Protein N-myristoylation plays a critical role in the mitochondrial localization of human mitochondrial complex I accessory subunit NDUFB7

The present study examined human N-myristoylated proteins that specifically localize to mitochondria among the 1,705 human genes listed in MitoProteome, a mitochondrial protein database. We herein employed a strategy utilizing cellular metabolic labeling with a bioorthogonal myristic acid analog in transfected COS-1 cells established in our previous studies. Four proteins, DMAC1, HCCS, NDUFB7, and PLGRKT, were identified as N-myristoylated proteins that specifically localize to mitochondria. Among these proteins, DMAC1 and NDUFB7 play critical roles in the assembly of complex I of the mitochondrial respiratory chain. DMAC1 functions as an assembly factor, and NDUFB7 is an accessory subunit of complex I. An analysis of the intracellular localization of non-myristoylatable G2A mutants revealed that protein N-myristoylation occurring on NDUFB7 was important for the mitochondrial localization of this protein. Furthermore, an analysis of the role of the CHCH domain in NDUFB7 using Cys to Ser mutants revealed that it was essential for the mitochondrial localization of NDUFB7. Therefore, the present results showed that NDUFB7, a vital component of human mitochondrial complex I, was N-myristoylated, and protein N-myrisotylation and the CHCH domain were both indispensable for the specific targeting and localization of NDUFB7 to mitochondria.

Using this strategy, we identified two mitochondrial membrane proteins, TOMM40 and SAMM50, as N-myristoylated membrane proteins 17,18 .Both of these proteins are β-barrel proteins that localize to the outer membrane of mitochondria.TOMM40 is a major component of the translocase of the mitochondrial outer membrane (TOM complex) that acts as a general import pore for most mitochondrial precursor proteins 19,20 .SAMM50 is a major component of the SAM complex required for the sorting and assembly of β-barrel proteins 21 .An analysis of the intracellular localization of non-myristoylatable G2A mutants revealed that protein N-myristoylation occurring on SAMM50, but not on TOMM40, plays a critical role in the mitochondrial targeting of this protein 18 .However, the function of protein N-myristoylation occurring on TOMM40 remains unclear.Based on these findings, protein N-myristoylation may mediate diverse roles in the intracellular localization and function of mitochondrial proteins.In the present study, we searched for human N-myristoylated proteins that specifically localize to mitochondria among the 1,705 human genes listed in MitoProteome, a mitochondrial protein database 22 .The results obtained revealed that four proteins, DMAC1, HCCS, NDUFB7, and PLGRKT, were N-myristoylated proteins that specifically localized to mitochondria.Among these proteins, DMAC1 and NDUFB7 were previously found to play critical roles in the assembly of complex I of the mitochondrial respiratory chain as an assembly factor and an accessory subunit, respectively 23 .An analysis of the intracellular localization of non-myristoylatable G2A mutants revealed that protein N-myristoylation occurring on NDUFB7 played a critical role in the mitochondrial localization of this protein.Furthermore, an analysis of the role of the CHCH domain (CHCHD) found in NDUFB7 using Cys to Ser mutants revealed that CHCHD was vital for the mitochondrial localization of NDUFB7.Therefore, the present results showed that NDUFB7, an essential component of human mitochondrial complex I, was N-myristoylated, and protein N-myristoylation and CHCHD were both indispensable for the specific targeting and localization of NDUFB7 to mitochondria.

