Regulation of Mitochondrial Complex I Biogenesis in Drosophila Flight Muscles

Abstract

The flight muscles of Drosophila are highly enriched with mitochondria, but the mechanism by which mitochondrial complex I (CI) is assembled in this tissue has not been described. We report the mechanism of CI biogenesis in Drosophila flight muscles and show that it proceeds via the formation of ∼315, ∼550, and ∼815 kDa CI assembly intermediates. Additionally, we define specific roles for several CI subunits in the assembly process. In particular, we show that dNDUFS5 is required for converting an ∼700 kDa transient CI assembly intermediate into the ∼815 kDa assembly intermediate. Importantly, incorporation of dNDUFS5 into CI is necessary to stabilize or promote incorporation of dNDUFA10 into the complex. Our findings highlight the potential of studies of CI biogenesis in Drosophila to uncover the mechanism of CI assembly in vivo and establish Drosophila as a suitable model organism and resource for addressing questions relevant to CI biogenesis in humans.

Keywords: Drosophila; complex I biogenesis/assembly; mitochondria.

Figure 1. Drosophila Flight Muscles Are Suitable For Studying Complex I Assembly

(A) Schematic representation of how the 44 different subunits of bovine CI are arranged to produce the L-shaped topology; adapted from (Guarani et al., 2014). The asterisk denotes subunits for which an ortholog was not identified in Drosophila by DIOPT. (B) Summary of the experimental procedure for studying CI assembly in Drosophila. Transgenic RNAi constructs to the nuclear-encoded subunits were expressed specifically in thoracic muscles using the mhc-Gal4 driver. Mitochondria were isolated from thoraxes of 1 week-old flies, solubilized in 1% digitonin, and analyzed by blue native polyacrylamide gel electrophoresis (BN-PAGE). (C) The constituents of each of the six major bands observed during BN-PAGE was analyzed by mass spectrometry. 38 subunits of Drosophila CI were confirmed by mass spectrometry. The 38 subunits correspond to 37 different orthologs of human CI. Two paralogs of human NDUFV1 were confirmed by mass spectrometry (see Table S1). See Table S2 for all the peptides identified in the six major bands shown. (D) BN-PAGE (left panel) and Silver staining (right panel) of samples from thoraxes following RNAimediated knockdown of complex I (CI), complex III (CIII), complex IV (CIV) and complex V (CV) proteins to confirm the identities of the bands. SupCI and CV2 denote a supercomplex of CI and a dimer of CV respectively. The exact RNAi constructs expressed starting from left to right were to the white gene (wildtype, WT), dNDUFV1 (CI), dNDUFS1 (CI), dUQCRC-2 (CIII), dUQCRC-Q (CIII), dCox5A (CIV), cyclope (CIV), dATPsyn-β (CV), and ATPsyn-b (CV). (E) Immunoblotting with anti-NDUFS3 and anti-ATPsynβ antibodies of native gels to detect CI and CV respectively. Note that band A is a doublet consisting predominantly of a dimer of CV, and a supercomplex of CI. (F) BN-PAGE (top panel) and CI in-gel enzyme activity (lower panel) indicate that most of CI exists as the holoenzyme in Drosophila melanogaster (DM) skeletal muscles, in contrast to cardiac, soleus, EDL and tibia muscles from mice where a significant portion of CI exists as a supercomplex.

Figure 2. Disruption of Several CI Core And Supernumerary Subunits Impair CI Assembly In Drosophila

BN-PAGE (A), Silver staining (B), and CI in-gel enzyme activity (C) of mitochondria isolated from thoraxes following RNAi-mediated knockdown of the CI proteins indicated (mhc-Gal4>dNDUFX^RNAi ). The values listed below each lane indicate the residual amount of CI normalized to the amount in the wildtype (mhc-Gal4>w¹¹¹⁸) lane.

