
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39289458
72681
10.1038/s41598-024-72681-9
Article
Characterization of Shy1, the Schizosaccharomyces pombe homolog of human SURF1
Luo Ying
Xu Yuanqi
Ahmad Fawad
Feng Gang fengg@njnu.edu.cn

Huang Ying yhuang@njnu.edu.cn

https://ror.org/036trcv74 grid.260474.3 0000 0001 0089 5711 Jiangsu Key Laboratory for Microbes and Genomics, School of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing, 210023 China
17 9 2024
17 9 2024
2024
14 2167819 2 2024
10 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Cytochrome c oxidase (complex IV) is the terminal enzyme in the mitochondrial respiratory chain. As a rare neurometabolic disorder caused by mutations in the human complex IV assembly factor SURF1, Leigh Syndrome (LS) is associated with complex IV deficiency. In this study, we comprehensively characterized Schizosaccharomyces pombe Shy1, the homolog of human SURF1. Bioinformatics analysis revealed that Shy1 contains a conserved SURF1 domain that links to the biogenesis of complex IV and shares high structural similarity with its homologs in Saccharomyces cerevisiae and humans. Our study showed that Shy1 is required for the expression of mtDNA-encoded genes and physically interacts with structural subunits and assembly factors of complex IV. Interestingly, Rip1, the subunit of ubiquinone-cytochrome c oxidoreductase or cytochrome bc1 complex (complex III), can also co-immunoprecipitate with Shy1, suggesting Shy1 may be involved in the assembly of the mitochondrial respiratory chain supercomplexes. This conclusion is further corroborated by our BN-PAGE analysis. Unlike its homologs, deletion of shy1 does not critically disrupt respiratory chain assembly, indicating the presence of the compensatory mechanism(s) within S. pombe that ensure mitochondrial functionality. Collectively, our investigation elucidates that Shy1 plays a pivotal role in the sustainability of the regular function of mitochondria by participating in the assembly of complex IV in S. pombe.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72681-9.

Keywords

Schizosaccharomyces pombe
mitochondria
cytochrome c oxidase
Shy1
SURF1
Subject terms

Microbiology
Fungi
Fungal biology
http://dx.doi.org/10.13039/501100012154 Graduate Research and Innovation Projects of Jiangsu Province KYCX23_1732 Luo Ying http://dx.doi.org/10.13039/501100004543 China Scholarship Council No. 202306860040 Luo Ying http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 31770810 Huang Ying issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

The mitochondrial respiratory chain (also called the electron transport chain, ETC) is pivotal for cellular energy metabolism. It serves as the core framework of mitochondrial oxidative phosphorylation1. ETC comprises four enzyme complexes, namely, NADH-ubiquinone oxidoreductase (complex I), succinate dehydrogenase (complex II), ubiquinone-cytochrome c oxidoreductase or cytochrome bc1 complex (complex III), and cytochrome c oxidase (complex IV), along with two mobile electron carriers, ubiquinone (CoQ) and cytochrome c (Cyt c). Situated within the inner mitochondrial membrane (IMM), these complexes contribute to the establishment of a continuous system of biochemical reactions2. Complex IV serves as the terminal enzyme of ETC, aiding in the oxidation of cytochrome c while simultaneously reducing O2 to H2O. Protons are translocated from the mitochondrial matrix to the intermembrane space (IMS) via a redox reaction3.

Mammalian complex IV is made of 14 subunits4. MT-CO1, MT-CO2, and MT-CO3 constitute the catalytic center of complex IV, encoded by mitochondrial DNA (mtDNA), whereas the other subunits are derived from nuclear DNA (nDNA). NUDFA4 is found to be a novel subunit weakly bound to complex IV which used to assign to complex I5,6. Mutations in certain nDNA-encoded subunits can disrupt the assembly of complex IV, resulting in a significant reduction in its activity. These subunits play a critical role in stabilizing MT-CO1, MT-CO2, and MT-CO3, as well as regulating the activity of complex IV7. The fission yeast Schizosaccharomyces pombe mitochondrial complex IV contains 13 subunits based on the model organism database (MOD) Pombase (https://www.pombase.org/). Cox1, Cox2 and Cox3 are encoded by mtDNA, and the other subunits are encoded by nDNA8.

Considerable investigation has been carried out concerning the formation of human complex IV in both normal and diseased cell lines. Presently, it is evident that the assembly of complex IV is facilitated by the integration of distinct subunit modules and is distinguished by each of the core subunits encoded by mtDNA9,10. A substantial portion of mitochondrial complex IV deficiency arises from mutations in genes that encode proteins primarily involved in stabilizing subunits of complex IV and its assembly intermediates11–13. SURF1 is the most prominent one whose functional absence will lead to Leigh syndrome12,14, which is a severe neurological disorder in subcortical brain typically linked to cytochrome c oxidase impairment15,16. While it is recognized that SURF1 participates in the activities associated with the stability, maturation, and assembly of the core MT-CO1 subunit, the precise molecular function of SURF1 within this process has yet to be fully elucidated. Notably, Saccharomyces cerevisiae SHY1 has the capacity to compensate for complex IV deficient mutants through complementation, thus establishing its eligibility as a critical IMM protein essential for mitochondrial respiration17. Subsequently, its direct involvement in the assembly of complex IV was identified, as evidenced by the defect in cell growth on nonfermentable carbon sources observed in the SHY1 mutant strain. This deficiency in SHY1 results in reduced levels of complex IV and a decrease in Cox118,19. In a complex IV mutant mouse model, injection of a safe dose of AAV9/hSURF1 vector can increase the expression of SURF1 mRNA, thereby alleviating surfeit locus protein 1 (SURF1)-related Leigh syndrome, and in no adverse reactions were found in tests on mice for up to a year20. The homolog of human SURF1 in Danio rerio was also studied by morpholino-mediated silencing of surf121. The results demonstrate that complex IV deficits are linked to endodermal tissue developmental abnormalities that result in compromised heart function and changed swimming patterns. However, the S. pombe homolog of human SURF1 has not been characterized yet.

In this study, we characterized Shy1, the S. pombe homolog of the human Leigh syndrome-related protein SURF1, and analyzed the protein structure, domain and motifs of Shy1 by comparing with its homologs in human and S. cerevisiae. BN-PAGE results showed that the abundance of the complex IV was markedly diminished in ∆shy1 strain. Analysis of protein-protein interactions indicated that Shy1 is inclined to interact with the structural subunits and additional assembly factors of complex IV, thus playing a role in the assembly process of complex IV. We then further studied the subcellular localization of Shy1 by fluorescent microscopy and treatment with Triton X-100 and proteinase K, the results showed that Shy1 is a IMM protein with two transmembrane domains. Our findings additionally revealed that Shy1 is indispensable for mitochondrial respiration and the maintenance of stable levels of the core subunits of the ETC. Collectively, our investigations elucidate that Shy1 is highly likely to play a pivotal role in the sustainability of the regular function of mitochondria by participating in the assembly of complex IV in S. pombe. Our study paves the way for future research on the molecular mechanism of Shy1 involved in complex IV assembly.