Identification of human N-myristoylated mitochondrial proteins from human genes listed in the MitoProteome protein database
We searched for human N-myristoylated mitochondrial proteins among the human genes listed in MitoProteome, a mitochondrial protein database, using a strategy established in our previous studies [14][15][16][17][18] .In this strategy, 131 genes with N-terminal Met-Gly motifs were extracted from the 1,705 human genes listed in the MitoProteome protein database 22 .After applying the N-terminal sequences of the proteins encoded by these genes to two protein N-myristoylation prediction programs, the MYR Predictor and Myristoylator 24,25 , 34 positively predicted genes were identified (Supplementary Table S1).Among these positively predicted genes, 12 candidate genes (BCAP31, CLN3, DMAC1/TMEM261, FLAD1, HCCS, LRRC10, MARC1/MTARC1, ME3, NDUFB7, NOL3, PLGRKT and TUSC3) for N-myristoylated mitochondrial proteins were selected and their susceptibility to protein N-myristoylation was evaluated using fusion proteins in which the 10 N-terminal amino acid residues were fused to a FLAG-tagged model protein, tGelsolin, as previously described 14 (Fig. 1A,B).The information about the 13 genes analyzed in this study was summarized in Supplementary Table S2.
In this experiment, N-myristoylatable tGelsolin-FLAG, its non-N-myristoylatable mutant (tGelsolin-G2A-FLAG), and the tGelsolin fusion protein of MIC19 (CHCHD3), a known N-myristoylated mitochondrial protein, were used as control proteins.To establish whether these fusion proteins are N-myristoylated, cellular metabolic labeling experiments on COS-1 cells using Alk-Myr, a bioorthogonal myristic acid analog, were performed [16][17][18] .In these experiments, after labeling cells transfected with cDNA coding C-terminally FLAG-tagged tGelsolinfusion proteins with Alk-Myr, the N-myristoylated protein was detected with click chemistry.As shown in the upper panels of Fig. 1C, efficient protein expression in COS-1 cells was equally observed for all the fusion proteins tested, including 3 control proteins, according to a Western blotting analysis.Metabolic labeling experiments revealed the efficient incorporation of Alk-Myr into the fusion proteins of 10 candidate genes (BCAP31, CLN3, DMAC1/TMEM261, HCCS, LRRC10, MARC1/MTARC1, ME3, NDUFB7, NOL3, and PLGRKT), similar to two N-myristoylatable control proteins (tGelsolin-FLAG and MIC19(N10)-tGelsolin-FLAG); however, this incorporation was not observed in the 2 fusion proteins of 2 candidate genes (FLAD1 and TUSC3), as was the case for the non-N-myristoylatable tGelsolin-G2A mutant (Fig. 1C, lower panels).
To clarify whether the full-length protein was N-myristoylated, we selected 7 genes (CLN3, DMAC1/ TMEM261, HCCS, MARC1/MTARC1, NDUFB7, NOL3, and PLGRKT) and their susceptibility to protein N-myristoylation was evaluated using the same strategy with the full-length cDNA coding for the C-terminally FLAG-tagged protein.The results obtained are shown in Fig. 2A.
In the left panel, all proteins, including three control proteins (tGelsolin-FLAG, tGelsolin-G2A-FLAG, and MIC19-FLAG), were expressed based on a Western blotting analysis using an anti-FLAG antibody.Metabolic labeling experiments revealed the incorporation of Alk-Myr in all 7 proteins encoded by the 7 tested genes, similar to the two N-myristoylatable control proteins (tGelsolin-FLAG and MIC19-FLAG), as shown in Fig. 2A right panel, indicating that the 7 proteins encoded by the 7 tested genes (CLN3, DMAC1/TMEM261, HCCS, MARC1/MTARC1, NDUFB7, NOL3, and PLGRKT) were N-myristoylated proteins.The characteristics of these proteins are summarized in Table 1.

DMAC1, HCCS, NDUFB7, and PLGRKT are N-myristoylated mitochondrial proteins
To assess the intracellular localization of the 7 proteins, an immunofluorescence analysis of COS-1 cells transfected with cDNA coding for the C-terminally FLAG-tagged protein was performed.In this analysis, MitoTracker Red and Hoechst 33342 were used as organelle markers for mitochondria and the nucleus, respectively.As shown in Fig. 2B, left panel and Supplementary Fig. S3, specific localization to mitochondria was observed with DMAC1, HCCS, NDUFB7, and PLGRKT, similar to MIC19, a known N-myristoylated mitochondrial protein.In contrast, selective localization to mitochondria was not observed with CLN3, MARC1, or NOL3.A line profile analysis Twelve candidate genes (BCAP31, CLN3, DMAC1/TMEM261, FLAD1, HCCS, LRRC10, MARC1/MTARC1, ME3, NDUFB7, NOL3, PLGRKT and TUSC3) were selected as N-myristoylated mitochondrial proteins from the human genes listed in MitoProteome, a mitochondrial protein database, and their susceptibility to protein N-myristoylation was evaluated by in vivo metabolic labeling in transfected COS-1 cells using fusion proteins in which the 10 N-terminal amino acid residues were fused to the FLAG-tagged model protein, tGelsolin.(A) Strategy to identify N-myristoylated mitochondrial proteins from human genes listed in the MitoProteome protein database.(B) Schematic representation of the generation of X(N10)-tGelsolin-FLAG with 10 N-terminal amino acids of the candidate protein X at its N terminus.(C) Analysis of the protein N-myristoylation of X(N10)-tGelsolin-FLAGs by metabolic labeling in transfected COS-1 cells.cDNAs coding X(N10)-tGelsolin-FLAGs were transfected into COS-1 cells.The expression of proteins was evaluated by Western blotting using an anti-FLAG antibody (upper panels).Protein N-myristoylation was evaluated by metabolic labeling with a myristic acid analog followed by click chemistry, as described in the Methods (lower panels).Images obtained by Western blotting or metabolic labeling were cropped.Uncropped full-length raw image data are shown in Supplementary Figure S1.