Figure 3. Proteomic Analyses And Immunoblotting Identify Assembly Intermediates Of CI

(A) Schematic of CI showing the three modules of the enzyme. The NADH Dehydrogenase module (N module) is located at the tip of the matrix arm, and is the site of NADH oxidation. Situated between the N module and the membrane arm, is the Q module, which is responsible for Ubiquinone reduction. The proton-conducting P module is in the membrane arm. (B) The current model of CI assembly in mammalian systems (reviewed in (Vartak et al., 2014). The assembly process begins with the formation of an assembly intermediate containing NDUFS2 and NDUFS3, which combines with NDUFS7 and NDUFS8. The subcomplex of NDUFS2, NDUFS3, NDUFS7 and NDUFS8 ultimately combines with ND1 to form the ~315 kDa assembly intermediate that is anchored to the membrane. The ~315 kDa subcomplex (also called the Q module) combines with an independently-formed ~370 kDa assembly intermediate to form an ~550 kDa assembly intermediate. This assembly intermediate which consists of the Q module and part of the P module grows by the addition of more subunits to form the ~815 kDa assembly intermediate, via mechanisms that are very poorly defined. The ~815 kDa assembly intermediate now consists of the complete Q and P modules. Finally, the N module is added to produce the 950kDa fully-assembled complex. Assembly factors or chaperones that assist in this process, but are not present in the fully assembled complex, have been omitted for clarity. (C) Western blot of samples obtained from thoraxes from pupae aged between 2 and 4 days after pupariation, and of flies from 0.5 hours to 48 hours post-eclosure to detect the assembly intermediates, fully assembled CI, and a supercomplex containing complex I (supCI) after BN-PAGE. The anti-NDUFS3 antibody strongly detects CI and supCI; and weakly detects the ~315 kDa, ~550 kDa and ~815 kDa assembly intermediates after a short exposure. However, after a longer exposure, the ~315 and ~550 kDa assembly intermediates can clearly be seen. In the right panel, the membrane was stripped and re-probed with anti-NDI. Anti-ND1 detects the ~315 kDa and ~550 kDa assembly intermediates, and a very faint band corresponding to CI. Note that in all 4 panels there is a general increase in accumulation of assembly intermediates, holoenzyme and CI-containing supercomplex with time. The CV antibody (anti- ATPsynß) was used as a loading control because commercially-available anti-CII antibodies we tested did not cross-react with Drosophila CII. (D) Proteomic analyses of assembly intermediates that form in the native gel sized between ~50 kDa and ~350 kDa. See Table S3 for all the peptides identified.

Figure 4. Specific Subunits Regulate the Biogenesis or Stability of Specific Assembly Intermediates Of CI

(A) The left panel depicts a schematic of the distribution of assembly intermediates on immunoblots as a result of RNAi-mediated disruption of various CI subunits. The right panel describes how various results can be interpreted. (B–D) Distribution of assembly intermediates in thoraxes dissected 24 hours after eclosion with transgenic RNAi expression of the CI subunits shown. The ~815 kDa assembly intermediate accumulates in thoraxes expressing transgenic RNAi to dNDUFS1, dNDUFV1, dNDUFA6 and dNDUFA12; the ~315 kDa assembly intermediate is decreased in thoraxes expressing transgenic RNAi of dNDUFS2, dNDUFS3, dNDUFS7 and dNDUFA5. In addition, another assembly intermediate accumulates in thoraxes expressing RNAi to dNDUFS5 and dNDUFC2 (denoted by * in B). In panels labeled long exposure, the region of the membrane just at or below CI was cut and imaged. (E and F) Distribution of assembly intermediates in thoraxes dissected 48 hours (E) and 72 hours (F) after eclosion with transgenic RNAi expression of the NDUFB subunits shown. RNAi-mediated knockdown of the expression of dNDUFB3 decreased the extent of accumulation of all the assembly intermediates; and the 550 kDa assembly intermediate accumulated when the expression of dNDUFB1, dNDUFB8 and dNDUFB11 were reduced. In addition, the extent of accumulation of the 315 kDa assembly intermediate was diminished following RNAi-mediated disruption of dNDUFB1, dNDUFB4, dNDUFB5, dNDUFB6 and dNDUFB10.

