XE07 mutations cause severe neurodegenerative phenotypes
To identify novel genes on the Drosophila
X chromosome that cause a neurodegenerative phenotype, we performed a large forward mosaic genetic screen using the flippase/flippase recognition target system (Xiong et al.,2012
; Yamamoto et al., 2012
) and identified a complementation group, XE07
, which exhibits degenerative electroretinogram (ERG) defects in clones. In response to a light pulse, 1-day-old flies display reduced amplitudes ( [bracket] and b), when compared with wild-type tissue. Aging the flies causes the defects to worsen, and the ERG amplitudes are almost completely abolished after 24 d (), showing that the photoreceptors (PRs) deteriorate with age. To determine whether there is a progressive morphological demise of the PR, we performed transmission EM (TEM) on mutant retinas of flies of different ages. Newly eclosed flies with XE07
mutant clones contain a normal number of rhabdomeres per ommatidium with the typical trapezoidal organization. However, the pigment cells that function as glial cells in the eye are vastly expanded and display highly aberrant morphological features (compare ). Upon aging for 10 d, some rhabdomeres are missing, and many PRs appear rough and/or vacuolated. The cell bodies of PRs are packed with electron-dense particles ( and S1 a
). At day 24, the rhabdomeres are barely recognizable or missing (), whereas the wild-type retinas still exhibit intact structure (Fig. S1 b). Hence, loss of XE07
causes a severe neurodegenerative phenotype. To assess whether this phenotype is light dependent, we raised the flies in complete darkness. Flies with mutant clones raised in darkness display less aberrant rhabdomeres per ommatidium than flies raised in a light/dark cycle (Fig. S1, a and c). Hence, the neurodegeneration associated with XE07
occurs in both the presence and absence of light and is only delayed in the dark.
Figure 1. XE07 mutants display progressive neurodegenerative features in PRs. (a) In response to a 1-s light pulse, 1-day-old XE07 mutant flies display reduced amplitudes (bracket) when compared with wild type. Aging these flies causes the defects to worsen, and (more ...)
TEM also revealed defects associated with mitochondria. Mitochondria of newly eclosed mutants are enlarged when compared with mitochondria in wild-type tissue (, compare with c, arrowheads and insets; and S1 d). At day 10, the mutant mitochondria are swollen and vacuolated, with dissociated cristae (, arrow and inset). At day 24, the overall morphology of PRs is so severely affected that one can hardly identify mitochondria ().
XE07 is the Drosophila homologue of human C8ORF38
To identify the molecular lesions of the XE07
alleles, we performed duplication and deficiency mapping. A lethal P
element insertion line (Bellen et al., 2004
) affecting CG15738
fails to complement the lethality of all six alleles, indicating that XE07
corresponds to CG15738
(Fig. S2 a
). Sanger sequencing identified three nonsense mutations (A, E, and H), two deletions (C and G), and a mutation in a potential splicing site (B) in CG15738
(). Mutant hemizygous males and trans-heterozygous females are pupal lethal (Fig. S2 b). Interestingly, mutant larvae have very extended larval stages and live for 7–10 d as third instars before pupation, whereas wild-type third instars typically pupate after 2 d. We therefore named our gene “sicily
” for severe impairment of CI with lengthened youth (). Finally, the ERG and TEM of the lamina associated with the six alleles do not reveal an obvious allelic series (unpublished data), suggesting that all alleles are similar loss-of-function mutations. We therefore decided only to characterize two alleles: sicilyC
(loss of the promoter and first two exons) and sicilyE
Figure 2. XE07 corresponds to CG15738 (sicily), the fly homologue of human C8ORF38. (a) Schematic representation of the molecular lesions of XE07 alleles. (b) Extent of a genomic rescue construct and its mCherry-tagged version. The 5′ of CG15738 contain (more ...)
The human homologue of Sicily, C8ORF38, is a mitochondrial CI assembly factor. Patients with mutations in C8ORF38
presented with Leigh syndrome and CI deficiency (Pagliarini et al., 2008
). Similar to its human homologue, Sicily possesses a predicted NMTS (11 aa). 70% of the protein consists of an evolutionarily conserved domain with unknown function (Fig. S2 c, black bar). Despite its similarity to the predicted evolutionary origin of squalene and phytoene synthase (~40% based on Pfam prediction), several key amino acids conserved in most squalene and phytoene synthases are absent in this domain (Robinson et al., 1993
), suggesting that it may not possess an enzymatic function.