Protein N-myristoylation plays a critical role in the mitochondrial targeting of NDUFB7
As shown in Fig. 3A, the high conservation of the N-myristoylation motif was observed among vertebrates by the interspecies alignment of the N-terminal sequences of DMAC1, HCCS, NDUFB7, and PLGRKT.
To clarify the role of protein N-myristoylation in the mitochondrial localization of these four proteins, nonmyristoylatable G2A mutants were generated and their susceptibility to protein N-myristoylation and intracellular localization were evaluated.As shown in Fig. 3B upper panel, the efficient expression of proteins was observed in the wild types and G2A mutants of four C-terminally FLAG-tagged proteins, except for the G2A mutant of DMAC1-FLAG.Metabolic labeling experiments revealed the efficient incorporation of Alk-Myr in wild-type proteins, whereas it was abolished in the G2A mutant (Fig. 3B, lower panel).When the intracellular localization of the G2A mutants of these four proteins was evaluated by an immunofluorescence analysis using an anti-FLAG antibody, marked differences were observed among the four proteins, as shown in Fig. 3C, left panel and Supplementary Fig. S5.Specific localization to mitochondria was observed for the G2A mutants of DMAC1, HCCS, and PLGRKT, similar to the wild-type proteins.The G2A mutant of NDUFB7 localized exclusively to the nucleus (Fig. 3C, left panel, Supplementary Fig. S5, and Supplementary Fig. S7A, upper panel).These results were further confirmed by the line profile analysis of the images obtained, as shown in Fig. 3C, right panel, and Supplementary Fig. S7A, lower panel.
However, in the case of MIC19, in which protein N-myristoylation was shown to be required for mitochondrial targeting, the G2A mutant localized to the cytoplasm (Fig. 3C, left panel, Supplementary Fig. S5, and Supplementary Fig. S8B, upper panel).This result was further confirmed by the line profile analysis of the images obtained, as shown in Fig. 3C, right panel.Therefore, protein N-myristoylation occurring on NDUFB7 played a critical role in mitochondrial localization, whereas that of DMAC1, HCCS, and PLGRKT did not affect the mitochondrial localization of these proteins.

Structure of NDUFB7
Similar to MIC19, NDUFB7 is a member of the CHCHD-containing protein family 31 .The structures of NDUFB7 and MIC19 are shown in Fig. 4.
In addition to the N-terminal N-myristoylation motif, NDUFB7 contains CHCHD at positions 56 to 98. CHCHD contains two Cx9C motifs at positions 59 to 69 and 80 to 90 that are predicted to form a helix-coil-helix structure, permitting the formation of intramolecular disulfide bonds 32 .Since the non-myristoylatable G2A mutant of NDUFB7 specifically localized to the nucleus, NDUFB7 was predicted to contain a nuclear localization signal-like sequence (NLSL).When the two public WWW server-based prediction programs for nuclear localization signals, cNLS Mapper (http:// nls-mapper.iab.keio.ac.jp/ cgi-bin/ NLS_ Mapper_ form.cgi) 33 and NucPred (http:// www.sbc.su.se/ ~macca llr/ nucpr ed) 34 , were applied to the amino acid sequence of NDUFB7, potential nuclear localization signals were predicted at positions 109 to 118 (LQRKKRREKK) and 111 to 115 (RKKRR), respectively.As shown in Fig. 4, the high conservation of the N-myristoylation motif, CHCHD, and NLSL was observed among vertebrates by the interspecies alignment of the protein sequence of NDUFB7.

Positive charge cluster localized to the C-terminal region functions as a nuclear localization signal of the non-myristoylatable G2A mutant of NDUFB7
Two public WWW server-based prediction programs for nuclear localization signals predicted a possible nuclear localization signal and the predicted nuclear localization signal overlapped at positions 111 to 115 (RKKRR).Therefore, we attempted to clarify whether the positive charge cluster at this position functioned as a nuclear Figure 2. DMAC1, HCCS, NDUFB7, and PLGRKT are N-myristoylated mitochondrial proteins.Seven proteins (CLN3, DMAC1, HCCS, MARC1, NDUFB7, NOL3, and PLGRKT) were selected from proteins in which protein N-myristoylation was detected in X(N10)-tGelsolin-FLAG, and their protein N-myristoylation and intracellular localization were evaluated using C-terminally FLAG-tagged full-length proteins.(A) Analysis of the protein N-myristoylation of CLN3, DMAC1, HCCS, MARC1, NDUFB7, NOL3, and PLGRKT expressed in transfected COS-1 cells by metabolic labeling.cDNAs coding C-terminally FLAG-tagged full-length proteins were transfected into COS-1 cells and cells were then labeled with a myristic acid analog.The expression of proteins was evaluated by Western blotting using an anti-FLAG antibody (left panel).Protein N-myristoylation was evaluated by metabolic labeling followed by click chemistry, as described in the Methods www.nature.com/scientificreports/localization signal.Two mutants in which amino acids at positions 111 to 115 of NDUFB7-FLAG and NDUFB7-G2A-FLAG were changed to Ala (NDUFB7-5KRtoA-FLAG and NDUFB7-G2A-5KRtoA-FLAG, respectively) were generated (Fig. 5A) and their susceptibility to protein N-myristoylation and their intracellular localization were evaluated.
As shown in Supplementary Fig. S6, efficient protein expression and protein N-myristoylation were equally observed on NDUFB7-FLAG and NDUFB7-5KRtoA-FLAG.In contrast, the level of protein expression of NDUFB7-G2A-5KRtoA-FLAG was lower than that of NDUFB7-G2A-FLAG.As expected, protein N-myristoylation was not observed on these two G2A mutants.As shown in Fig. 5B upper left panel, NDUFB7-5KRtoA-FLAG specifically localized to mitochondria, similar to wild-type NDUFB7-FLAG.In contrast, NDUFB7-G2A-5KRtoA-FLAG localized to the cytoplasm, whereas NDUFB7-G2A-FLAG specifically localized to the nucleus (Fig. 5B lower left panel and Supplementary Fig. S7, A, B upper panels).These results were further confirmed by the line profile analysis of the images obtained, as shown in Fig. 5B, right panels and Supplementary Fig. S7,  A B, lower panels.

