Disease-causing mutations in subunits of OXPHOS complex I affect certain physical interactions

Mitochondrial complex I (CI) is the largest multi-subunit oxidative phosphorylation (OXPHOS) protein complex. Recent availability of a high-resolution human CI structure, and from two non-human mammals, enabled predicting the impact of mutations on interactions involving each of the 44 CI subunits. However, experimentally assessing the impact of the predicted interactions requires an easy and high-throughput method. Here, we created such a platform by cloning all 37 nuclear DNA (nDNA) and 7 mitochondrial DNA (mtDNA)-encoded human CI subunits into yeast expression vectors to serve as both ‘prey’ and ‘bait’ in the split murine dihydrofolate reductase (mDHFR) protein complementation assay (PCA). We first demonstrated the capacity of this approach and then used it to examine reported pathological OXPHOS CI mutations that occur at subunit interaction interfaces. Our results indicate that a pathological frame-shift mutation in the MT-ND2 gene, causing the replacement of 126 C-terminal residues by a stretch of only 30 amino acids, resulted in loss of specificity in ND2-based interactions involving these residues. Hence, the split mDHFR PCA is a powerful assay for assessing the impact of disease-causing mutations on pairwise protein-protein interactions in the context of a large protein complex, thus offering a possible mechanistic explanation for the underlying pathogenicity.


Results
The split mDHFR protein complementation assay offers a robust platform for following protein-protein interactions in human complex I. To assess pairwise interactions between CI subunits, we fused the coding sequences (CDSs) of all nuclear DNA (nDNA)-and recoded mitochondrial DNA (mtDNA)-encoded human complex I subunits (N = 44) to the 5′ end of that part of mDHFR gene encoding a C-terminal fragment (termed the CI-F3 constructs). In addition, 37 CI subunits were fused to the 5′ end of that part of mDHFR gene encoding a N-terminal fragment (termed the CI-F1,2 constructs) (Fig. 1). Notably, unlike the traditional yeast-two-hybrid system which utilizes a split transcription factor, the split mDHFR system was chosen as it is better adapted to screen for interactions between membrane-bound proteins 26 . Secondly, since the baker's yeast lost the multi-subunit CI during evolution our assay is less prone to false negative results that could stem from interactions between our human proteins with endogenous yeast proteins. In our hands, 8 subunits were not Figure 1. Workflow of the split-mDHFR-based screen for testing pairwise interactions between complex I subunits. Upper panel shows the construction of the haploid yeast split mDHFR libraries which harbor complex I subunit sequences, followed by mating. This allows to identify candidate interacting proteins, which lead to increase in yeast colony size after seeding on MTX+ plates. Then, the screen results were compared to published structural data to identify testable interactions. Notably, mammalian CI structure shown is based on (PDB ID: 5XTD) 15  www.nature.com/scientificreports www.nature.com/scientificreports/ successfully cloned into the latter constructs (i.e, ND6, NDUFA9, NDUFA12, NDUFB9, NDUFS1, NDUFS4). While plasmids containing the CI-F3 constructs were inserted into MATa yeast, those plasmids containing the CI-F1,2 constructs were transformed into MATα yeast strains (Fig. 1, Supplementary Table S1) to enable rapid mating and the creation of diploids. The coding sequences of the mtDNA-encoded subunits (i.e., ND1-ND6 and ND4L) were edited to comply with yeast cytosolic codon usage. The construct harboring the mDHFR F1,2 fragment also harbored a YFP reporter gene inserted between the CI subunit sequence and the F1,2 fragment; this enabled tracing intracellular localization of the resultant fusion protein in yeast. Approximately 23 out of the 37 human CI subunits cloned in this plasmid showed mitochondrial associated signals (Supplementary Material, Fig. S1). Five additional subunits were localized to membrane-embedded organelles such as the endoplasmic reticulum or the plasma membrane (Supplementary Material, Fig. S1). These results suggest that although the experiment was performed in the baker's yeast which totally lacks an endogenous CI, most human complex I subunits retained their mitochondrial localization, or membrane association, thus supporting the potential physiological relevance of our experiments. Notably, to avoid false positive results we focused our analysis only on interactions that (A) share the same/adjacent subcellular compartment, and (B) are consistent with the published mammalian complex I structures. To further reduce false-positive interactions, we excluded two highly over-expressed subunits (i.e., NDUFA2 and NDUFA5) from further analyses. To assess interactions between the various subunits, the haploid yeast strains were mated in a high-throughput manner (see Materials and Methods), thus creating 1,628 independent CI-F1,2/CI-F3 diploid strains. The resultant colony sizes of these diploid strains were measured as a proxy for growth rate in the presence of methotrexate (MTX), thus selecting for yeast growth only when complementation of mDHFR activity had occurred (Fig. 1). This experiment revealed 94 significantly positive (feasible) pairwise interactions (Fig. 2). In seeking candidate subunit interactions able to report on the impact of pathological mutations, we focused on 19 interactions that were consistent with the CI structural models from bovine and ovine heart 13,14 (Fig. 1). Such interactions were also consistent with recently published human CI structures 15 .