Genomic rescue constructs () rescue the lethality (Fig. S2 b), ERG defects (), and retinal degeneration () associated with the loss of sicily
for all alleles. Ubiquitous expression of the CG15738
cDNA using the GAL4/upstream activation sequence (UAS) system (Brand and Perrimon, 1993
), driven by daughterless
also rescues the lethality and phenotypes associated with all alleles (unpublished data), demonstrating that sicily
indeed corresponds to CG15738
. In addition, ubiquitous expression of human C8ORF38
cDNA also rescues the lethality and ERG defects (; and S2 b), indicating that the function of sicily/C8ORF38
is conserved between flies and human.
To determine whether sicily
affects CI function, we performed the ETC assay and found that the CI activity is severely compromised in sicily
mutants when compared with wild type (). To further test whether sicily
affects the protein level of CI subunits, we immunoblotted purified mitochondria of sicily
mutants with eight different antibodies raised against human CI subunits. Only two, NDUFS3 (Bratic et al., 2011
) and NDUFA10 (ND42 in Drosophila
), cross-reacted and were specific. Both NDUFS3 and ND42 are severely down-regulated in sicily
mutants, whereas other mitochondrial proteins, including Porin (Graham et al., 2010
) and cytochrome c
, are unaltered or slightly increased (). These data indicate that sicily
affects CI, in agreement with previous findings for C8ORF38
(Pagliarini et al., 2008
; McKenzie et al., 2011
; Zurita Rendón and Shoubridge, 2012
CI is responsible for most cellular ROS production in mitochondria (Brand, 2010
), and impairment in CI activity often results in oxidative stress (Owusu-Ansah et al., 2008
; Owusu-Ansah and Banerjee, 2009
). To test this, we performed the aconitase assay, an indicator of ROS-mediated oxidative damage (Yan et al., 1997
), and found that aconitase activity is severely decreased in sicily
mutants, whereas the in vitro reactivated aconitase activity remains unaltered (), suggesting that the ROS levels are chronically increased in sicily
mutant cells (Emptage et al., 1983
). We also confirmed that the ROS level is increased in sicily
mutant tissues based on MitoSOX staining (Invitrogen; unpublished data; Yarosh et al., 2008
). ROS damages the proper folding of proteins that can activate Hsp60 (Yoneda et al., 2004
; Bayat et al., 2012
). As shown in , Hsp60 is up-regulated, suggesting that sicily
mutants may contain misfolded mitochondrial proteins.
Sicily interacts with the preproteins of ND42 and NDUFS3 before their mitochondrial import
To study the function of Sicily, we performed a yeast two-hybrid screen and identified ND42, a CI subunit, as a Sicily interactor (). We confirmed this interaction by coimmunoprecipitation (IP; co-IP; ). In these co-IP assays, we noticed that both Sicily and ND42 display two isoforms (, large [l] vs. small [s]). However, based on annotations, neither Sicily nor ND42 has more than one translational variant in Drosophila
, suggesting that the isoforms are caused by posttranslational modifications. As NMTSs are cleaved from preproteins after mitochondrial import (Marc et al., 2002
), we hypothesized that the two isoforms correspond to the uncleaved preproteins in cytoplasm and cleaved proteins in mitochondria, respectively. Indeed, the predicted length of the NMTS in Sicily is 11 aa (1.2 kD) and the NMTS of ND42 is 47 aa (5.1 kD), in agreement with the difference between the two isoforms.
Figure 3. Sicily interacts with CI subunits in cytoplasm. (a) Sicily interacts with ND42 in a yeast two-hybrid assay. Yeast transfected with DNA are plated on SD-Trp-Leu-His media supplemented with X-α-gal. (b) Sicily and ND42 co-IP. S2 cells transfected (more ...)
To further test this hypothesis, we separated the cytoplasmic and mitochondrial fractions and found that the larger isoforms of both Sicily and ND42 are enriched in cytoplasm, whereas the smaller isoforms are enriched in mitochondria (). We then purified both isoforms of ND42 and subjected them to mass spectrometry analysis and identified five readings of peptide aa 16–37 from the larger isoform but none from the smaller isoform of ND42 (). As this peptide (aa 16–37) is derived from the predicted NMTS (aa 1–47), these data indicate that the NMTS is present in the larger isoform. We also repeat this experiment with Sicily. However, we were unable to obtain enough protein for mass spectrometry analysis.