CHCHD plays a critical role in the mitochondrial localization of NDUFB7
The Mia40/CHCHD4 import pathway has been shown to mediate the import of (CX 9 C) 2 motif-carrying substrates into the intermembrane space (IMS) through outer membrane-localized TOM translocase 35 .The formation of two disulfide bonds among the (CX 9 C) 2 motif in the substrates catalyzed by the oxidase Mia40/ CHCHD4 plays a critical role in this transport pathway 31 .Regarding NDUFB7, two intra-molecular disulfide bridges between two Cys pairs (Cys59/Cys90 and Cys69/Cys80) were proposed to be formed by this system 32 .Therefore, we attempted to elucidate the role of CHCHD in NDUFB7 in the mitochondrial localization of this protein.To achieve this, two mutants in which Cys residues at positions 59 and 69 in the wild type and G2A mutant of NDUFB7 were changed to Ser (NDUFB7-CtoS-FLAG and NDUFB7-G2A-CtoS-FLAG, respectively) were generated (Fig. 6A and Supplementary Fig. S8A) and their susceptibility to protein N-myristoylation and their intracellular localization were evaluated.
As controls for this analysis, two MIC19 mutants in which the corresponding Cys residues at positions 183 and 193 in the wild type and G2A mutant of MIC19-FLAG were changed to Ser (MIC19-CtoS-FLAG and MIC19-G2A-CtoS-FLAG, respectively) were generated.As shown in Supplementary Fig. S9, efficient protein expression and effective protein N-myristoylation were equally observed on NDUFB7-FLAG and NDUFB7-CtoS-FLAG.In contrast, the protein expression level of NDUFB7-G2A-CtoS-FLAG was significantly lower than that of NDUFB7-G2A-FLAG.Protein N-myristoylation was not observed on NDUFB7-G2A-FLAG as expected.As shown in Fig. 6B, the CtoS mutant of NDUFB7-FLAG (NDUFG7-CtoS-FLAG) did not localize to mitochondria, but specifically localized to the Golgi apparatus (TGN), as was the case with MIC19-CtoS-FLAG.These results were further confirmed by the line profile analysis of the images obtained, as shown in Fig. 6C.In contrast, the CtoS mutant derived from the G2A mutant of NDUFB7 (NDUFB7-G2A-CtoS-FLAG) localized to the nucleus, as was the case with NDUFB7-G2A-FLAG (Supplementary Fig. S8B).Regarding the CtoS mutant of MIC19-G2A-FLAG (MIC19-G2A-CtoS-FLAG), a cytosolic distribution was observed, similar to MIC19-G2A-FLAG, as shown in Supplementary Fig. S8B upper panels.These results indicated that CHCHD played a critical role in the mitochondrial localization of NDUFB7, as was the case for MIC19.Furthermore, protein N-myristoylation and CHCHD were both required for the proper mitochondrial targeting and localization of NDUFB7.
Table 1.Proteins found to be N-myristoylated in the present study.The characteristics of seven proteins found to be N-myristoylated by the experiments shown in Fig. 2