Two disease-associated point mutations, affecting residues at the interaction interface of ND2 and ND5, had no effect on the pairwise subunit interaction. Next, we employed our assay to assess the impact of disease-associated human gene variants on physical interactions between the encoded proteins. First, we screened the literature for CI disease-associated mutations that affect residues found at physical interaction interfaces of two CI subunits, according to the available structures (Table 1, Supplementary File S1) 27,28 . Two missense mutations at nucleotide positions 4917G and 4640A are associated with Leber's hereditary optic neuropathy (LHON) in separate families 27,29 . Both of the resulting amino acid substitution (i.e. N150D and I57M, respectively) occur in ND2 trans-membrane alpha-helixes (TMHs), which in the structure are adjacent to a ND5 subunit TMH, and hence potentially alter the interaction interface (Fig. 3A). Furthermore, the 4460A mutation was found in a Russian family from Novosibirsk who exhibited poor oxygen utilization in cybrids 29 . The 4917G nucleotide substitution, an ancient mutation that defines the mtDNA genetic background haplogroup T, was associated with LHON only in the presence of the R2-JT haplogroup cluster-defining 4216C mutation 27 . We introduced these two SNPs either separately or together into the ND2 re-coded sequence within the F3 mDHFR fragment-containing construct. We then mated the MATa yeast strains harboring these mutant constructs with a MATα strain harboring the ND5-F1,2 construct and conducted a dilution drop-test to assess the impact of these mutations on the ND2-ND5 interaction. Neither of the two single mutants showed any significant change in terms of yeast growth on selective media (containing MTX), suggesting that no significant impact on the ND2-ND5 interaction had occurred (Fig. 3B). Notably, this result does not reflect reduced resolution of our protein complementation assay, as it was previously successfully used to assess the impact of point mutations on protein-protein interactions 30 . In addition, the double mutant construct was not expressed to a detectable level in yeast, thus precluding any analysis of this combination of mutations ( A two base-pair deletion causing an MT-ND2 frame shift mutation leads to the emergence of interaction promiscuity. We next examined the impact of a double adenine deletion within an adenine triplet at mtDNA nucleotide positions 5132 to 5134, causing a frame shift in the MT-ND2 sequence. This deletion was reported in a 28-year-old patient with mitochondrial myopathy and severe exercise intolerance 31 . Such frameshift led to replacement of the 126 C-terminal residues harboring three TMHs in ND2 by 30 highly hydrophilic residues (Fig. 4A). We cloned the ND2 deletion mutant into the ND2-F3 construct and introduced the resulting plasmid into the MATa yeast strain, which was in turn mated with MATα strains containing one of four ND2 interactors (i.e., NDUFC2, ND4, ND4L and ND5) (Fig. 4B) fused to the F1,2 mDHFR fragments. Here too, we performed dilution drop-test experiments for all four mating-pairs and compared the relative growth rates of the diploid strains containing the ND2 mutants with that of diploid strains harboring wild type ND2 (Fig. 5A). Our results indicated unexpected increased growth rates of the ND2 mutant strains when mated with NDUFC2-and ND4-expressing strains, while no significant change in growth rate was evident when the ND2 mutant strain was mated with ND4L or ND5 (Fig. 5B). However, while inspecting the control diploid strains, harboring mutated ND2 and an empty (i.e., not encoding any CI subunits) F1,2 construct, we noticed much higher growth as compared to the diploid strain harboring wild type ND2 and the empty F1,2 vector (Fig. 5B). This suggests that the ND2 mutant had become a promiscuous interacting protein.
However, promiscuity did not explain the increase in interaction strength in all cases; ND4L exhibited a stronger interaction with the mutant ND2, as compared to the empty vector control (Fig. 5B). A close inspection of the CI structure revealed ND4L to be the only subunit tested that interacts with the N-terminal portion of ND2 (Fig. 4B). This suggests that the specific interaction of ND2 and ND4L, which is dictated by their interaction interface, is not affected by the deletion mutation in ND2.