Materials and methods

Strains and media

S. pombe strains used in this study are listed in Supplementary Table S1 online. Strains expressing Shy1 C-terminal tagging of endogenous S. pombe genes were carried out by in-frame integration of the respective tagging cassette obtained by overlap extension PCR into WT (yHL6381) strain. Strains expressing the FLAG-tagged Shy1 (Shy1-FLAG) and HA-tagged proteins are constructed using overlapping PCR, the method to distinguish Cox1101 and Cox1102 is shown in Supplementary Fig. S1 online. S. pombe cells were grown in YES rich medium with 3% glucose for fermentative growth or 3% glycerol and 0.1% glucose for respiratory growth22,23. The standard protocols for the genetic manipulation of the S. pombe genome were from previously published procedures22.

Prediction and alignment of the protein structure

Structural predictions of S. pombe Shy1, S. cerevisiae SHY1 and human SURF1 were conducted by AlphaFold24,25 with default parameters (https://alphafold.ebi.ac.uk/). TM-align26 (https://zhanggroup.org/TM-align/) was used for the structural alignments, the structural similarity was evaluated using the TM‐score and the root‐mean‐squared deviation (RMSD) value. A TM‐score ranging from 0.5 to 1 signifies a comparable fold between the two protein structures under examination. Conversely, an elevated RMSD value signifies a significant dissimilarity between the structures.

Proteins data set and sequence analyses

The protein sequences of S. pombe Shy1, S. cerevisiae SHY1 and human SURF1 were obtained from GenBank. Subsequently, all protein sequences were aligned using the MUSCLE algorithm27. The analysis was performed with MEGAX28. Sequence similarities were rendered using ESPript 3.029. Domains were identified by the Conserved Domain Database (CDD https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and Simple Modular Architecture Research Tool (SMART http://smart.embl-heidelberg.de/) analyses. The motifs were detected by the MEME Suite (https://meme-suite.org/meme/tools/meme) (Version 5.5.4). Mitochondrial presequence was predicted using MitoFates30 (https://mitf.cbrc.pj.aist.go.jp/MitoFates/cgi-bin/top.cgi).

Purification of mitochondria, subfractionation of mitochondria and protease treatment of mitochondria

Purification of S. pombe mitochondria were performed using lytic enzymes from Trichoderma harzianum (Sigma), the standard protocols were described previously31. The crude mitochondria can be further purified according to the reference32. Mitochondrial extracts were collected by centrifugation at a speed of 12,000 g for 10 min. A protein concentration of 5 µg/µL mitochondrial extracts were resuspended in the SEM Buffer mitochondria were resuspended. And then treated with 50 µg/mL proteinase K and 0.2% Triton X-100 for 20 min on ice and was stopped by 1 mM PMSF33. Trichloroacetic acid (TCA) was added to precipitate the proteins with a final concentration of 25%, then washed by iced acetone. The protein extracts were detected by Western blotting with corresponding antibodies. Mitochondrial soluble proteins were analyzed by sodium carbonate treatment according to protocols described34.

Western blot analysis

Alkaline extraction was used for whole cell protein extracts35. The proteins were separated using 1× protein loading buffer and then subjected to SDS-PAGE analysis followed by Western blotting. Subsequently, the protein bands were transferred onto a nitrocellulose filter membrane (GE Healthcare). The blots were subsequently probed with specific antibodies. Protein bands were visualized using the Odyssey near-infrared fluorescence scanner (LI-COR Biosciences). Primary antibodies against anti-Cob1, anti-Cox1, anti-Cox2, anti-Cox3, anti-Atp6, and anti-Hsp60 were prepared as described previously36.

Fluorescence microscopy

Overnight cultures of strains expressing Shy1 tagged with green fluorescent protein（GFP）were collected for fluorescence microscopy. For MitoTracker (Thermo-Fisher Scientific, USA) staining, cells were collected and first washed and then suspended in 1× PBS (Phosphate Buffered Saline) containing 50 nM MitoTracker Red. Subsequently, incubation was carried out at 37 °C for 15 min, followed by washing with 1× PBS for three times. The green and red fluorescence signals were captured using a Zeiss Axio Imager A1 microscope (Zeiss, Jena, Germany) with excitation wavelengths of 488 nm and 579 nm, respectively.

Quantitative real-time RT-PCR

The wild type and ∆shy1 strain were cultured overnight in YES medium at 30 °C and then diluted into fresh YES to an OD600 of 0.2. Cells were collected after 6-hour growth. Total RNA was extracted using an E.Z.N.A. Yeast RNA Kit (OMEGA BIO-TEK, China). RNA was reversed transcribed using HiScript III RT SuperMix for qPCR (Vazyme Biotech, China). Real-time quantitive PCR (qPCR) was carried out using Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech, China). The primers for qPCR were listed in Supplementary Table S2 online. All reactions were performed in triplicate. Data were analyzed using StepOne™ software (Thermo Fisher Scientific, USA). The CT values were normalized against levels of actin (act1) transcript from the same preparations. Fold changes in mRNA levels of ∆shy1 strain relative to wild type were calculated using the 2–∆∆CT method.

Immunoprecipitation assay

The S. pombe Shy1 is predicted to interact with 10 proteins according to STRING database (https://string-db.org), including 2 complex IV structural subunits (Cox5 and Cox6) and 6 complex IV assembly factors (Mss51, Cox1101, Cox1102, Cox17, Pet117, Sco1). The immunoprecipitation assay was performed to examine the physical interaction between Shy1 and these proteins. Cells expressing chromosomally encoded FLAG-tagged Shy1 and HA-tagged Cox5, Cox6, Mss51, Cox1101, Cox1102, Cox17, Pet117 and Sco1 were constructed using an overlapping PCR approach. Cells were collected and suspended in binding buffer (20 mM Tris-HCI, pH 8, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM PMSF and complete protease inhibitor), and disrupted with glass beads (Sigma-Aldrich) using the FastPrep-24 bead beater. Proteins were precipitated with anti-FLAG beads. The beads were washed with binding buffer, wash buffer 1 (10 mM Tris-HCI, pH 8, 68.5 mM NaCl, 0.1% Nonidet P-40, 5% glycerol) and wash buffer 2 (5 mM Tris-HCI, pH 8, 34.25 mM NaCl, 0.1% Nonidet P-40, 5% glycerol) for two times, respectively. The bound proteins were eluted with SDS-PAGE loading buffer, and subjected to immunoblot analysis with anti-FLAG and anti-HA Abs.