Discussion
In an attempt to identify novel human N-myristoylated proteins from human cDNA clones in human cDNA resources, we previously found that two mitochondrial outer membrane proteins, SAMM50 and TOMM40, were N-myristoylated integral membrane proteins 18 .The protein N-myristoylation of SAMM50, but not TOMM40, was also shown to be required for mitochondrial targeting.In the present study, we searched for human N-myristoylated mitochondrial proteins in which protein N-myristoylation was required for their specific localization to mitochondria.Among the 1,705 human genes listed in MitoProteome, 12 candidate genes for N-myristoylated mitochondrial proteins were selected and their susceptibility to protein N-myristoylation and their intracellular localization were evaluated, as previously described [14][15][16][17][18]36 . As  result, 7 proteins (CLN3, DMAC1, HCCS, MARC1, NDUFB7, NOL3, and PLGRKT) were found to be N-myristoylated proteins.The endogenous protein N-myristoylation occurring on these 7 proteins expressed in HeLa cells have been reported in recent reports 37,38 .Among them, four proteins, DMAC1, HCCS, NDUFB7, and PLGRKT, were identified as N-myristoylated proteins that specifically localized to mitochondria (Fig. 2).None of these proteins contain predicted cleavable presequences that direct mitochondrial localization.Among these proteins, DMAC1, HCCS, and NDUFB7 have been shown to be mitochondrial proteins.DMAC1 and NDUFB7 were found to play important roles in the assembly of complex I of the mitochondrial respiratory chain as an assembly factor and an accessory subunit, respectively 23 .Both DMAC1 and NDUFB7 localize to the inner membrane and associate with complex I of the mitochondrial respiratory chain 23,39 .The import pathway for NDUFB7 has been characterized.NDUFB7 was first synthesized by cytosolic ribosomes in the cytoplasm, and imported into intermembrane space through outer membrane-localized TOM complex in an unfolded reduced form, then folded and oxidized by the disulfide relay-dependent Mia40/CHCHD4 import machinery 39,40 .The import pathway for DMAC1 has not been characterized so far.
HCCS localizes to the intermembrane space and functions as a lyase that catalyzes the covalent linking of the heme group to the cytochrome C apoprotein to produce mature functional cytochrome C 27 .
The localization and import pathway of HCCS has been well characterized in yeast 41 .HCCS was synthesized in the cytosol and directly imported to intermembrane space through outer membrane-localized TOM complex, then localized to the outer surface of the inner membrane.
Different from DMAC1, NDUFB7 and HCCS, PLGRKT has been shown to be a cell surface receptor for plasminogen that regulates cell surface plasminogen activation 30 .The presence and biological function of PLGRKT in mitochondria have not yet been reported.Thus, intramitochondrial localization and import pathway for PLGRKT have not been characterized yet.
An analysis of the intracellular localization of non-myristoylatable G2A mutants indicated that protein N-myristoylation occurring on NDUFB7, an accessory subunit of human mitochondrial respiratory chain complex I, played a vital role in the mitochondrial localization of this protein (Fig. 3).NDUFB7 is a member of the CHCHD-containing family of proteins (CHCHD proteins), which are imported into the mitochondrial IMS, and it has been proposed to stabilize assembled mitochondrial respiratory chain complex I by binding to its surface 32 .Mitochondrial respiratory chain complex I is the largest complex in the oxidative phosphorylation system.It comprises 14 central subunits, 31 accessory subunits, and at least 15 assembly factors 40 .Significant advances have recently been made in elucidating the structure and assembly steps of complex I. NDUFB7 is an accessory subunit of module P D -b.A critical role has been suggested for NDUFB7 in the assembly of complex I in the yeast Yarrowia lipolytica 42 .Furthermore, from in vitro experiments, the absence of any of the accessory subunits of the P D -b module may critically affect the assembly and function of human complex I 23,42,43 .
In fully assembled complex I, the module P D -b is composed of the central subunit ND5 and accessory subunits NDUFB2, NDUFB3, NDUFB7, NDUFB8, and NDUFB9 40,43 .Deleterious changes in proteins of the P D -b module have been described and associated with complex I disorders [44][45][46] .Regarding NDUFB7, a recent study reported that an intronic mutation leading to a cryptic splice site in the NDUFB7 gene caused a mitochondrial disorder 47 .In this case, an intronic mutation in NDUFB7 caused severe congenital lactic acidosis and hypertrophic cardiomyopathy.The detected mutation resulted in a significant reduction in the NDUFB7 protein and reduced complex I activity.It was also revealed that complementation studies with the expression of wild-type NDUFB7 in patient fibroblasts normalized complex I function.Therefore, NDUFB7 plays an important role in the formation and function of complex I.
The CHCHD-containing family of proteins are proteins that localize to the mitochondrial IMS and contain (CX 3 C) 2 or (CX 9 C) 2 motifs 31 .These CHCHD proteins are specifically transported into mitochondria by the disulfide relay-dependent Mia40/CHCHD4 import machinery 35 .They are involved in mitochondrial biogenesis, bioenergetics, dynamics, and quality control in the IMS 31 .Similar to other CHCHD proteins, CHCHD is required for the mitochondrial localization of NDUFB7; however, the present study revealed that NDUFB7 required protein N-myristoylation in addition to CHCHD.G2A mutant in which protein N-myristoylation was inhibited did not localize to mitochondria, but specifically localized to the nucleus (Figs. 3, 5).The nuclear localization of the G2A mutant of NDUFB7 was directed by the NLSL RKKRR, located at positions 111-115 (Fig. 5).This result indicated that protein N-myristoylation-mediated mitochondrial localization is preferential over nuclear localization mediated by the NLSL RKKRR.