Discussion
The availability of structures of multi-subunit protein complexes enables predicting the potential impact of mutations on subunit interactions. Such predictions can serve to design experiments that test the impact of such mutations. Nevertheless, such experiments cannot easily employ classical structural biology methodologies, which  Asp110Val (77) Arg118Gln (85) Arg138Gln (105) Ala224Val (191) Arg323Gln (290) NDUFS3 Arg199Trp (163) Pro223Leu (187) NDUFS8 Arg54Trp (20) Glu63Gln (29) Arg77Trp (43) Pro79Leu (45) Pro85Leu (51) Arg94Cys (60) Arg138His (104) Ala159Asp ( www.nature.com/scientificreports www.nature.com/scientificreports/ are not compatible with high-throughput approaches and are less accessible to most molecular biologists. Hence, we established an assay to enable such assessment to be carried out easily. Specifically, we used the split mDHFR protein complementation assay (PCA) to assess the impact of mutations at interaction interfaces between CI subunits on protein-protein interactions. First, we cloned all known human CI subunits (N = 44) into constructs   www.nature.com/scientificreports www.nature.com/scientificreports/ harboring either of both parts of split mDHFR, thus establishing a resource which enables testing for all pairwise interactions. Such a resource offers the obvious advantage of accessibility and relative ease-of-use, relying on an assay based on yeast mating and measurement of growth rate on selective medium (containing methotrexate). The main disadvantage, especially in the case of a multi-subunit protein, is that the PCA is limited to assessment of pairwise interactions. Additionally, in OXPHOS CI, several subunits are simultaneously added during each stage of the assembly process 32,33 . This suggests that many interactions between subunit pairs likely depend on the presence of additional subunits; naturally, an assay designed to test pairwise interactions is blind to such interactions, which will generate false negative results. Moreover, some of the observed interactions may reflect transient interactions that occur during the assembly process of complex I, yet cannot be currently verified due to relative data sparsity. Additionally, we cannot exclude that false negative rate of subunit interactions was also influenced by protein misfolding. These may explain, at least in part, why only 19 interactions in our assay were consistent with the 3D structures. Once human CI will be reconstituted in yeast at least some of these caveats could be addressed, while also taking into account assembly factors. Regardless, our observed interactions www.nature.com/scientificreports www.nature.com/scientificreports/ contained core subunits, which play a major role in the activity of CI and reflect the usefulness of our tool for assessing the impact of mutations on such interactions.
As proof-of-concept, we focused our experimental assessment on three changes, considering two disease-associated SNPs and one frameshift mutation, all occurring in the mtDNA-encoded ND2 subunit. The first SNP, 4640A, was originally found in a Russian family from Novosibirsk. The 4640A mutation resulted in poor oxygen utilization by cybrids, although these experiments did not reveal any reduction in the specific activity of CI, relative to control lymphoblast cells 29 . Close inspection of this mutation showed that it altered an amino acid position with poor evolutionary conservation. Additionally, the 4640A mutation appears to associate with the U3b sub-haplogroup in the human mtDNA phylogenetic tree. Similarly, the 4917G mutation associates with both the T and N1b haplogroups. Haplogroup T associates with LHON 34 , and with reduced sperm motility in the Spanish population 35 and haplogroup N1b altered the susceptibility of Ashkenazi Jewish type 2 diabetes patients to develop complications 36 . Notably, haplogroup T cybrids possess a higher capability to cope with oxidative stress in the form of an H 2 O 2 challenge 37 . Taken together, the impact of SNPs that co-occur with certain mtDNA genetic backgrounds cannot be easily distinguished from the linked set of mutations. One should thus take into consideration the fact that our observed lack of impact of both the 4640 and 4917 mutations on CI subunit interactions may either attest to their minor functional effect, suggesting that their impact is not manifested at the level of protein-protein interactions, or that this reflects the resolution limitations of the PCA. These possibilities cannot be resolved at this point and requires additional experiments.