Blue native PAGE (BN-PAGE)

BN-PAGE was performed to analyze the assembly of ETC complexes III and IV as described34. Briefly, 2 mg mitochondria were lysed by detergents digitonin (DG) or n-Dodecyl-β-D-maltopyranoside (DDM). The mitochondrial membranes were solubilized by adding either 4 mg Digitonin/mg protein to maintain the supercomplexes or 1.6 mg DDM/mg protein to separate the individual complexes. The DG-solubilized and DDM-solubilized mitochondrial complexes were analyzed by a linear 3–12% or 5–10% gradient gels. 100 µg of protein per sample was loaded on a gradient native gel. Proteins were transferred to nitrocellulose membranes (GE Healthcare) and immunoblotted with indicated Abs. The molecular weights of the complexes were eveluated by the High Molecular Weight Native Electrophoresis Protein Marker II (Real Times, China).

Determination of mtDNA copy number

The mtDNA copy number was determined as described36. Briefly, yeast cells were inoculated into YES and cultured overnight at 30 °C. Cells were diluted into fresh YES medium to an OD600 of 0.2 and then harvested for gDNA extraction in mid-log phase. qPCR was performed using KAPA HiFi HotStart Uracil + ReadyMix (Roche, Pleasanton, CA, USA) following the manufacturer’s instructions. The median of CT values for mtDNA-encoded genes (cob1, cox1, cox3, and atp9) and nuclear DNA-encoded genes (spo12, ace2, and exg1) were used to estimate mtDNA and nuclear DNA levels, respectively. The fold change of the mtDNA copy number of mutant compared to that of WT strain (whose value was set to 1) was calculated using Eq. 2−∆∆Ct.

Enzyme activity assay

The complex III (CoQ-cytochrome c reductase) activity was determined using the mitochondrial respiration complex III activity assay kits (Solarbio Science & Technology Co, Ltd) following the manufacturer’s instructions.

Results

S. pombe Shy1 is structurally similar to both S. cerevisiae SHY1 and human SURF1

Shy1 (SPBC1215.01) is annotated as a cytochrome c oxidase assembly protein on S. pombe database Pombase, it comprises 290 amino acids, with a predicted molecular weight of 33 kDa and an isoelectric point (pI) of 10.67. Shy1 is 24% identical and 36% similar to S. cerevisiae SHY1 and 27% identical and 37% similar to human SURF1. Given that proteins with moderate sequence similarity can adopt analogous three-dimensional structures and execute comparable functions, we firstly compare the tertiary structures of S. pombe Shy1, S. cerevisiae SHY1, and human SURF1. The tertiary structures of these proteins were predicted by AlphaFold and shown as ribbon patterns in Fig. 1a, b and c. Subsequently, TM-align was used to compare the structures of S. pombe Shy1 with its homologs. As shown in Fig. 1d, e and S. pombe Shy1 harbors a TM‐score of 0.73 and 0.71, and an RSMD value of 2.86 Å and 3.21 Å with its homologs in S. cerevisiae and human respectively, indicating that S. pombe Shy1 is highly structurally similar to both S. cerevisiae SHY1 and human SURF1 and they may perform similar functions.

Fig. 1 S. pombe Shy1 is structurally similar to both S. cerevisiae SHY1 and human SURF1. (a, b, c) Ribbon representations of the predicted three-dimensional structure of S. pombe Shy1 (a), S. cerevisiae SHY1 (b) and human SURF1 (c). The structural prediction was performed using AlphaFold24,25. Figures are generated using PyMOL (The PyMOL Molecular Graphics System, Version 2.5, Schrödinger, LLC.). Secondary structure elements of proteins are colored with blue to red running from the N‐ to C‐terminus. The N and C termini are labeled. (d) Alignment of the predicted structures of S. pombe Shy1 and S. cerevisiae SHY1. The structures of S. pombe Shy1 and S. cerevisiae SHY1 are colored in red and blue, respectively. (e) Alignment of the predicted structures of S. pombe Shy1 and human SURF1. The structures of S. pombe Shy1 and human SURF1 are colored in red and blue, respectively. TM, template modeling. Structural alignment was performed using TM‐align26.

3.2 The missense mutations residues in SURF1 resulted in Leigh Syndrome are completely conserved inS. pombe Shy1 (G103, I210 and Y247).

The patients with a specific deficit of complex IV often have missense mutations in SURF1, whereas SURF1 is not completely required for the activity of complex IV14,37,38. Three missense mutations of SURF1 have been discovered in Leigh Syndrome patients, G124E, I246T, and Y274D, which are three conserved residues16,39. Multiple sequence alignment of Shy1 and Shy1 orthologs revealed that Gly103 and Tyr247 are completely conserved residues in Shy1 homologs, and Ile210 is a conserved hydrophobic residue. SMART database search predicted 2 transmembrane domains (indicated by bars) in Shy1 (Fig. 2a). Gly103 and Ile210 exist in the IMS domain, whereas Tyr247 is located in the second transmembrane domain (Fig. 2a and b).

Fig. 2 A schematic view of Shy1. (a) The amino acid sequences of S. pombe Shy1, S. cerevisiae SHY1 and human SURF1 were obtained from GenBank. All proteins sequences were aligned by MUSCLE27. The analysis was performed using the neighbor-joining method with MEGAX28,55. Sequence similarities were rendered using ESPript 3.029. Bars indicate the predicted transmembrane domains. The arrowheads indicate amino acid residues mutated in LS patients. (b) Two predicted transmembrane domains (TM1 and TM2) are indicated along with the IMS (188 residues) and the N (38 residues)- and C (24 residues)- terminal domains. Marked residues represent SURF1 substitutions that cause pathogenic mutations. (c) The network of S. pombe Shy1 protein-protein interaction is shown by nodes and edges using STRING database with default setting. The edges indicate both functional and physical protein associations. (d) The majority of predicted proteins are either the structural subunits or assembly factors of complex IV. Cox10 is a protoheme IX farnesyltransferase and Etp1 is a mitochondrial [2Fe-2S] cluster assembly ferredoxin Etp1/heme O monooxygenase Cox15 fusion protein, both of which are involved in the heme A biosynthetic process.