Figure 5. Identification Of An ~700 kDa Assembly Intermediate Of CI In Drosophila

(A) Top Panel: Immunoblots of samples obtained from wildtype and mhc>dNDUFS5^RNAi thoraxes of flies aged for 6 hours after eclosure depicting co-migration of the ~700 kDa intermediate and CV. In the left and middle panels, anti-NDUFS3 antibodies detect the fully assembled CI, the ~700 kDa subcomplex, as well as other assembly intermediates in dNDUFS5^RNAi thoraxes. Note that in the middle panel, the region of the membrane just below CI was cut and imaged. In the right panel, anti-ATPsynβ detects the CV monomer (700kDa) and dimer as shown. Lower Panel: Mitochondrial protein complexes from wildtype and mhc>dNDUFS5^RNAi thoraxes were resolved by BN-PAGE and the region corresponding to the ~700 kDa assembly intermediate (i.e. CV, demarcated) was cut out, subjected to tryptic digestion, and analyzed by label-free quantitative LC-MS/MS. (B) Immunoblots from samples obtained after 6 hours, 12 hours and 24 hours post eclosure from thoraxes where NDUFS1, NDUFS3, NDUFS5 and NDUFV1 were knocked down as a result of transgenic RNAi exression. Note that the ~815 kDa assembly intermediate accumulates as a result of disruption of NDUFS1 and NDUFV1, and the ~700 kDa assembly intermediate stalls and accumulates in NDUFS5 mutants at all time points. Importantly, upon prolonged exposure of the immunoblot, a band corresponding to the ~700 kDa assembly intermediate can also be observed in wild-type samples (denoted with the * in the lower panel), which confirms that it is an authentic, albeit transient assembly intermediate. (C) The accumulation of the ~815 kDa assembly intermediate was significantly attenuated in mhc>dNDUFS5^RNAi,dNDUFS1^RNAi thoraxes relative to mhc>dNDUFS1^RNAi thoraxes; instead there is an accumulation of the ~700 kDa assembly intermediate. Similar results were obtained when samples from mhc>dNDUFS5^RNAi,dNDUFV1^RNAi thoraxes were compared to samples from mhc>dNDUFV1^RNAi thoraxes. (D) Proteomic changes in the gel slice sample from wildtype and mhc>dNDUFS5^RNAi thoraxes corresponding to the ~700 kDa assembly intermediate. Relative protein abundance among biological samples is expressed by spectral counts on a log scale. Several CI subunits and CIAFs, most notably components of the MCIA complex are upregulated in the ~700 kDa assembly intermediate. However, the amount of dNDUFA10 (denoted with an asterisk) is reduced in mhc>dNDUFS5^RNAi thoraxes relative to wild type. See Table S4 for all the peptides identified

Figure 6. CI Assembly In Drosophila Involves An Assembly Intermediate Containing Several Membrane-Associated Accessory Subunits

(A) Mitochondrial protein complexes from wildtype, mhc>dNDUFS5^RNAi and mhc>dNDUFV1^RNAi thoraxes were separated by BN-PAGE and the region corresponding to the accumulated assembly intermediate (demarcated) was cut out, subjected to tryptic digestion, and analyzed by label-free quantitative LC-MS/ MS. (B) Proteomic changes in the gel slice samples from wildtype, mhc>dNDUFS5^RNAi and mhc>dNDUFV1^RNAi thoraxes. Relative protein abundance among biological samples is expressed by spectral counts on a log scale. The color scale bar indicates the range of protein expression levels. See additional information in Table S5. (C) Schematic representation highlighting the membrane subunits that are upregulated in the gel slice from the mhc>dNDUFS5^RNAi and mhc>dNDUFV1^RNAi thoraxes.

Figure 7

An assembly intermediate consisting of dNDUFS2, dNDUS3, dNDUFS7, dNDUFS8 and dNDUFA5 are combined in essentially one step to form the Q module, which is anchored to the membrane by ND1. Subsequently, an independently-formed subcomplex comprising of membrane-associated subunits (Partial P1) is conjugated to the Q module, and possibly other subunits, to form an assembly intermediate comprised of the Q module and part of the P module (Q + Partial P2). This grows by the addition of more subunits to form a transient assembly intermediate of ~700kDa (Q + Partial P3). We propose that dNDUFS5 is then incorporated at this step, to promote incorporation or stabilization of dNDUFA10. Subsequently, the transient ~700 kDa assembly intermediate is rapidly converted to the ~815 kDa assembly intermediate, consisting of the complete P and Q modules (Q + P). Finally, the N module is added to produce the CI holoenzyme.