria and cytochrome C in the assembly of the apoptosome and caspase activation has been established for mammalian cells, the existence of a comparable function for cytochrome C in invertebrates remains controversial. Drosophila possesses two cytochrome c genes, cyt-c-d and cyt-c-p. We show that only cyt-c-d is required for caspase activation in an apoptosis-like process during spermatid differentiation, whereas cyt-c-p is required for respiration in the soma. However, both cytochrome C proteins can function interchangeably in respiration and caspase activation, and the difference in their genetic requirements can be attributed to differential expression in the soma and testes. Furthermore, orthologues of the apoptosome components, Ark (Apaf-1) and Dronc (caspase-9), are also required for the proper removal of bulk cytoplasm during spermatogenesis. Finally, several mutants that block caspase activation during spermatogenesis were isolated in a genetic screen, including mutants with defects in spermatid mitochondrial organization. These observations establish a role for the mitochondria in caspase activation during spermatogenesis. Introduction Apoptosis is a morphologically distinct form of active cellular suicide that serves to eliminate unwanted and potentially dangerous cells (Thompson, 1995; Jacobson et al, 1997; Hengartner, 2000; Meier et al, 2000a; Baehrecke, 2002; Nelson and White, 2004). The key enzymes responsible for the execution of apoptosis are an evolutionarily conserved family of cysteine proteases known as caspases (Salvesen, 2002; Degterev et al, 2003; Abraham and Shaham, 2004). Caspases are present in an inactive or weakly active state in virtually all cells of higher metazoans, and their activity is carefully regulated by both activators and inhibitors (Song and Steller, 1999; Salvesen and Abrams, 2004). In vertebrates, the mitochondria play an important role in the control of apoptosis: they release cytochrome C and other pro-apoptotic proteins in response to various death signals (Zou et al, 1997; Green and Reed, 1998; Benedict et al, 2000; Larisch et al, 2000; Wang, 2001). In the cytosol, cytochrome C binds to Apaf-1 (Zou et al, 1997) which in turn promotes the assembly of a multiprotein complex, termed the ‘apoptosome’, and caspase-9 activation (Rodriguez and Lazebnik, 1999; Adams and Cory, 2002; Cain et al, 2002; Salvesen and Renatus, 2002). In the ensuing ‘caspase cascade’, many intracellular substrates are cleaved and apoptosis is executed (Slee et al, 1999; Riedl and Shi, 2004). However, the exact physiological role of cytochrome C for caspase activation remains to be determined, and a recent report on a mutant cytochrome c that fails to activate Apaf-1 in the mouse suggests that cytochrome C is required for caspase activation in only some mammalian cell types (Hao et al, 2005). In invertebrates, any role of cytochrome C for the activation of caspases has remained highly controversial (Kornbluth and White, 2005). Whereas RNAi experiments in Drosophila S2 cells have failed to reveal a role for cytochrome C in apoptosis, other reports suggest that cytochrome C may promote caspase activation (Dorstyn et al, 2002, 2004; Zimmermann et al, 2002). Drosophila contains two Apaf-1 isoforms: one with a WD40 repeat domain, the target for cytochrome C binding, and another lacking this domain, similar to Caenorhabditis elegans Ced-4. The large isoform can directly bind cytochrome C in vitro and promote cytochrome C-dependent caspase activation in lysates from developing embryos (Kanuka et al, 1999). Furthermore, an overt alteration in the cytochrome C immuno-staining can be detected in doomed cells in some Drosophila tissues, and the mitochondria from apoptotic cells can activate cytosolic caspases (Varkey et al, 1999). Finally, disruption of one of the two Drosophila cytochrome c genes, cyt-c-d, is associated with a failure to activate caspases in an apoptosis-like process during sperm terminal differentiation in Drosophila (Arama et al, 2003). In this process, also known as spermatid individualization, the majority of cytoplasm and cellular organelles are eliminated from the developing spermatids in an apoptosis-like process that requires caspase activity (Arama et al, 2003). However, it was suggested that the mutants used in our previous study may also affect other genes located in the vicinity of the cyt-c-d locus (Huh et al, 2004). Here, in order to rigorously address