A known CHCHD protein that requires protein N-myristoylation for mitochondrial localization is MIC19 (CHCHD3), an N-myristoylated mitochondrial inner membrane-binding protein that interacts with the luminal surface of the MICOS complex and is involved in the formation of cristae 18,48 .In addition to MIC19, protein N-myristoylation has been proposed to occur on another CHCHD protein, MIC25 (CHCHD6), which localizes to the mitochondrial IMS.A phylogenic analysis indicated that human MIC19 and MIC25 resulted from a gene duplication at the root of vertebrates 49 .Therefore, they are both orthologous to MIC19 in yeast and likely the result of whole genome duplication 50 .Regarding the mitochondrial localization of MIC19 and MIC25, neither a www.nature.com/scientificreports/canonical cleavable N-terminal mitochondrial targeting signal (presequence) nor another specific mitochondrial targeting signal has been identified.However, as for MIC19, protein N-myristoylation and CHCHD were both essential for the proper mitochondrial localization of this protein 48 .MIC25 was predicted to be N-myristoylated; however, direct biochemical evidence for protein N-myristoylation has not yet been reported.We previously showed that exogenously and endogenously expressed MIC19 and MIC25 were efficiently N-myristoylated 18 .Overexpressed MIC19 specifically localized to mitochondria, whereas the non-myristoylatable G2A mutant of MIC19 localized to the cytoplasm, confirming the requirement of protein N-myristoylation for the mitochondrial localization of MIC19.However, the majority of overexpressed N-myristoylated MIC25 was detected in the cytoplasm, but not in mitochondria.A similar cytosolic localization was observed for the non-myristoylatable G2A mutant of MIC25.Therefore, protein N-myristoylation did not appear to play a critical role in the mitochondrial localization of MIC25.The mechanisms responsible for differences in the role of protein N-myristoylation in the mitochondrial localization of MIC19 and MIC25 remains unclear.
A mechanism for the protein N-myristoylation-dependent mitochondrial localization of Mic19 was recently proposed using yeast Mic19 51 .This study demonstrated that the CHCHD-mediated mitochondrial translocation of Mic19 was inhibited by an unfolded domain in its N-terminal region, called the DAF domain.Protein N-myristoylation circumvented this inhibitory effect by mediating the binding of MIC19 to Tom20 in the TOM complex and to the mitochondrial outer membrane, thereby achieving the efficient mitochondrial localization of MIC19.It remains unclear whether protein N-myristoylation plays a similar role with respect to NDUFB7.However, since there is no DUF domain-like unfolded domain in the N-terminal region of NDUFB7, it is unlikely that the protein N-myristoylation of NDUFB7 circumvented the inhibitory effects of the DUF domain-like unfolded domain on mitochondrial localization.In addition, the non-myristoylatable G2A mutant of NDUFB7 was shown to localize to the nucleus rather than to the cytoplasm, different from the case for MIC19 (Fig. 5B lower panel, Supplementary Fig. S5, Supplementary Fig. S7A).Furthermore, the nuclear localization of the non-myristoylatable G2A mutant of NDUFB7 was mediated by the NLSL RKKRR, located at positions 111 ~ 115 (Fig. 5B lower panel, Supplementary Fig. S7).Therefore, protein N-myristoylation in NDUFB7 appears to function in the inhibition of nuclear localization mediated by the NLSL RKKRR rather than circumventing the suppressive effects of the DUF domain on mitochondrial localization, as observed for Mic19.It currently remains unclear why a positive charge amino acid cluster, such as RKKRR, is present in NDUFB7.In some proteins, positively charged amino acids play a role in protein-protein interactions and the assembly of protein complexes.For example, electrostatic interactions between positively and negatively charged residues play critical roles in the structure and function of mammalian respiratory complex I 39,52 .Therefore, the amino acid sequence RKKRR in NDUFB7 may be involved in the function of NDUFB7 as an accessory subunit of respiratory chain complex I.
In MIC19, protein N-myristoylation played a critical role not only in mitochondrial localization, but also in interactions with SAMM50 localized to the mitochondrial outer membrane 48 .In the case of NDUFB7, protein N-myristoylation was first reported on bovine NDUFB7, and this modification was proposed to function as a membrane anchor to the mitochondrial inner membrane 53 .Concerning the protein-protein interactions mediated by protein N-myristoylation of NDUFB7, recent study on the structure of mammalian mitochondrial complex I indicated that the N-terminal myristoyl group of NDUFB7 is bound in a groove on ND5 formed by TMH12ND5, TMH13ND5 and TMH15ND5 39 .Thus, it is quite probable that the protein-protein interactions mediated by protein N-myristoylation are involved in the function of NDUFB7 as an accessory subunit of respiratory chain complex I.
Schematic representation of possible roles of protein N-myristoylation and the CHCH domain in the mitochondrial targeting and localization of NDUFB7 is shown in Fig. 7.
NDUFB7 was first synthesized by cytosolic ribosomes in the cytoplasm, targeted to the mitochondrial outer membrane, and then translocated across the outer membrane through outer membrane-localized TOM translocase (TOM complex) by a protein N-myristoylation-dependent mechanism.The Mia40/CHCHD4 import Figure 3. Protein N-myristoylation plays a critical role in the mitochondrial localization of NDUFB7.To clarify the role of protein N-myristoylation in the mitochondrial localization of DMAC1, HCCS, NDUFB7, and PLGRKT, non-myristoylatable G2A mutants were generated and their susceptibility to protein N-myristoylation and intracellular localization were evaluated.(A) Interspecies alignment of the N-terminal sequences of DMAC1, HCCS, NDUFB7, and PLGRKT.N-myristoylation motifs are shown in purple in N-terminal sequences.(B) Analysis of protein N-myristoylation of the WT and non-myristoylatable G2A mutants of DMAC1, HCCS, NDUFB7, and PLGRKT.cDNAs coding the WT and non-myristoylatable G2A mutants of four proteins were transfected into COS-1 cells.The expression of proteins was evaluated by Western blotting using an anti-FLAG antibody (upper panel).Protein N-myristoylation was evaluated by metabolic labeling with a myristic acid analog followed by click chemistry, as described in the Methods (lower panel).Arrows indicate the position of the expressed proteins.The images obtained by Western blotting or metabolic labeling were cropped.Uncropped full-length raw image data are shown in Supplementary Figure S4.(C) Analysis of the intracellular localization of the non-myristoylatable G2A mutant of DMAC1, HCCS, NDUFB7, and PLGRKT.The intracellular localization of the G2A mutant of the four proteins was assessed by an immunofluorescence analysis of COS-1 cells transfected with cDNA coding the C-terminally FLAG-tagged G2A mutant using an anti-FLAG antibody.MIC19-G2A-FLAG was used as a control non-myristoylatable G2A mutant.Hoechst and MitoTracker Red were used as organelle markers for the nucleus and mitochondria, respectively.Experiments were repeated 3 times and similar results were obtained.Representative data are shown.Abbreviation used: PC, phase contrast image.The results of the line profile analysis are shown.In the line profile analysis, corresponding line-scan graphs of the relative fluorescence intensities of green anti-FLAG fluorescence (green line) and red MitoTracker red fluorescence (red line) along the white line indicated in the merged images are shown.