In our co-IP assays, we also observed that the larger isoforms of Sicily and ND42 preferentially coprecipitate with each other (). This is not because of a generic bias of the IP, as most of the precipitated proteins consist of the smaller isoforms (, second and fourth boxes). These data indicate that the preproteins of Sicily and ND42 interact with each other in the cytoplasm, before their mitochondrial import.
To provide additional evidence that Sicily is able to interact with ND42 outside mitochondria, we engineered a Sicily protein lacking its predicted NMTS (aa 1–11), which we will refer to as Sicily-MTS. Sicily-MTS is mostly localized to cytoplasm ( compare with c; and S3
) and exhibits a similar binding affinity to ND42 as full-length Sicily (). To test whether Sicily-MTS rescues the phenotypes caused by loss of Sicily, we expressed Sicily-MTS in sicily
mutants. Ubiquitous expression of Sicily-MTS, driven by da-GAL4
, rescues the lethality, CI defects (), and decreased aconitase activity () in sicily
mutants. Together, these data indicate that the cytoplasmic role of Sicily is essential for CI function and that there is a dramatic rescue when compared with . However, rescued animals display some subtle defects: a partial loss of “off” transient in their ERGs (), enlarged mitochondria at day 1 (, arrowhead and box), and some loss of PRs at day 10 ().
Figure 4. Cytoplasmic Sicily (Sicily-MTS) rescues defects caused by loss of Sicily.
sicily mutant males that lack the protein express Sicily-MTS under the control of the da-GAL4 driver, a ubiquitous driver. (a) These flies express wild-type levels of ND42 and NDUFS3 (more ...)
If Sicily regulates CI solely through ND42, overexpression of ND42 in sicily
mutants might rescue some of the defects. However, this is not the case, as overexpression of ND42 does not rescue the lethality or retinal degeneration in sicily
mutants, nor does it restore the abundance of NDUFS3 (unpublished data), suggesting that Sicily may affect multiple CI subunits in addition to ND42. Indeed, we also observe that Sicily interacts with NDUFS3 in co-IP assays and that the preprotein of Sicily again preferentially interacts (). Because NDUFS3 is a component that has been implicated in an early phase of CI assembly (Antonicka et al., 2003
), it is likely that most CI subunits are affected when Sicily is lost.
Sicily is localized to cytoplasm and mitochondria in vivo
To determine where Sicily is localized in vivo when expressed at endogenous levels, we generated five antibodies. However, none recognized endogenous Sicily. We therefore tagged our genomic rescue construct with different tags: HA, V5, or mCherry. Unfortunately, the HA-tagged construct did not rescue the lethality. In addition, though the V5-tagged construct rescues the lethality and phenotypes, we were unable to detect the V5 epitope with either immunoblot or immunofluorescent staining in vivo, potentially because the endogenous level of Sicily is very low. The mCherry-tagged genomic rescue construct rescued the lethality and ERG defects of all alleles (unpublished data), showing that it is functional. Using a DsRed antibody (Takara Bio Inc.), we found that Sicily-mCherry is expressed in the ventral nerve cord (), larval brain (not depicted), motor neuron axons (), imaginal discs (), and muscles (not depicted). Hence, the gene is expressed in many tissues.
Figure 5. Sicily is present in both cytoplasm and mitochondria. (a–a″) Third instar posterior ventral nerve cord of sicily mutants rescued with a genomic Sicily-mCherry construct costained with anti-DsRed (a) and anti-ATP synthase α subunit (more ...)
To determine whether Sicily is localized to cytoplasm and/or mitochondria in vivo, we costained Sicily-mCherry with two mitochondrial markers, ATP synthase (complex V [CV]) α subunit (; Baqri et al., 2009
) and mitochondria-EYFP (not depicted; LaJeunesse et al., 2004
). Sicily is only partially colocalized with these mitochondrial markers in all tissues observed, and a significant amount of protein is not colocalized but often juxtaposed to mitochondria. To confirm that Sicily is present in cytoplasm, we separated the mitochondrial and cytoplasmic fractions of sicily
mutant flies rescued by genomically encoded Sicily-mCherry and immunoblotted them with the DsRed antibody. However, because of the low abundance of Sicily, we were unable to detect the endogenous protein on Western blot after fractionation (unpublished data). We therefore used an RFP nanobody (ChromoTek) to immunoprecipitate the Sicily-mCherry expressed by the genomic rescue construct (Fig. S3, c and d) in both fractions. These camel nanobodies have been shown to be very efficient for IPs of tagged proteins (Fig. S3 c; Neumüller et al., 2012
). As shown in Fig. S3 d, Sicily-mCherry is found in both fractions, showing that Sicily is present in both cytoplasm and mitochondria.