this issue, we conducted a series of genetic and transgenic rescue experiments that unequivocally establish a role of cytochrome C for caspase activation during Drosophila spermatogenesis. First, we isolated a point mutation in cyt-c-d that is defective in caspase activation. Next, we demonstrated that transgenic expression of cyt-c-d restores effector caspase activation and rescues all the sterility phenotypes associated with various cyt-c-d mutant alleles. We also investigated the possibility that cyt-c-p functions specifically in respiration, whereas cyt-c-d plays a role in caspase regulation. To our surprise, we found that expression of either cyt-c-d or cyt-c-p can restore caspase activation in cyt-c-d-deficient spermatids, demonstrating that both proteins are functionally equivalent. Other apoptosome proteins in Drosophila, Ark (Apaf-1) and Dronc (caspase-9) are also required for spermatid individualization, and their mutant phenotypes are similar to spermatids with a block in caspase activity. Surprisingly, however, we can still detect some active caspase-3 staining in these mutant testes, suggesting that cytochrome-C-d may function in yet other unknown pathways to promote caspase-3 activation. Finally, we have identified several mutants affecting spermatid mitochondria that provide a strong link between mitochondrial organization and caspase activation during sperm development. Results Mutations in cyt-c-d block caspase activation during spermatid individualization In order to identify genes required for caspase activation during spermatid differentiation in Drosophila, we sought to identify mutants that lacked CM1 staining, which detects the active form of the effector casepase drICE (Baker and Yu, 2001). For this purpose, we screened an existing collection of more than 1000 male-sterile mutant lines defective in spermatid individualization that were previously identified among a collection of about 6000 viable mutants generated in the laboratory of Dr Charles Zuker (Koundakjian et al, 2004; Wakimoto et al, 2004). We stained dissected testes from each line with CM1 (see Supplementary data) and identified 33 lines that were CM1-negative. However, the vast majority of male-sterile lines remained CM1-positive, even though many displayed severe defects in spermatid individualization (e.g. Figure 1F). Therefore, consistent with our earlier observations (Arama et al, 2003), caspase activation at the onset of spermatid individualization appears to be independent of other aspects of sperm differentiation, such as the assembly of the individualization complex or its movement. One of the mutants, line Z2-1091, failed to complement the sterility of bln1, a P-element insertion in cyt-c-d, and was CM1-negative as a homozygote, in trans to a small deletion removing the cyt-c-d locus (Df(2L)Exel6039), or in trans to the cyt-c-dbln1 allele (Figure 1C–E). In contrast, Z2-1091 complemented the lethality of K13905, a P-element insertion in cyt-c-p, and K13905 complemented the sterility of Z2-1091. Genomic sequence analyses of the transcription units of both cyt-c-d and cyt-c-p in Z2-1091 flies revealed a point mutation of TGG → TGA at codon 62 in cyt-c-d, causing a change of Trp62 into a stop codon that results in a truncation of almost half of the protein (Figure 1H). We will henceforth refer to this allele as cyt-c-dZ2−1091. Given the molecular nature of cyt-c-dZ2−1091, it is very unlikely that this allele affects the function of genes adjacent to cyt-c-d (see below). Figure 1.(A–F) Mutations in cyt-c-d block caspase activation and spermatid individualization. Visualization of active drICE with anti-cleaved caspase-3 antibody (CM1; green) in wild-type (A), cyt-c-dbln1 (B), cyt-c-dZ2−1091 (C), cyt-c-dZ2−1091/DF(2L)Exel6039 (D), cyt-c-dZ2−1091/cyt-c-dbln1 (E), and Z2-2468 (F). Whereas CM1-positive elongated spermatid cysts at different individualization stages can be readily seen in wild-type testes (A; white arrow pointing at a CB), no CM1-staining was detected in spermatids of flies homozygous for the P-element allele, bln1 (B) and the point mutation allele, Z2-1091 (C). Similarly, spermatids of Z2-1091 flies either trans-heterozygous to the small deficiency Df(2L)Exel6039 (D) or to the bln1 allele (E) also displayed no CM1 staining. In contrast, the vast majority of male-sterile mutants with spermatid individualization defects display strong CM1 positive cysts (e.g. Z2-2468; F). To visualize