Unlike the tested point mutations, a pathological MT-ND2 frameshift mutation had a dramatic impact on the pattern of subunit interactions. Our results suggest that the MT-ND2 frameshift mutant generated a promiscuous interacting protein, specifically in interactions involving the region encoded downstream to the frameshift. Such a molecular phenotype might explain the appearance of the disease at the relatively high (90%) heteroplasmy level seen in the patient affected by the deletion mutation 25 . It is also possible that at high heteroplasmy levels, the translation product of the mutated MT-ND2 gene interacts with many members of the mitochondrial proteome, thus interfering with the proper ND2 interaction pattern, thereby causing the mitochondrial dysfunction observed in the clinic. Additionally, our results suggest that interaction of the N-terminal region of ND2 with the ND4L subunit was not affected, suggesting that the promiscuous interaction phenotype is limited to interactions involving the C-terminal region of ND2, thus excluding the possibility of a general mis-folding effect of ND2. These results provide, for the first time, a possible molecular mechanistic explanation for the pathophysiology of the ND2 frameshift mutation.
As CI structure and assembly pathway are very complex, studies of the phenotypic impact of human disease-causing mutations and disease-associated polymorphisms have largely relied on previous measurements of CI enzymatic activity 2,29,38 or on earlier work on the assembly of the holoenzyme. Still, little is known to date of the effects of human mutations on specific pairwise interactions between CI subunits. We speculate that protein-protein interactions between CI subunits that occur independently of additional subunits are important for the structure and proper assembly of CI.
A recent RNA-seq analysis from multiple human tissues revealed that certain CI subunits show tissue-dependent expression patterns 39 , thus questioning the generality of structural analysis of the complex from specific tissues, such as the heart or liver. With this in mind, our analysis of CI subunit interactions is not linked to a particular tissue, and hence is less influenced by tissue-dependent subunit composition considerations.
In summary, we provide a resource of clones for all known human CI subunits ready to be tested for pairwise protein-protein interactions using the split mDHFR PCA. Our experimental assessment of the impact of mutations on pairwise CI subunit interactions serve as a proof-of-concept and may encourage using this approach to study the impact of specific mutations on other multi-subunit protein complexes, such as the ribosome. Our finding of a profound impact of a MT-ND2 frameshift mutation on interactions with partners revealed in the crystal structure of the complex provides the first direct explanation of the molecular mechanism underlying the phenotypic impact of this mutation. This discovery increases the motivation to recruit the system established in the current study to screen for the impact of other mutations of interest on confirmed subunit interactions.

Materials and Methods
Yeast growth media. The complete recipes for yeast media used in the current study are detailed in Supplementary Material, Table S2.
Preparation of split mDHFR libraries. Two 2-micron plasmids were used for human CI subunit library preparation. Plasmid p340 includes amino acid sequence-coding motifs at the cloning site for a linker peptide (GlyGlyGlyGlySer)X2 followed by the ORF of the yellow fluorescent protein (YFP)-coding gene, followed by another (GlyGlyGlyGlySer)X2 peptide linker and DNA encoding mDHFR N' terminal fragments (F1,2). Expression of this fusion construct was under the transcriptional control of the GAP promoter and CYC1 terminator. Plasmid pMS141 includes amino acid sequence-coding motifs at the cloning site for a linker peptide (GlyGlyGlyGlySer)X2 followed by DNA encoding a mDHFR C' terminal fragment (F3), and is expressed under the transcriptional control of the TEF promoter and ADH1 terminator. In both plasmids, a SpeI restriction site was fused to the 5′ codon of the constructs (Supplementary Material, Fig. S3).
ORFs of nDNA-encoded human CI subunits were amplified from commercial human cDNA samples (See Supplementary Material, Table S3 for the list of sources of human RNA). ORFs of the mtDNA-encoded subunits were recoded to match the cytoplasmic translation code (Supplementary Material, Table S4), generated commercially according to our design and cloned into pUC57 plasmids (OriGene). To create the two libraries, namely a F3 library in strain BY4741 [MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0] and a YFP-F1,2 library in strain BY4742 [MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0], the ORF encoding each subunit was amplified using primers harboring 5′ tails of ~50 bp homology to either plasmid p340 or pMS141 (primers are listed in Supplementary Material, (2019) 9:9987 | https://doi.org/10.1038/s41598-019-46446-8 www.nature.com/scientificreports www.nature.com/scientificreports/ Table S5). PCR amplification of the inserts was performed using Phusion DNA polymerase (Thermo-Scientific), with the following amplification protocol: Two min at 98 °C followed by 35 cycles of 30 sec at 98 °C, 30 sec at the melting temperature of the primers used and 72 °C for an elongation time calculated as template length in kilo base pairs multiplied by 30 sec. To perform homologous recombination-based cloning in yeast, 15 µL of the PCR reaction were used for co-transformation with the relevant plasmid into yeast haploid strains (plasmids p340 and pMS141 were used for transformation into the BY4742 and BY4741 strains, respectively). To improve the efficiency of the cloning process in yeast, the plasmids were linearized by digestion with SpeI, and 200 ng of the digested plasmids were used for co-transformation with the PCR fragments. The yeast transformations were performed as previously described 40 . Following co-transformation of the inserts and the linearized plasmids, the yeast were grown on selective plates, namely SD-His agar plates for plasmid p340-transformed cells and YPD agar plates with cloNAT for plasmid pMS141-transformed cells, for a minimum of 48 h at 30 °C. Colonies (N = 8 to 10) were picked and streaked onto a similar second selective plate and grown overnight at 30 °C.