Prediction of protein N-myristoylation using prediction programs
Two public WWW server-based prediction programs for protein N-myristoylation, MYR Predictor (http:// mendel.imp.ac.at/ myris tate/ SUPLp redic tor.htm) 24 and Myristoylator (http:// www.expasy.org/ tools/ myris toyla tor/) 25 , were used to predict protein N-myristoylation.The entire amino acid sequences deduced from the nucleotide sequences of the ORFs were used as the query.

Plasmid construction
The nucleotide sequences of oligonucleotides used for plasmid construction are summarized in Supplementary Tables S3, S4, and S5.The plasmids pcDNA3-tGelsolin-FLAG, pcDNA3-FLAG, and pcDNA3-TGN46-EGFP were constructed as previously described 58,59 .pcDNA3 plasmids containing the cDNAs coding tGelsolin fusion proteins with the 10 N-terminal amino acids of the proteins listed in the MitoProteome protein database at the N terminus (pcDNA3-X(N10)-tGelsolin-FLAG) were constructed using the oligonucleotides listed in Table S3 and the plasmids pcDNA3-tGelsolin-FLAG and pcDNA3-FLAG, as described in Table S6.pcDNA3 plasmids,

Figure 1 .
Figure 1.Identification of N-myristoylated mitochondrial proteins from human genes listed in the MitoProteome protein database.Twelve candidate genes (BCAP31, CLN3, DMAC1/TMEM261, FLAD1, HCCS, LRRC10, MARC1/MTARC1, ME3, NDUFB7, NOL3, PLGRKT and TUSC3) were selected as N-myristoylated mitochondrial proteins from the human genes listed in MitoProteome, a mitochondrial protein database, and their susceptibility to protein N-myristoylation was evaluated by in vivo metabolic labeling in transfected COS-1 cells using fusion proteins in which the 10 N-terminal amino acid residues were fused to the FLAG-tagged model protein, tGelsolin.(A) Strategy to identify N-myristoylated mitochondrial proteins from human genes listed in the MitoProteome protein database.(B) Schematic representation of the generation of X(N10)-tGelsolin-FLAG with 10 N-terminal amino acids of the candidate protein X at its N terminus.(C) Analysis of the protein N-myristoylation of X(N10)-tGelsolin-FLAGs by metabolic labeling in transfected COS-1 cells.cDNAs coding X(N10)-tGelsolin-FLAGs were transfected into COS-1 cells.The expression of proteins was evaluated by Western blotting using an anti-FLAG antibody (upper panels).Protein N-myristoylation was evaluated by metabolic labeling with a myristic acid analog followed by click chemistry, as described in the Methods (lower panels).Images obtained by Western blotting or metabolic labeling were cropped.Uncropped full-length raw image data are shown in Supplementary FigureS1.
Figure2.DMAC1, HCCS, NDUFB7, and PLGRKT are N-myristoylated mitochondrial proteins.Seven proteins (CLN3, DMAC1, HCCS, MARC1, NDUFB7, NOL3, and PLGRKT) were selected from proteins in which protein N-myristoylation was detected in X(N10)-tGelsolin-FLAG, and their protein N-myristoylation and intracellular localization were evaluated using C-terminally FLAG-tagged full-length proteins.(A) Analysis of the protein N-myristoylation of CLN3, DMAC1, HCCS, MARC1, NDUFB7, NOL3, and PLGRKT expressed in transfected COS-1 cells by metabolic labeling.cDNAs coding C-terminally FLAG-tagged full-length proteins were transfected into COS-1 cells and cells were then labeled with a myristic acid analog.The expression of proteins was evaluated by Western blotting using an anti-FLAG antibody (left panel).Protein N-myristoylation was evaluated by metabolic labeling followed by click chemistry, as described in the Methods (right panel).Arrows indicate the position of the expressed proteins.The images obtained by Western blotting or metabolic labeling were cropped.Uncropped full-length raw image data are shown in Supplementary FigureS2.(B) Analysis of the intracellular localization of CLN3, DMAC1, HCCS, MARC1, NDUFB7, NOL3, and PLGRKT by an immunofluorescence microscopic analysis.The intracellular localization of seven proteins was assessed by an immunofluorescence analysis of COS-1 cells transfected with cDNA coding C-terminally FLAGtagged full-length proteins using an anti-FLAG antibody.MIC19-FLAG was used as a control mitochondrial N-myristoylated protein.Hoechst and MitoTracker Red were used as organelle markers for the nucleus and mitochondria, respectively.Experiments were repeated 3 times and similar results were obtained.Representative data are shown.Abbreviation used: PC, phase contrast image.The results of the line profile analysis are shown.In the line profile analysis, corresponding line-scan graphs of the relative fluorescence intensities of green anti-FLAG fluorescence (green line) and red MitoTracker red fluorescence (red line) along the white line indicated in the merged images are shown. https://doi.org/10.1038/s41598-023-50390-z https://doi.org/10.1038/s41598-023-50390-zwww.nature.com/scientificreports/machinery then mediated the oxidation-dependent retention of NDUFB7 in the intermembrane space.The NDUFB7 localized in the intermembrane space functioned as an accessory subunit of complex I of the mitochondrial respiratory chain (Complex I).In this mechanism, protein N-myristoylation and CHCH-domain were both required for the proper mitochondrial localization of NDUFB7.However, the relative roles of protein N-myristoylation and CHCH-domain in the intracellular localization and function of NDUFB7 remained to be elucidated.Further analyses are required to fully elucidate the role of protein N-myristoylation in the intracellular localization and function of NDUFB7.