all the spermatids, the testes were counter-stained with phalloidin that binds F-actin (red). (G) Caspase-3-like (DEVDase) activity is detected in wild-type testes, and is blocked either after treatment with the caspase-3 inhibitor Z-VAD.fmk or in cyt-c-dZ2−1091−/− mutant testes. DEVDase activity, presented as relative luminescence units (RLU), was determined on Ac-DEVD-pNA substrate in 62 wild-type (yw) or cyt-c-dZ2−1091−/− mutant testes treated with Z-VAD or left untreated (DMSO). Readings were obtained every 2 min, and each time interval represents an average (mean±s.e.m.) of five readings (for more details also see the Supplementary data). Note that the levels of DEVDase activity in cyt-c-dZ2−1091−/− mutant testes are highly similar to the corresponding levels in wild-type testes that were treated with Z-VAD. (H) Alignment of the predicted protein sequences of cytochrome C-d (c-d) and cytochrome C-p (c-p). Identical residues are indicated in the consensus (cons.) line. Cytochrome C-d and cytochrome C-p share 72% identity and 82% similarity. The tryptophan (W; red) at position 62 of cytochrome C-d is mutated to a stop codon in the Z2-1091 allele. (I) RT–PCR analyses of cyt-c-d and cyt-c-p expression. After reverse transcription with adult flies RNA, PCR was performed using two sets of specific primers spanning the unique 5′ (upper panel) and 3′ (lower panel) UTRs of both cyt-c-d and cyt-c-p. Whereas cyt-c-d and cyt-c-p are expressed in wild-type flies (YW), only cyt-c-p is expressed in the bln1 flies, confirming that bln1 is a null allele of cyt-c-d. Download figure Download PowerPoint Effector caspases, such as drICE, can display DEVD cleaving activity (Fraser et al, 1997). Therefore, we asked whether wild-type adult testes also contain DEVDase activity, and whether this activity is affected in cyt-c-d mutant testes. Lysates of wild-type testes indeed display detectable levels of DEVDase activity, which were significantly reduced upon treatment with the potent DEVDase inhibitor Z-VAD.fmk (Figure 1G). Importantly, this activity was highly reduced in cyt-c-dZ2−1091 mutant testes (Figure 1G). These results provide independent evidence for effector caspase activity in wild-type sperm, and they support a role of cytochrome C-d in caspase activation in this system. Because of the cytological proximity between cyt-c-d and cyt-c-p (241 bp maximum between the end of the 3′ UTR of cyt-c-d and the beginning of the 5′ UTR of cyt-c-p) there is a possibility that the bln1 P-element insertion in cyt-c-d might also interfere with the expression of cyt-c-p (Huh et al, 2004). In order to determine whether cyt-c-p expression was altered in cyt-c-dbln1, we performed RT–PCR analyses with RNA from wild-type (yw) and cyt-c-dbln1 adult flies using two sets of primers for each gene specific for either the 5′ UTRs (upper panel of Figure 1I) or 3′ UTRs (lower panel of Figure 1I) of cyt-c-d and cyt-c-p. In agreement with our previous Northern results, no cyt-c-d RNA was detected in cyt-c-dbln1 flies, confirming that bln1 is a null allele of cyt-c-d. In contrast, cyt-c-p is expressed in both wild-type and cyt-c-dbln1 flies. Both cyt-c-d and cyt-c-p can rescue caspase activation, spermatid individualization, and the sterility of bln1 and Z2–1091 adult males Although the sequence of cyt-c-d and cyt-c-p proteins is highly conserved, they are not identical (Figure 1H). In addition, mutations in each gene display distinct phenotypes (Arama et al, 2003). This raises the possibility that both proteins may have distinct functions in respiration (cytochrome C-p) and caspase activation/apoptosis (cytochrome C-d). To test this hypothesis, we first asked whether expression of cyt-c-p in developing spermatids is able to substitute for the loss of cyt-c-d. In order to drive expression of transgenes in the male germ line, we constructed an expression vector composed of the hsp83 promoter followed by the 5′ and 3′ UTRs of cyt-c-d, which are important for the proper temporal regulation of cyt-c-d translation in spermatids (Figure 2A, I; see Supplementary data; Arama and Steller, unpublished), and next we inserted the coding regions of either cyt-c-d (Figure 2A, II) or cyt-c-p (Figure 2A, III) between both UTRs and generated transgenic flies with these constructs. At least three independent transgenic lines for each of these constructs were crossed to cyt-c-dbln1 or cyt-c-dZ2−1091 flies, and the presence of the appropriate