Subsequently, protein-protein interaction of Shy1 was analyzed using the STRING database (https://string-db.org), it was determined that Shy1 interacts with 10 proteins (Cox10, Cox1101, Cox1102, Cox6, Sco1, Etp1, Cox17, Cox5, Pet117, Mss51), among of which two proteins are the structural subunits of complex IV (Cox5 and Cox6), six proteins are the assembly factors of complex IV (Mss51, Pet117, Sco1, Cox17, Cox1101 and Cox1102) (Fig. 2d), indicating that Shy1 is likely to interact with other proteins and form a complex to function in the process of the complex IV assembly. The remaining two proteins are Cox10 and Etp1. Cox10 is a protoheme IX farnesyltransferase and Etp1 is a mitochondrial [2Fe-2 S] cluster assembly ferredoxin Etp1/heme O monooxygenase Cox15 fusion protein, both of which are involved in the heme A biosynthetic process. The protein-protein interaction network is shown in Fig. 2c. The proteins and the interaction scores are listed in Table 1. CDD and SMART analyses predicted that both S. pombe Shy1 and its orthologs in S. cerevisiae and human contain a SURF1 domain that is linked to the biogenesis of the complex IV of ETC15 (Fig. 3a). Using the MEME Suite, 3 highly conserved motifs were identified in Shy1 and Shy1 orthologs. These three motifs are important for the function of complex IV as they contain the two conserved residues Gly103 and Tyr247 (Fig. 3b), Gly103 and Tyr247 are marked with stars in the sequence logos for motifs 1–3 (Fig. 3c).

Fig. 3 Both S. pombe Shy1 and its homologs in S. cerevisiae and human contain a SURF1 domain and 3 conserved motifs. (a) Locations of SURF1 domain in S. pombe Shy1, S. cerevisiae SHY1 and human SURF1. Domains were identified by CDD and SMART analyses. (b) Locations of the conserved motifs in S. pombe Shy1 and its homologs. (c) Sequence logos for Motifs 1–3. The sequence logos of the motifs derived from S. pombe Shy1, S. cerevisiae SHY1 and human SURF1 were generated using the MEME Suite. Height indicates conservation level for each amino acid. The amino acid residues are highlighted in accordance with their biochemical properties. The conserved amino acids related to Leigh Syndrome in human marked with stars.

Table 1 STRING database protein interaction scores of S. pombe Shy1.

Protein Name	UniProt accession number	Interaction scores	Description	
Shy1	Q9Y810		cytochrome c oxidase assembly protein Shy1; A member of the SURF1 family required for efficient assembly of complex IV	
Cox10	Q9Y7Y4	0.991	protoheme IX farnesyltransferase, which converts protoheme IX and farnesyl diphosphate to heme O	
Cox1101	Q9UTM2	0.953	cytochrome c oxidase assembly protein cox11-1, which is a member of the COX11/CtaG family	
Cox1102	Q86ZU7	0.939	cytochrome c oxidase assembly protein cox11-2, which is a member of the COX11/CtaG family	
Cox6	Q9UTF6	0.904	cytochrome c oxidase subunit 6, which is a component of the complex IV	
Sco1	O42899	0.899	a copper chaperone transporting copper to the Cu(A); a member of the SCO1/2 family	
Etp1	Q10361	0.893	electron transfer protein 1.	
Cox17	Q9P7Z7	0.861	complex IV copper chaperone, which delivers copper ions to the Cu(A) site of complex IV; a member of the COX17 family	
Cox5	O74988	0.83	cytochrome c oxidase polypeptide 5, which is a component of the complex IV	
Pet117	C6Y4C1	0.809	mitochondrial protein Pet117 involved in the assembly of cytochrome c oxidase; the PET117 family	
Mss51	Q9UTB4	0.741	Mss51 has a dual role in Cox1 synthesis, acting as both a translational activator and assembly factor for the Cox1 (By similarity); a member of the MSS51 family	

S. pombe Shy1 physically interacts with complex IV structural subunits and assembly factors

S. pombe Shy1 was predicted to interact with two structural subunits (Cox5 and Cox6) and six assembly factors (Mss51, Pet117, Sco1, Cox17, Cox1101 and Cox1102) of complex IV. To determine the physical interaction between Shy1 and these proteins, the Shy1-FLAG and other HA-tagged proteins strains were constructed for the immunoprecipitation assay. Our results showed that Shy1 physically interacts with complex IV structural subunits Cox5 and Cox6 (Fig. 4a). Mss51 and Cox14 are the translational regulators of Cox1, they were co-isolated with Shy1 in S. cerevisiae 40,41. Here we also tested whether Cox14 interacts with Shy1 in S. pombe. Among these assembly factors of complex IV, Mss51, Pet117, Sco1, Cox14 were identified in the Shy1-FLAG eluate (Fig. 4a and b), elucidating that Shy1 interacts with other proteins to form a complex to be involved in the assembly of complex IV. However, Cox17, Cox1101 and Cox1102 were not detectable in the Shy1-FLAG eluate (Fig. 4b and c), the possible reason might be the low expression level of Cox17 or the interaction between them might be transient.The untagged strains are served as the negative control for immunoprecipitation (Fig. 4d). All tags strains used for immunoprecipitation assay do not affect mitochondrial function (Supplementary Fig. S2–S4 online).

Fig. 4 Shy1 physically interacts with complex IV structural subunits and assembly factors. (a) Shy1 interacts with complex IV structural subunits (Cox5 and Cox6), complex IV assembly factor Mss51 and complex III subunit Rip1. Overnight cultures of WT expressing Shy1-FLAG and HA-tagged Mss51, Rip1, Cox5 and Cox6 were diluted with fresh YES medium to an OD600 of 0.2, cells were harvested after 12 h growth and lysed by glass beads beating and subjected to anti-c-FLAG IP. Extracts and immunoprecipitates (IP) were analyzed by Western blotting using anti-FLAG and anti-HA Abs. actin (Act1) was detected as a loading control. (b) Shy1 interacts with complex IV assembly factors Pet117, Sco1 and Cox14. Cells expressing Shy1-FLAG and HA-tagged Pet117, Sco1, Cox14 and Cox17 were collected and subjected to anti-FLAG IP. Extracts and IP were analyzed by Western blotting. (c) Cox1101-HA and Cox1102-HA are not detectable in Shy1-FLAG IP extracts. Cells expressing Shy1-FLAG and Cox1101-HA or Cox1102-HA were harvested and subjected to anti-FLAG IP. Extracts and IP were analyzed by Western blotting using indicated Abs. (d) WT and Shy1-FLAG single tagged strains were served as the negative control of IP assay. Original blots are presented in Supplementary Fig. S6-S8 online.

Shy1 co-isolates with the complexe III subunit Rip1 

In order to determine whether Shy1 is involved in the formation of respiratory chain supercomplexes, we also tested the physical interaction of Shy1 and Rip1, a structural subunit of ubiquinone-cytochrome c oxidoreductase (complex III). To this end, a strain expressing Shy1-FLAG and Rip1-HA was conducted and immunoprecipitated with anti-FLAG beads. Surprisingly, Rip1 was indeed detectable in Shy1-FLAG eluate (Fig. 4a), since Shy1 has been implicated to be associated with the assembly of complex IV. Here, our results showed that Shy1 is physically linked to Rip1, the structural subunit of complex III, suggesting that Shy1 co-isolated with complexes III and IV of the mitochondrial respiratory chain.