Sicily chaperones ND42 outside mitochondria
As the preproteins of Sicily and ND42 interact with each other, Sicily may stabilize the ND42 preprotein in cytoplasm and/or target it to mitochondria. If the latter hypothesis is correct, ND42 preprotein should accumulate in cytoplasm in sicily mutants. However, this is not observed (). Moreover, when overexpressed in sicily mutants, most ND42 is cleaved and localized to mitochondria (), further supporting that Sicily is not required to target ND42 to mitochondria. Hence, we propose that the cytoplasmic role of Sicily is to prevent the degradation of, rather than to target, ND42 preproteins to mitochondria.
Figure 6. Sicily stabilizes ND42 in cytoplasm. (a) Immunoblots of whole body extract from sicily mutants expressing ND42-HA with a genomic construct. The overall level, including the level of ND42-HA preprotein, is down-regulated in sicily mutants, when compared (more ...)
To further test this hypothesis, we engineered an ND42 protein that lacks its NMTS (ND42-MTS) and coexpressed it with or without Sicily. We found that ND42-MTS is mostly localized to cytoplasm ( compared with c–c″) and that coexpression of Sicily does not alter this localization (; and ), providing compelling evidence that Sicily does not target ND42 to mitochondria. On the other hand, coexpressing Sicily or Sicily-MTS leads to an increase of ND42-MTS levels (). These data strongly suggest that Sicily stabilizes ND42 and that it does not require its mitochondrial localization to perform this function. To confirm that Sicily stabilizes ND42 posttranslationally, we in vitro translated ND42 (full length) with or without Sicily and observed that ND42 is up-regulated when co-translating Sicily or Sicily-MTS (). These data show that Sicily prevents the degradation of ND42 preproteins.
As ND42 is down-regulated in the absence of Sicily, we next explored how ND42 is degraded in cytoplasm. We treated cells expressing ND42-MTS with a proteasome inhibitor, MG132, or a lysosomal inhibitor, bafilomycin A1, and found that ubiquitinated ND42-MTS is up-regulated when the proteasomal, but not the lysosomal, function is blocked (). These data suggest that the cytoplasmic ND42 is degraded mainly through the ubiquitin–proteasomal pathway. In summary, our data indicate that Sicily interacts with and stabilizes the preproteins of ND42 in cytoplasm.
Akin to Sicily, loss of ND42 causes CI defects and neurodegeneration
ND42 is an integral membrane protein of eukaryotic evolutionary origin, as it is not found in prokaryotes. Its human homologue, NDUFA10
, has been found to be mutated in a case of Leigh syndrome with a CI deficiency (Hoefs et al., 2011
). To probe the role of ND42
in CI function and neurodegeneration, we knocked down ND42
using two UAS-RNAi
lines and showed that both RNAi lines lead to a severe knockdown of the protein (). The loss of ND42 also leads to the loss of NDUFS3 () and reduced CI activity (), indicating that ND42 is an essential subunit of CI. Furthermore, larvae expressing the ND42
RNAi also exhibited evidence of elevated ROS () and an up-regulation of Hsp60 (), similar to loss of sicily
. Finally, knockdown of ND42 in the visual system causes a severe retinal degeneration, as observed in sicily
mutants (; and S4
). In summary, loss of ND42, a CI subunit, and loss of Sicily lead to very similar biochemical and morphological defects.
Figure 7. Loss of ND42 phenocopies loss of Sicily. (a) Immunoblots of CI subunits, Porin, and Hsp60 in mitochondrial fractions purified from third instar larvae expressing ND42 RNAi. CI subunits are almost completely lost, and Hsp60 is up-regulated. The arrowhead (more ...)