transgene was confirmed by genomic PCR (Figure 2F and data not shown). To validate expression of the transgenes, we performed RT–PCR analysis with testes RNA in a cyt-c-dbln1−/− background (Figure 2G and H). Finally, we examined the ability of these transgenes to rescue caspase activation, spermatid individualization, and male sterility in cyt-c-dbln1 and cyt-c-dZ2−1091 flies. As a control, transgenic flies containing ‘empty vector’ (including the hsp83-promotor with the 5′ and 3′ UTRs of cyt-c-d but without a coding region) were also generated (Figure 2A, I and G). As expected, no caspase activation was detected in testes of these control flies (Figure 2C, compare to the wild-type in Figure 2B). On the other hand, a transgene with the cyt-c-d open reading frame (ORF) fully rescued CM1-staining, spermatid individualization, and male fertility (Figure 2D, note the reappearance of cystic bulges (CBs) and waste bags (WBs), white and yellow arrows, respectively). This firmly establishes that both the caspase and sterility phenotypes seen in cyt-c-dbln1 and cyt-c-dZ2−1091 mutant flies are strictly due to the loss of cytochrome c function, with no detectable contribution from adjacent genes. Figure 2.Both cyt-c-d and cyt-c-p can rescue the male sterile phenotypes of cyt-c-d−/− flies. (A) Schematic structure of the rescue constructs for cyt-c-d−/− male sterile flies. The promoter region (dark blue) and a portion of the 5′ UTR (light blue) of the hsp83 gene were fused to the 5′ UTR followed by the 3′ UTR sequences of cyt-c-d and served as a control (I). The precise coding region sequences of either cyt-c-d (II) or cyt-c-p (III) were subcloned in between the 5′ and 3′ UTRs. (B–E) Ectopic expression of either cyt-c-d or cyt-c-p rescues caspase activation, spermatid individualization, and sterility of cyt-c-dbln1−/− male flies. Similar to WT (B), CM1-positive spermatids (green), CBs (white arrows), and WBs (yellow arrows) are readily detected in transgenic lines of the cyt-c-dbln1−/− background expressing either cyt-c-d (D) or cyt-c-p (E) coding regions. In contrast, no CM1-positive cysts are found in cyt-c-dbln1−/− flies that ectopically express the control construct of cyt-c-d 5′–3′ UTRs alone (C). To visualize all the spermatids and the ICs, the testes were counter-stained with phalloidin that binds F-actin (spermatids are in weak red; ICs are in strong red or yellow and associated with CM1-positive spermatids; note that remnants of the testis sheath layer autofluoresce in strong red in B). Scale bars 200 μm (B–E, upper panels), and 100 μm (D, E, lower panels). (F) Integrations of the appropriate constructs into the genome were confirmed by genomic PCR analyses. The relative locations of the primers, which are indicated with forward black and reverse red (cyt-c-d; A, II) or reverse orange (cyt-c-p, A, III) arrows were used to amplify the fragments seen in the upper and middle panels in (F), respectively. For loading control, the dcp-1 gene was amplified (lower panel in F). (G, H) Transcriptional expression from the transgenes was confirmed by RT–PCR analyses on RNA from testes of the indicated genotypes. The relative locations of the primers are indicated with black arrows in (A). Representative figures demonstrating exogenous expression of cyt-c-d 5′-3′ UTR sequences alone (G), and the exogenous expression of cyt-c-d coding region flanked by its UTR sequences (H). ‘RT+Taq’ and ‘Taq’ indicate reactions with reverse transcriptase or without it, respectively, to control for possible genomic DNA contamination. (H) Primers corresponding to the unique 5′ and 3′ UTRs of cyt-c-p and primers corresponding to the exogenous cyt-c-d sequences (see Materials and methods and black arrows in A) were used in the same reaction. Download figure Download PowerPoint We next tested the ability of cyt-c-p to functionally substitute for the loss of cyt-c-d. To our surprise, transgenic expression of cyt-c-p was equally effective in rescuing all defects in cyt-c-dbln1 or and cyt-c-dZ2−1091 males (Figure 2E). We conclude that both proteins have similar biochemical properties to promote caspase activation and spermatid individualization. cyt-c-p is mainly somatic, whereas cyt-c-d is almost exclusively restricted to the male germ line Our rescue results raise the question of why cyt-c-d−/− males are sterile if both cytochrome c genes are functionally equivalent. One possible explanation is distinct expression of the two