S. pombe Shy1 is localized in the mitochondrial inner membrane

S. pombe Shy1 was predicted possessing mitochondrial presequence using MitoFates30 (https://mitf.cbrc.pj.aist.go.jp/MitoFates/cgi-bin/top.cgi). To confirm the localization of Shy1, we firstly added a GFP tag to the C-terminal of Shy1 and investigated the location of Shy1-GFP in the cell by fluorescence microscopy. The results showed that Shy1 is colocalized with mitochondria (Fig. 5a). To further determine the subcellular localization of Shy1, strain expressing Shy1-TAP was constructed under the control of their own promoters. In order to determine whether a ‘TAP’ tag impairs the protein functionality, the spot assay was performed on both fermentative and nonfermantative medium. The results showed that strain expressing Shy1-TAP does not affect the growth on both fermentative and nonfermantative conditions (Fig. 5a). The mitochondria were extracted from the Shy1-TAP strain, and Shy1 was detected in the same fraction with Hsp60, the mitochondrial protein marker (Fig. 6b), and nDNA-encoded Sla1 was detected as a non-mitochondrial protein marker42. The isolated mitochondria were subjected to treatment with proteinase K and Triton X-100 to ascertain the localization of Shy1 in the outer membrane36. Notably, the level of the mitochondrial outer membrane protein Tom20-FLAG was markedly diminished solely following the proteinase K treatment (Fig. 6c), whereas Shy1 could be detected, suggesting that Shy1 does not reside in the mitochondrial outer membrane. Then alkaline treatment was performed to determine whether Shy1 is a soluble or peripheral membrane protein34. The protein extracts of different fractions were analyzed by Western blotting. The analysis revealed the presence of the mitochondrial matrix protein Hsp60 (SPAC12G12.04) in the supernatant fraction, while the mitochondrial inner membrane protein Cox2 was detected in the pellet fraction. Similarly, Shy1-TAP was detected in the pellet fraction (Fig. 6d). Collectively, our findings strongly indicate that Shy1 is affixed to the mitochondrial inner membrane.

Fig. 5 Shy1 is required for mitochondrial respiration and the assembly of complex IV. (a) WT and Δshy1 strains were grown in rich medium under fermentative or nonfermentative conditions, the plates were cultured at 30 °C for up to 3 days. (b) qRT-PCR analysis of the steady-state levels of mature mt-RNAs in WT and Δshy1 strains. Levels of mature mt-RNAs in Δshy1 cells were normalized to the S. pombe actin gene act1 and expressed as fold change over control WT strain yHL6381 (set to 1). The statistical significance was determined by the Student’s t-test using the GraphPad Prism software (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗P < 0.001). (c). Analysis of mtDNA copy number in WT and ∆shy1 cells. Changes in mtDNA copy numbers were determined by measuring the relative mtDNA/nuclear DNA ratio by qPCR, and expressed as relatively mtDNA copy numbers normalized to that of the WT cells (which is set to 1). Three independent experiments were performed. The error bars represent the SD of triplicates. Statistical significance was determined by the student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). (d).Western blotting analysis of mtDNA-encoded proteins. Mitochondrial extracts were analyzed by Western blotting with specific Antibodies. Hsp60 servesed as a loading control. Original blots are presented in Supplementary Fig. S10 online.

Fig. 6 Shy1 is localized in the mitochondrial inner membrane. (a) S. pombe WT cells expressing a GFP-tagged Shy1 from the endogenous shy1 locus were examined by fluorescence microscopy. MitoTracker Red was used as a mitochondrial marker. (b) Mitochondria were isolated from cells expressing Shy1-TAP. T, Total cell extracts; Mt, mitochondria fraction; and PMS, post mitochondrial supernatants. (c) Purificated mitochondria were treated with proteinase K and Triton X-100 as described, and followed by Western blotting with specific antibodies. (D) Isolated mitochondria were treated by sodium carbonate. Proteins were analyzed by Western blotting for Shy1-TAP, Hsp60 and Cox2 were detected as the mitochondrial matrix protein and the IMM protein marker, respectively. Original blots are presented in Supplementary Fig. S8-S9 online.

Shy1 is required for mitochondrial respiration

In order to ascertain the role of S. pombe Shy1, ∆shy1 strain was constructed and spotting assays were performed to evaluate the impact of its deletion on cell growth. The cell growth of the ∆shy1 mutant exhibited a notable decline when cultivated in glycerol medium in comparison to the wild-type (WT) strain (Fig. 5a). Conversely, the growth reduction was marginal when cultured in glucose medium, which supports fermentative growth and thus necessitates moderate respiratory activity43. To investigate whether the accumulation of mt-mRNAs and mt-rRNAs were affected by shy1 deletion, total RNA was extracted from WT and ∆shy1 cells, and qRT-PCR was performed using specific primer pairs. Our results showed that the levels of cob1, cox1, cox2, cox3, atp6, atp8 and atp9 RNAs were reduced in ∆shy1 cells (Fig. 5b). Furthermore, the abundance of mt-rRNAs (rns and rnl) was also reduced in shy1 deletion cells compared to WT cells, suggesting that deletion of shy1 affects the overall levels of the expression of the mtDNA-encoded genes. To further investigate the possible causes of reduced mitochondrial RNAs, the copy number of mitochondrial DNA was measured. In order to quantitatively evaluate potential changes in total mtDNA levels in the ∆shy1 mutant, we assessed the mtDNA to nuclear DNA ratios using real-time quantitative PCR (qPCR). These ratios were then compared to those in WT cells, following the methodology described in previous reports44. As shown in Fig. 5c, the mtDNA copy number is slightly increased in ∆shy1 cells compared to that of in WT strain, indicating the observed reduction in mitochondrial RNA is not attributed to a decrease in mtDNA levels. Therefore, we hypothesized that the loss of shy1 may affect mitochondrial RNA stability, leading to a reduction in their steady-state levels.

Subsequently, a Western blot analysis was conducted to investigate whether the absence of shy1 influenced the steady-state levels of core subunits of the ETC encoded by mtDNA. Our results showed that the expression of Cob1, Cox1, Cox2, Cox3, and Atp6 were greatly reduced in ∆shy1 cells (Fig. 5d). Whether this phenomenon is caused by protein synthesis or protein degradation still needs to be further investigated. Our findings indicated that deletion of shy1 impairs mitochondrial respiration primarily by affecting the expression of genes encoded by mtDNA.