Hsp90 interacts with Sicily to chaperone ND42
Because our data indicate that Sicily interacts with and stabilizes CI preproteins in the cytoplasm, we reasoned that it may act as a chaperone. As Sicily does not show homology to known chaperones, it may stabilize ND42 by recruiting other chaperones as a cofactor. To determine whether any cytosolic chaperones interact with Sicily, we performed in vivo co-IPs of Sicily-mCherry from whole flies using the RFP nanobody (Neumüller et al., 2012
) followed by mass spectrometry analysis to identify interactors. We found several chaperones that interact with Sicily, including Hsp90 (CG1242). Hsp90 is a specialized chaperone that only functions at a late stage of substrate folding (Young et al., 2001
). Specific cochaperones are required to stabilize its substrates, or these partially folded proteins are degraded (Johnson et al., 1998
; Young et al., 2001
). Hence, Sicily may be a cochaperone of Hsp90 to stabilize ND42. Indeed, Hsp90 binds to Sicily in an in vitro co-IP assay (), and Hsp90 coimmunoprecipitates ND42 only when Sicily is coexpressed (), indicating that Sicily, ND42, and Hsp90 are in a complex and that Sicily is required for Hsp90 to interact with ND42. To test whether ND42 facilitates the Hsp90–Sicily interaction, we compared co-IPs of Hsp90 and Sicily in the absence and presence of ND42 and found that the affinities are similar (), suggesting that ND42 does not affect the Hsp90–Sicily interaction. Together, these data indicate that Sicily is a primary binding partner of Hsp90, whereas ND42 is a secondary binding partner.
Figure 8. Hsp90 is required for Sicily to chaperone ND42. (a) Co-IP of Hsp90 and Sicily. (b) Co-IP of Hsp90 and ND42. Hsp90 interacts with ND42 only when Sicily is coexpressed. (c) Comparing co-IP of Hsp90 and Sicily in the presence or absence of ND42. ND42 does (more ...)
To confirm that the interaction between ND42 and Hsp90 occurs in cytoplasm through Sicily, we performed co-IPs and found that the coimmunoprecipitated ND42 is the isoform with the NMTS (), suggesting that cytoplasmic ND42 forms a complex with Hsp90 and Sicily. To test whether Hsp90 directly or indirectly interacts with ND42, we cross-linked the complex with different cross-linkers ( and not depicted). We observed that ND42 is present in cross-linked protein complexes (), whereas the level of native full-length ND42 is decreased (, third lane). In addition, less native full-length ND42 protein is immunoprecipitated when the proteins are cross-linked (, fifth and sixth lanes compared with fourth lane), suggesting that the ND42–Hsp90 interaction is not indirect. However, we were unable to detect ND42 in a cross-linked complex after IP (, sixth lane compared with third lane), suggesting that cross-linking eliminates their ability to be immunoprecipitated.
Hsp90 contains an ATPase domain that is required to stabilize its substrate. Inhibition of its ATPase activity by geldanamycin (GA), a drug that has been shown to be specific for Hsp90, leads to the proteasomal degradation of Hsp90 substrates and has been used to identify substrates for Hsp90 (Neckers, 2002
; Weihofen et al., 2008
; Okiyoneda et al., 2010
). To determine whether the ATPase activity of Hsp90 is required for ND42 stability, we treated S2 cells expressing Sicily-V5 and ND42-HA with GA. As shown in , cells treated with GA exhibit a significant loss of ND42-HA as well as endogenous NDUFS3 (Fig. S5
), whereas levels of unrelated proteins, Porin and a transfection control, GFP, are unaltered, indicating that Hsp90 activity is required for the stability of ND42 and NDUFS3. Interestingly, cells treated with GA also exhibit a loss of Sicily, suggesting that Sicily also requires Hsp90 for its stability, unlike canonical Hsp90 cochaperones or substrates.
To confirm that Hsp90 stabilizes Sicily and CI subunits in vivo, we analyzed protein levels of Sicily and CI in Hsp90 knockdown flies. As shown in , flies expressing Hsp90 RNAi #1 exhibit severely decreased levels of Sicily, ND42, and NDUFS3 compared with controls. In addition, these flies are third instar lethal at 25°C and have extended larval stages, similar to sicily mutants. These data indicate that Hsp90 is also required for the abundance of Sicily and ND42 in vivo. In addition, flies expressing a weaker Hsp90 RNAi line #2 are viable but exhibit a less severe decrease in Sicily, ND42, and NDUFS3 levels upon aging (unpublished data). However, loss of ND42 does not negatively affect Sicily levels (), indicating that ND42 is not required for the stability of Sicily. This is consistent with the notion that ND42 is a secondary binding partner of the Sicily–Hsp90 complex ().