genes, namely that cyt-c-d is testis-specific, whereas cyt-c-p may be restricted to the soma. To examine this possibility, we investigated the distribution of transcripts from both cytochrome c genes in the testis and the soma. For this purpose, comparative RT–PCR experiments were performed using specific primers in the unique 5′ and 3′ UTR sequences of cyt-c-d and cyt-c-p (Figure 3A). While cyt-c-p was highly expressed in the soma, cyt-c-d was only weakly expressed there (represented by adult females that lack testes). On the other hand, cyt-c-d expression was much higher in testes than cyt-c-p (Figure 3B). We attribute the low levels of cyt-c-p in testes to the somatic cells present in this tissue (see below). Furthermore, although the expression of cyt-c-d in the soma of both males and females is much lower than the levels of cyt-c-p, cyt-c-d levels are much higher in adult males than in females, suggesting that the male germ cells provide the main contribution of cyt-c-d in the adult (Figure 3C). Our results suggest that the distinct phenotypes of cyt-c-d and cyt-c-p are mainly due to their restricted differential expression in the testis and the soma, respectively. Figure 3.cyt-c-d is mainly expressed in the testis, while in contrast cyt-c-p is mainly expressed in the soma. (A) Schematic structure of the Drosophila cytochrome c genes. cyt-c-d and cyt-c-p display similar genomic organization of two exons (thick black and gray bars, respectively) separated by a relatively large intron (thin bar). In both of them, the coding region is restricted to the second exon (between the ATG and the stop codons). The locations of the primers used in the comparative RT–PCR experiments in (B–E) are indicated by arrows. (B) Analysis of cyt-c-d versus cyt-c-p expression in the testis and the soma. The above primers (arrows in A) to amplify either a 543-bp cyt-c-d fragment or a 483-bp cyt-c-p fragment were added to one reaction master-mix. The reaction was stopped at different cycle points to identify the linear amplification phase (20, 25, and 30 cycles are indicated). Note that the relative expression levels of cyt-c-d (strong) and cyt-c-p (weak) in the testis are switched in the soma, which is represented by adult female flies. (C) In the soma of both males and females, the expression levels of cyt-c-d are much lower than the levels of cyt-c-p. However, cyt-c-d levels are higher in adult males than in females. Note the PCR cycle number at which a band first becomes visible. (D) The expression of cyt-c-d is restricted to the male germ cells. While the expression of cyt-c-p is not affected in sons of oskar agametic testes, no cyt-c-d expression was detected. (E) cyt-c-d is expressed in premeiotic cells comprising the larval testis but not in ovaries. (F) cyt-c-p is exclusively expressed in first instar WT larva and is almost completely absent from the cyt-c-pk13905−/−. Download figure Download PowerPoint In addition to germ cells, the testis also contains somatic cells, such as the testicular wall, muscles cells, and cyst cells. To determine which testicular cell types express cyt-c-d, we first performed comparative RT–PCR analyses with RNA from reproductive tracts of oskar male mutants that are defective in germline development and lack germ cells in the adults. While both cytochrome c genes were expressed in wild type, only cyt-c-p was detected in the germ-cell-less reproductive tracts of sons of oskar−/− (Figure 3D). This indicates that cyt-c-d expression is restricted to the germ cells of the adult male. Next, we investigated the developmental stage at which cyt-c-d is expressed in the male germ line. For this purpose, we took advantage of the fact that testes of adult flies and third instar larvae differ in their repertoire of germ cells. While adult testes contain germ cells in a variety of developmental stages, the most developmentally advanced germ cells present in third instar larval testes are premeiotic spermatocytes. Interestingly, the patterns of cyt-c-d expression in both adult and larval testes are identical (Figure 3E), demonstrating that cyt-c-d mRNA accumulates before the entry of spermatocytes into meiosis. The activation of apoptotic effector caspases, as visualized by CM1-staining, is not restricted to the male germ cells but can also be detected in nurse cells during oogenesis (Peterson et al, 2003). We considered the possibility that caspase activation in this system is also