Deletion of shy1 primarily affects the assembly of complex IV

S. pombe Shy1 is predicted as a complex IV assembly factor, to determine whether deletion of shy1 affects the assembly of complex III, complex IV of ETC and ATP synthase (V), the abundance of complex III, complex IV, ATP synthase and supercomplexes were tested by BN-PAGE in WT and shy1 deletion cells. Cob1, Cox1 and Atp6 are the structural subunits of complex III, complex IV and ATP synthase, respectively. The mitochondria were extracted and solubilized with detergents digitonin (DG), which maintains the supercomplexes, or n-Dodecyl-β-D-maltopyranoside (DDM), which separates the individual complexes. When mitochondria were solubilized with DG, the supramolecular complexes such as III2IV2, III2IV and Vn could be detected with specific antibodies (Fig. 7a). Here, three bands of monomeric complex IV were detected using anti-Cox1 antibody, we will refer to these complexes as COA complexes for cytochrome c oxidase assembly intermediates. In S. cerevisiae, the intermediate-sized 250 kD, 300 kD and 450 kD complexes also represent complex IV subcomplexes40,46. The levels of supercomplexes III2IV2 and III2IV were found to be lower in ∆shy1 cells compared to WT cells, while the abundance of COA complexes increased (Fig. 7a), indicating that the formation of supercomplexes involving complex IV is influenced by deletion of shy1. In contrast, the abundance of III2 and V complexes was largely unchanged in ∆shy1 cells (Fig. 7a). Additionally, we assessed the enzyme activity of complex III in both WT and ∆shy1 cells to understand the impact of shy1 deletion on complex III. Our results revealed a slight decrease in complex III enzyme activity in ∆shy1 cells compared to WT cells (Supplementary Fig. S5 online), suggesting that shy1 affects complex III in ways other than its assembly. When DDM was used to solubilize the mitochondria, the dimeric complex III (III2) and monomeric complex IV (COA complexes) could be detected (Fig. 7b). The abundance of COA complexes was slightly diminished by shy1 deletion, which differed somewhat from the results seen with DG treatment, this discrepancy could be due to DDM being a more aggressive detergent compared to DG, leading to the degradation of some intermediates. Notably, the steady-state level of Cob1 was dramatically reduced (Fig. 5d) whereas the amount of dimer of complex III did not change (Fig. 7). This scenario can occur due to several reasons, the most likely reason is that the assembly of complex III might be regulated post-translationally, and these regulatory mechanisms could mitigate the effects of reduced Cob1 expression47. The similar principle applies to the degradation of Atp6, which does not affect the overall structure of ATP synthase. Overall, these data suggest that deletion of shy1 primarily affects the assembly of complex IV.

Fig. 7 Analysis of the assembly of ETC complex III and IV in ∆shy1 strain. (a, b). Mitochondria were extracted from WT and ∆shy1 strains. 2 mg mitochondria were solubilized by detergent DG, which maintains the supercomplexes, or DDM, which separates the individual complexes. 100 µg of protein per sample was loaded on a linear 3–12% (a) or 5–10% (b) gradient gel. The protein complexes were detected by BN-PAGE and Western blotting using indicated abs. Hsp60 was detected as a loading control. High molecular weight markers (kDa) are indicated. Cob1, Cox1 and Atp6 are the structural subunits of complex III, complex IV and ATP synthase respectively. COA complexes refer to the cytochrome c oxidase assembly intermediates. Original blots are presented in Supplementary Fig. S11-S12 online.

Discussion

Mitochondrial complex IV plays a vital role in electron transport and ATP synthesis. The composition of complex IV in both mammalian and yeast systems includes three core subunits encoded by mtDNA and eight to eleven subunits encoded by nDNA3,48. Leigh Syndrome, a rare neurometabolic disorder resulting from mutations in SURF1, is characterized by complex IV deficiency. LS has been linked to two distinct groups of SURF1 alleles15. In patients with LS, several missense mutations of SURF1 have been identified, including G124E, I246T, and Y274D. The homologs of SURF1 in other species have also been reported to be associated with the activity of complex IV.

S. cerevisiae SHY1 is able to suppress the complex IV deficient mutants and is identified as an essential IMM protein that is required for mitochondrial respiration17. The ΔSHY1 strain shows a growth defect on the glycerol carbon source. The null mutation of SHY1 results in the reduction of Cox1 protein level and the deficiency of complex IV. A Surf1 knock-out (KO) mouse displayed a complex IV activity less than 50% in different tissues49. The fruit fly homolog of human SURF1 in has also been studied. Adult flies with pan-neuronal knockdown (KD) exhibit brain-specific deficiency in cytochrome c oxidase, resulting in decreased locomotor ability, reduced excitatory junctional potential (EJP) brought on by nerve stimulation and compromised light responsiveness50.

In this study, we conducted a comprehensive characterization of the counterpart of the human Leigh syndrome-related protein SURF1 in S. pombe. To gain a better understanding of Shy1, we first conducted some bioinformatics analyses on this gene. TM-align25 based on TM-score indicated that S. pombe Shy1 is highly structurally similar to its homologs in human and S. cerevisiae and that they may perform similar functions. Protein sequence analysis predicted that Shy1 contains a SURF1 domain which is linked to the biogenesis of the complex IV, implying that Shy1 is probably involved in the process of assembling complex IV. Protein-protein interaction analysis suggested that Shy1 is likely to interact with some of the structural subunits and assembly factors of complex IV to function in the process of complex IV assembly.

To further investigate whether Shy1 participates in the assembly of complex IV by interacting with these proteins, immunoprecipitation assay was performed and our results showed that not all proteins predicted to interact with Shy1 show physical interactions with it, although the majority do, the possible reason might be the low expression levels of the certain proteins or their interactions with Shy1 are transient. Similar to its counterpart in S. cerevisiae, Shy1 physically interacts with the Cox1 translational regulators Mss51 and Cox1446. Surprisingly, Rip1, a structural subunit of complex III, is also identified in Shy1-FLAG eluate (Fig. 4a), suggesting that Shy1 might be involved in the formation of mitochondrial respiratory chain supercomplexes (complexes III and IV). Our results of BN-PAGE experiments further confirmed this finding (Fig. 7a). Collectively, our investigations elucidate that Shy1 is highly likely to play a pivotal role in the assembly of complex IV by interacting with its structural subunits and assembly factors in S. pombe. However, the specific mechanism of the assembly processes still remains to be determined.

To investigate whether deletion of shy1 affects the expression of the mitochondrial genome, we conducted a series of experiments focusing on various aspects of mitochondrial gene expression. Our results showed that Shy1 is required for mitochondrial respiration (Fig. 5a). Additionally, we observed a significant reduction in the RNA levels of core subunits of the ETC complexes and rRNA in ∆shy1 cells compared to that of WT cells (Fig. 5b). To identify the possible causes of this reduction, the mtDNA copy number was measured in both WT and ∆shy1 cells. Unexpectedly, the mtDNA copy number in ∆shy1 cells was found to be slightly higher than that in WT cells (Fig. 5c), which indicates that shy1 does not affect RNA levels by altering mtDNA copy number, instead, it is more likely to influence the RNA stability. The steady-state levels of proteins encoded by mtDNA were also decreased (Fig. 5d). Considering our BN-PAGE findings (Fig. 7a), it appears that the decrease in protein levels is most likely due to post-translational regulation rather than an issue with protein synthesis. Overall, our study reveals that shy1 affects mitochondrial function by influencing both the RNA and protein levels of mitochondrial gene expression.