To validate successful cloning, a crude extract containing plasmid DNA was prepared by boiling small patches of the streaked colonies in 50 µL of 20 mM NaOH at 100 °C for 25 minutes and centrifugation for 2 min at 2,500 × g. The supernatant was used as template in a 10 µL PCR amplification using primers from the plasmid sequence flanking the insert (see Supplementary Material, Table S6). PCR products of appropriate size were Sanger-sequenced at the Ben-Gurion University sequencing unit. The sequences obtained were compared with the RefSeq sequences of the subunit-encoding ORFs (see Supplementary Material, Table S6 for CI subunits RefSeq protein accession numbers).
Split mDHFR screen. All 44 human CI subunits were cloned into plasmid pMS141 (mDHFR fragment F3) and 37 of the subunits were also cloned into plasmid p340 (mDHFR fragments F1,2). To test for all possible pairwise interactions between CI subunits, seven haploid strain selection plates were prepared, harboring 1,536 colonies each. Three SD-His agar plates were prepared for plasmid p340-transformed BY4742 cells and four YPD agar plates were prepared with cloNAT for plasmid pMS141-transformed BY4741 cells. The haploid strain libraries were seeded onto haploid selection plates and grown at 30 °C for 48 h, replicated on a second identical haploid selection plate and grown for an additional 24 h to reduce contamination. The libraries were then mated so that each of the plasmid p340-transformed BY4742 clones (three 1,536 clone plates) could be crossed with each of the four plasmid pMS141-transformed BY4741 plates, and vice-versa, yielding a total of 12 mating plates. Mating was performed while growing the yeast on non-selective YPD agar plates at 30 °C overnight to allow for the differently selected haploid strains to mate. These plates were subsequently replicated onto diploid selection SD-His agar plates with cloNAT. The resulting yeast colonies were grown overnight at 30 °C and then, to reduce haploid contamination, were replicated onto a second diploid selection plate and once again grown overnight at 30 °C. Each of the 12 diploid selection plates from the second round of diploid replication were replicated onto SD agar plates containing MTX and placed in a 30 °C incubator for 5 days (which in our hands gave clearer results than previously suggested 4 days). All yeast plate replicates and mating experiments were performed using a RoToR bench top colony arrayer (Singer Instruments, UK). The split mDHFR PCA results are semi quantitative, as protein-protein interactions are supported by significantly larger colony size from our negative control, similar to our positive control.
Plate distribution of the haploid strains was designed so that crosses of the BY4741 and BY4742 strains would result in a minimum of 9 replicates of each of tested subunit A-F1,2 x subunit B-F3 interactions, with an additional 15 colonies representing the control population for each tested interaction. Here, 9 subunit A-F1,2 x F3 colonies did not contain an insert and 6 colonies were F1,2 without insert x subunit B-F3 crosses. Each haploid plate harbored control colonies in which the construct did not include an insert.

Validation of F1,2 fragment expression and human CI subunits localization in yeast. BY4742
yeast clones with human CI subunits that were cloned into the p340 construct were grown over night in 30 °C SD-His medium in 15 ml falcons with shaking, back diluted (OD 600 = 0.1) into 96 well plate and grown for 4 hours in 30 °C SD-His medium with shaking. Notably, the yeast were grown in 30 °C rather than in 37 °C to ovoid increased membrane propensity to be leaky, which likely influence growth and mask the influence of protein complementation on such growth. Then, yeasts were transferred into a 384 well plate for imaging in a Zeiss 710 Confocal Microscope. YFP expression was validated and the pattern of YFP distribution within the yeast cells were compared with the distribution patterns observed at Breker et al. 41 to determine protein localization within the yeast.