influenced by cyt-c-d. However, no abnormalities during oogenesis were detected in cyt-c-d−/− flies and the females are fertile (data not shown). Consistent with this idea, comparative RT–PCR analysis of adult ovaries revealed expression of cyt-c-p but not cyt-c-d (Figure 3E). cyt-c-d expression is not detectable in early larva but can rescue the lethality of cyt-c-p−/− mutant flies l(2)k13905 flies contain a P-element insertion in the 5′ UTR of cyt-c-p and die as late embryos or early first instar larva (Arama et al, 2003). Using RT–PCR, we found that only cyt-c-p expression was detected in early first instar wild-type larvae, while a dramatic reduction was observed in the cyt-c-pk13905 mutants (Figure 3F). These results are consistent with the phenotypes of cyt-c-d (viable but male sterile) and cyt-c-p (early lethal) mutants. Lethality of cyt-c-pk13905 homozygotes as well as trans-heterozygotes to Df(2L)Exel6039, a deletion in the region that includes both cyt-c-p and cyt-c-d, is consistent with the idea that cyt-c-p encodes the major cytochrome C responsible for respiration (Inoue et al, 1986). We investigated whether cyt-c-d could also function in respiration and rescue the early lethality of cyt-c-p−/− flies. Both cytochrome C proteins were ectopically expressed in cyt-c-pk13905 mutants using the GAL4-UAS system (Brand and Perrimon, 1993). The Tub-Gal4 driver line was used to drive cyt-c-p and cyt-c-d expression throughout the lifespan of the fly. Notably, one copy of either the UAS-cyt-c-p or the UAS-cyt-c-d transgenes together with one copy of the driver completely rescued the lethality of cyt-c-pk13905/Df(2L)Exel6039 flies (Figure 4). We conclude that both cytochrome C proteins of Drosophila can function in electron transfer/respiration. The complete absence of cyt-c-p from the rescued adult flies is consistent with the idea that cyt-c-pk13905 is a null allele of cyt-c-p. We attribute the faint expression of cyt-c-p in cyt-c-pk13905 homozygote and cyt-c-pk13905/Df(2L)Exel6039 trans-heterozygote mutants detected in early first instar larvae only after 30 PCR cycles (Figure 3F and data not shown) to remnants of maternal contribution. This also explains how cyt-c-p−/− mutant embryos can reach the early first instar larval stage without any zygotic contribution. Finally, we could not rescue the lethality of flies homozygous for the cyt-c-pk13905 allele, suggesting that the k13905 chromosome carries an additional unrelated lethal mutation. Figure 4.Both cyt-c-p and cyt-c-d can completely rescue the lethality/respiration defect of cyt-c-p−/− embryos. A premix of RT–PCR reaction was designed to amplify endogenous cyt-c-p and/or transgenic cyt-c-d from testes of wild-type (WT) or rescued cyt-c-p−/− adult flies that express transgenic cyt-c-d under the control of the tubulin promoter (cyt-c-pk13905/Df(2L)Exel6039; tub-Gal4/UAS-cyt-c-d). To confirm that the rescued flies are of the right genotypes, we performed RT–PCR analyses with wild-type and the rescued adult flies using specific primers for the endogenous cyt-c-p as well as the transgenic cyt-c-d. Note that the strong cytochrome c transcript expression in cyt-c-p−/− adult flies originated from the cyt-c-d transgene. Download figure Download PowerPoint Immunoreactivity of the cytochrome C-d protein increases at the onset of spermatid individualization To study the pattern of cytochrome C-d expression in the testis, polyclonal antibodies were raised against four peptides covering the entire length of the protein (see Supplementary data). Consistent with our findings that no cyt-c-d RNA is expressed in cyt-c-dbln1 homozygote flies, almost no signal was detected after staining testes of this mutant with the anti-cytochrome C-d antibody (Figure 5B). Staining wild-type testes with this antibody revealed a grainy pattern of cytochrome C-d signal along the entire length of elongating spermatids and elongated spermatids (Figure 5A). Once an individualization complex (IC) was assembled in the vicinity of the nuclei, an increase in cytochrome C-d staining was detected with the highest intensity found next to the IC (arrowheads in Figure 5C). During the caudal translocation of the IC, a significant portion of cytochrome C-d is depleted from the newly individualized part of the spermatids (arrow in Figure 5C) into the CB (arrowhead in Figure 5D). Eventually, the newly formed WBs accumulate high levels of cytochrome C-d (Figure 5E). Figure 5.Exp