The analysis of the assembly of ETC complexes and ATP synthase indicates that deletion of shy1 primarily affects the assembly of complex IV and that Shy1 is involved in the formation of supercomplexes containing complex IV (Fig. 7). From our BN-PAGE results, it is evident that deletion of shy1 does not affect the assembly of complex III. However, our enzyme activity assays indicate a decrease in complex III activity (Supplementary Fig. S5 online). This discrepancy could be due to compensatory mechanisms maintaining the assembly of complex, highlighting the complexity of the regulatory network involved in complex assembly. In addition, we observed that the steady-state levels of Cob1 and Atp6 were dramatically reduced (Fig. 5d) whereas the amount of the complex did not change (Fig. 7). This scenario can occur due to several reasons, the most likely reason is that the assembly of the complexes might be regulated post-translationally, and these regulatory mechanisms could mitigate the effects of reduced Cob1 or Atp6 expression47. Other reasons, such as compensatory mechanisms, which means the loss of subunits in respiratory complexes can be compensated by upregulating other subunits, can also contribute to this phenomenon51. In addition, the threshold effect in mitochondrial complex assembly can also explain this observation, it refers to the phenomenon where mitochondrial function can tolerate a reduction in the levels of specific subunits up to a certain critical point, beyond which the function significantly declines52. These potential explanations highlight the complexity of mitochondrial protein regulation and the need for further investigation.

Characterizing Shy1 in S. pombe provides significant insights into mitochondrial function due to its role as a homolog of human SURF1 and yeast SHY1, both crucial for the assembly of complex IV. Shy1 in S. pombe, unlike human SURF1 and S. cerevisiae SHY1, does not critically disrupt electron transport chain assembly when deleted, indicating the presence of compensatory mechanisms within S. pombe that ensure mitochondrial functionality. To further explore the specific compensatory mechanisms, RNA sequencing can be used to identify upregulated genes associated with mitochondrial complex assembly. In contrast, mutations in SURF1 lead to Leigh Syndrome in humans, characterized by severe reductions in complex IV activity, demonstrating its indispensable role in mitochondrial respiration53. Similarly, SHY1 is essential for complex IV assembly in S. cerevisiae, and its absence results in significant functional deficits and impaired respiration18. These differences underscore the importance of Shy1 characterization to understand the evolutionary conservation and functional flexibility within mitochondrial systems. By comparing Shy1 with its homologs, we can explore alternative assembly pathways and potential therapeutic targets for mitochondrial disorders, enhancing our grasp of mitochondrial biology and pathology, paving the way for novel strategies to address mitochondrial disorders54.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Acknowledgements

L.Y. discloses support for the research of this work from the Graduate Research and Innovation Program of Jiangsu Province (KYCX23_1732) and China Scholarship Council (No. 202306860040). H.Y. discloses support for the research of this work from the National Natural Science Foundation of China (31770810). We are grateful to Qinglong Yang, Xiao Yuan and Alia Ebrahim for their technical support on bioinformatics analysis in this study.

Author contributions

L.Y. designed the experiments. L. Y. and X. Y. performed the experiments. L.Y. and A.F. conducted bioinformatics analyses. L.Y. prepared the manuscript. L.Y., X. Y., A.F., F.G. and H.Y. read, reviewed and approved the final version of the manuscript and supervised this study.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Consent to participate