Statistical analysis of the yeast colonies. Colony area size was measured using Balony 42 . Firstly, colony sizes were normalized to the median size of all colonies within their row and column on each plate. Then, a one-tailed Mann Whitney U (MWU) test was performed with the alternative hypothesis of the form P(case > control) > P(control > case). An interaction was considered positive only if the 'case' group was significantly larger than both the 'control' groups described above (i.e., colonies resulting from mating of subunit A-F1,2 × F3 without insert and from F1,2 without insert × subunit B-F3). When colony area size was equal to zero, the colony was excluded from further statistical analysis to avoid technical bias.
Drop test for evaluating the effect of the ND2 frameshift mutation. Haploid strains were seeded onto haploid selection plates (SD-His agar plates for strain BY4742 containing the F1,2 constructs with DNA encoding ND4, ND4L, ND5 or NDUFC2 or insert-less and YPD agar plates with cloNAT for strain BY4741 containing the F3 constructs with DNA encoding ND2-WT, ND2-DEL (frameshift mutant), ND2-I57M, ND2-N150D or ND2-I57M + N150D or insert-less), grown at 30 °C for 48 h, replicated onto a second identical haploid selection plate and grown for an additional 24 h to reduce contamination. Mating was performed while (2019) 9:9987 | https://doi.org/10.1038/s41598-019-46446-8 www.nature.com/scientificreports www.nature.com/scientificreports/ growing the yeast on non-selective YPD agar plates at 30 °C overnight. These plates were subsequently replicated on diploid selection SD-His agar plates with cloNAT. The resulting yeast colonies were grown overnight at 30 °C and then, to reduce haploid contamination, were replicated onto a second diploid selection plate and once again grown overnight at 30 °C. After validating expression of the ND2 variants, the diploid strains were grown in SD liquid medium containing MTX (to reduce nucleotide pools) and SD liquid medium containing DMSO (control) for 24 h at 30 °C with shaking. These yeast strains were diluted to OD 600 = 0.1 in fresh SD liquid medium containing MTX or DMSO, respectively, and each of the diluted strains was further diluted 7 times at a 1:5 ratio. Five μL of each diluted diploidic strain were placed on a SD agar plate containing MTX or DMSO and placed in a 30 °C incubator for 5 days. To avoid reaching a growth asymptote, images were taken at days 2-5. Growth was measured as colony color intensity (which equals biomass density)-the intensity of the white color given by the yeast colony, over background. The overall highest differences between colonies at the same dilution resulting from the different matings were spotted on day 3 with the smallest intra-mating variations at the seeding density of OD 600 = 0.1. As the only measurable background was upon mating the insert-less-F1,2 control with either ND2-WT-or ND2-DEL-expressing cells, we tested for significant growth over background by comparing the growth of the interactor-F1,2 X ND2-WT/ND2-DEL cross with the appropriate control mating of the insert-less-F1,2 X ND2-WT/ND2-DEL cross, respectively. Such pairwise comparisons were made using a two-tailed, unpaired Student's t-test with equal variance.
Validation of F3 fragment expression by western blot. Diploid strain colonies representing all matings performed for the drop-test were picked from the second diploid selection plates (see above) and grown overnight in SD-His liquid medium supplemented with cloNAT at 30 °C with shaking. The strains were diluted to OD 600 = 0.1 and grown overnight in SD-His liquid medium supplemented with cloNAT until mid-log phase (OD 600 = 0.6-0.8). Approximately 2.5 OD 600 units (3.125-4.167 ml) of the cells were centrifuged at 1000 g for 5 min, resuspended in 100 mM NaOH and incubated for 5 min at room temperature. The resulting cell extracts were centrifuged at 16,000 × g for 3 min and the protein-containing pellet was resuspended in SDS-containing loading buffer and incubated at 70 °C for 10 min. Thirty µg aliquots were loaded onto 4-20% polyacrylamide gels and the separated proteins were transferred to a polyvinylidene difluoride membrane at ~330 mA for 1 h. The membrane was then subjected to a standard blotting protocol with primary rabbit anti-mDHFR(F3) antibodies (Sigma-Aldrich D0942) diluted 1:1000 and horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies. Antibody binding was visualized with an enhanced chemiluminescent solution (WesternBright Sirius, advansta, BGU) for 5 min and image analysis (LAS-3000 luminescent image analyzer, Fujifilm). Such analysis revealed an additional ~10 kDa band in the ND2-WT strains that was absent from the ND2-DEL strains, probably reflecting some level of unfused F3 expression product (Supplementary Material, Fig. S4).