Figure 4 .
Figure 4. Structure of NDUFB7.Schematic representation of the structures of MIC19 and NDUFB7.Interspecies alignments of the N-myristoylation motif, CHCH domain, and nuclear localization signal-like (NLSL) sequence of NDUFB7 are shown.N-myristoylation motifs were boxed.Four conserved Cys residues in the CHCH domain were indicated by the blue characters in the amino acid sequences.Conserved RKKRR sequences in the NLSL sequence were indicated by the red characters in the amino acid sequences.

Figure 5 .
Figure 5. Positive charge cluster localized in the C-terminal region functions as a nuclear localization signal of the non-myristoylatable G2A mutant of NDUFB7.To establish whether the positive charge cluster at positions 111 to 115 of NDUFB7 function as a nuclear localization signal, two mutants in which amino acids at this positions of NDUFB7-FLAG and NDUFB7-G2A-FLAG were changed to Ala (NDUFB7-5KRtoA-FLAG and NDUFB7-G2A-5KRtoA-FLAG, respectively) were generated (Fig. 5A), and their susceptibility to protein N-myristoylation and their intracellular localization were evaluated.The results obtained on protein expression and protein N-myristoylation from a Western blotting analysis and metabolic labeling experiments, respectively, are shown in Supplementary Figure S6.(A) Structure of NDUFB7 mutants lacking the C-terminal positive charge cluster.(B) Analysis of the intracellular localization of NDUFB7 mutants lacking the C-terminal positive charge cluster.The intracellular localization of NDUFB7-FLAG, NDUFB7-5KRtoA-FLAG, NDUFB7-G2A-FLAG, and NDUFB7-G2A-5KRtoA-FLAG was assessed by an immunofluorescence analysis of COS-1 cells transfected with cDNA coding these four proteins using an anti-FLAG antibody.Hoechst and MitoTracker Red were used as organelle markers for the nucleus and mitochondria, respectively.Experiments were repeated 3 times and similar results were obtained.Representative data are shown.Abbreviation used: PC, phase contrast image.The results of the line profile analysis are shown.In the line profile analysis, corresponding line-scan graphs of the relative fluorescence intensities of green anti-FLAG fluorescence (green line) and red MitoTracker red fluorescence (red line) along the white line indicated in the merged images are shown.

Figure 7 .
Figure 7. Schematic representation of possible roles of protein N-myristoylation and the CHCH domain in the mitochondrial targeting and localization of NDUFB7.NDUFB7 was synthesized by cytosolic ribosomes in the cytoplasm, targeted to the mitochondrial outer membrane, and then translocated across the outer membrane through outer membrane-localized TOM translocase (TOM complex) by a protein N-myristoylation-dependent mechanism.The Mia40/CHCHD4 import machinery then mediated the oxidation-dependent retention of NDUFB7 in the intermembrane space.NDUFB7 functioned as an accessory subunit of complex I of the mitochondrial respiratory chain (Complex I).In this mechanism, protein N-myristoylation and CHCH-domain were both required for the proper mitochondrial localization of NDUFB7. ) are summarized.