All authors read and approved the final version of the manuscript for publication.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Vercellino I Sazanov LA The assembly, regulation and function of the mitochondrial respiratory chain Nat Rev Mol Cell Biol. 2022 23 141 161 10.1038/s41580-021-00415-0 34621061
Vercellino, I. & Sazanov, L. A. The assembly, regulation and function of the mitochondrial respiratory chain. Nat Rev Mol Cell Biol. 23, 141–161 (2022).34621061
2. Rich PR Maréchal A The mitochondrial respiratory chain Essays Biochem. 2010 47 1 23 10.1042/bse0470001 20533897
Rich, P. R. & Maréchal, A. The mitochondrial respiratory chain. Essays Biochem. 47, 1–23 (2010).20533897
3. Brischigliaro M Zeviani M Cytochrome c oxidase deficiency Biochim. Biophys. Acta Bioenerg. 2021 1862 148335 10.1016/j.bbabio.2020.148335 33171185
Brischigliaro, M. & Zeviani, M. Cytochrome c oxidase deficiency. Biochim. Biophys. Acta Bioenerg. 1862, 148335 (2021).33171185
4. Tsukihara T The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A Science 1996 272 1136 1144 10.1126/science.272.5265.1136 8638158
Tsukihara, T. et al. The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science 272, 1136–1144 (1996).8638158
5. Pitceathly RDS Taanman JW NDUFA4 (Renamed COXFA4) is a cytochrome-c oxidase subunit Trends Endocrinol. Metab. 2018 29 452 454 10.1016/j.tem.2018.03.009 29636225
Pitceathly, R. D. S. & Taanman, J. W. NDUFA4 (Renamed COXFA4) is a cytochrome-c oxidase subunit. Trends Endocrinol. Metab. 29, 452–454 (2018).29636225
6. Zong S Structure of the intact 14-subunit human cytochrome c oxidase Cell Res. 2018 28 1026 1034 10.1038/s41422-018-0071-1 30030519
Zong, S. et al. Structure of the intact 14-subunit human cytochrome c oxidase. Cell Res. 28, 1026–1034 (2018).30030519
7. Fornuskova D Novel insights into the assembly and function of human nuclear-encoded cytochrome c oxidase subunits 4, 5a, 6a, 7a and 7b Biochem. J. 2010 428 363 374 10.1042/BJ20091714 20307258
Fornuskova, D. et al. Novel insights into the assembly and function of human nuclear-encoded cytochrome c oxidase subunits 4, 5a, 6a, 7a and 7b. Biochem. J. 428, 363–374 (2010).20307258
8. Harris MA Fission stories: Using PomBase to understand Schizosaccharomyces pombe biology Genetics 2022 2022 220
Harris, M. A. et al. Fission stories: Using PomBase to understand Schizosaccharomyces pombe biology. Genetics 2022, 220 (2022).
9. Signes A Fernandez-Vizarra E Assembly of mammalian oxidative phosphorylation complexes I-V and supercomplexes Essays Biochem. 2018 62 255 270 10.1042/EBC20170098 30030361
Signes, A. & Fernandez-Vizarra, E. Assembly of mammalian oxidative phosphorylation complexes I-V and supercomplexes. Essays Biochem. 62, 255–270 (2018).30030361
10. Stiburek L Hansikova H Tesarova M Cerna L Zeman J Biogenesis of eukaryotic cytochrome c oxidase Physiol. Res. 2006 55 Suppl 2 S27 41 10.33549/physiolres.930000.55.S2.27 17298220
Stiburek, L., Hansikova, H., Tesarova, M., Cerna, L. & Zeman, J. Biogenesis of eukaryotic cytochrome c oxidase. Physiol. Res. 55(Suppl 2), S27-41 (2006).17298220
11. Fernandez-Vizarra E Zeviani M Mitochondrial disorders of the OXPHOS system FEBS Lett. 2021 595 1062 1106 10.1002/1873-3468.13995 33159691
Fernandez-Vizarra, E. & Zeviani, M. Mitochondrial disorders of the OXPHOS system. FEBS Lett. 595, 1062–1106 (2021).33159691
12. Ghezzi D Zeviani M Human diseases associated with defects in assembly of OXPHOS complexes Essays Biochem. 2018 62 271 286 10.1042/EBC20170099 30030362
Ghezzi, D. & Zeviani, M. Human diseases associated with defects in assembly of OXPHOS complexes. Essays Biochem. 62, 271–286 (2018).30030362
13. Rak M Mitochondrial cytochrome c oxidase deficiency Clin. Sci. (Lond). 2016 130 393 407 10.1042/CS20150707 26846578
Rak, M. et al. Mitochondrial cytochrome c oxidase deficiency. Clin. Sci. (Lond). 130, 393–407 (2016).26846578
14. Tiranti V Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency Am. J. Hum. Genet. 1998 63 1609 1621 10.1086/302150 9837813
Tiranti, V. et al. Mutations of SURF-1 in Leigh disease associated with cytochrome c oxidase deficiency. Am. J. Hum. Genet. 63, 1609–1621 (1998).9837813
15. Zhu Z SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome Nat. Genet. 1998 20 337 343 10.1038/3804 9843204
Zhu, Z. et al. SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome. Nat. Genet. 20, 337–343 (1998).9843204
16. Poyau A Missense mutations in SURF1 associated with deficient cytochrome c oxidase assembly in Leigh syndrome patients Hum. Genet. 2000 106 194 205 10746561
Poyau, A. et al. Missense mutations in SURF1 associated with deficient cytochrome c oxidase assembly in Leigh syndrome patients. Hum. Genet. 106, 194–205 (2000).10746561
17. Mashkevich G Repetto B Glerum DM Jin C Tzagoloff A SHY1, the yeast homolog of the mammalian SURF-1 gene, encodes a mitochondrial protein required for respiration J. Biol. Chem. 1997 272 14356 14364 10.1074/jbc.272.22.14356 9162072
Mashkevich, G., Repetto, B., Glerum, D. M., Jin, C. & Tzagoloff, A. SHY1, the yeast homolog of the mammalian SURF-1 gene, encodes a mitochondrial protein required for respiration. J. Biol. Chem. 272, 14356–14364 (1997).9162072
18. Barrientos A Korr D Tzagoloff A Shy1p is necessary for full expression of mitochondrial COX1 in the yeast model of Leigh’s syndrome EMBO J. 2002 21 43 52 10.1093/emboj/21.1.43 11782424
Barrientos, A., Korr, D. & Tzagoloff, A. Shy1p is necessary for full expression of mitochondrial COX1 in the yeast model of Leigh’s syndrome. EMBO J. 21, 43–52 (2002).11782424
19. Nijtmans LG Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast FEBS Lett. 2001 498 46 51 10.1016/S0014-5793(01)02447-4 11389896
Nijtmans, L. G. et al. Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast. FEBS Lett. 498, 46–51 (2001).11389896
20. Ling Q Rioux M Hu Y Lee M Gray SJ Adeno-associated viral vector serotype 9-based gene replacement therapy for SURF1-related Leigh syndrome Mol. Ther. Methods Clin. Dev. 2021 23 158 168 10.1016/j.omtm.2021.09.001 34703839
Ling, Q., Rioux, M., Hu, Y., Lee, M. & Gray, S. J. Adeno-associated viral vector serotype 9-based gene replacement therapy for SURF1-related Leigh syndrome. Mol. Ther. Methods Clin. Dev. 23, 158–168 (2021).34703839
21. Baden KN Murray J Capaldi RA Guillemin K Early developmental pathology due to cytochrome c oxidase deficiency is revealed by a new zebrafish model J. Biol. Chem. 2007 282 34839 34849 10.1074/jbc.M703528200 17761683
Baden, K. N., Murray, J., Capaldi, R. A. & Guillemin, K. Early developmental pathology due to cytochrome c oxidase deficiency is revealed by a new zebrafish model. J. Biol. Chem. 282, 34839–34849 (2007).17761683
22. Moreno S Klar A Nurse P Molecular genetic analysis of fission yeast Schizosaccharomyces pombe Methods Enzymol. 1991 194 795 823 10.1016/0076-6879(91)94059-L 2005825
Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).2005825
23. Malecki M Functional and regulatory profiling of energy metabolism in fission yeast Genome Biol. 2016 17 240 10.1186/s13059-016-1101-2 27887640
Malecki, M. et al. Functional and regulatory profiling of energy metabolism in fission yeast. Genome Biol. 17, 240 (2016).27887640
24. Jumper J Highly accurate protein structure prediction with AlphaFold Nature 2021 596 583 589 10.1038/s41586-021-03819-2 34265844
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).34265844
25. Varadi M AlphaFold Protein Structure Database: Massively expanding the structural coverage of protein-sequence space with high-accuracy models Nucleic Acids Res. 2022 50 D439 d444 10.1093/nar/gkab1061 34791371
Varadi, M. et al. AlphaFold Protein Structure Database: Massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 50, D439-d444 (2022).34791371
26. Zhang Y Skolnick J TM-align: A protein structure alignment algorithm based on the TM-score Nucleic Acids Res. 2005 33 2302 2309 10.1093/nar/gki524 15849316
Zhang, Y. & Skolnick, J. TM-align: A protein structure alignment algorithm based on the TM-score. Nucleic Acids Res. 33, 2302–2309 (2005).15849316
27. Edgar RC MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucleic Acids Res. 2004 32 1792 1797 10.1093/nar/gkh340 15034147
Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).15034147
28. Stecher G Tamura K Kumar S Molecular evolutionary genetics analysis (MEGA) for macOS Mol. Biol. Evol. 2020 37 1237 1239 10.1093/molbev/msz312 31904846
Stecher, G., Tamura, K. & Kumar, S. Molecular evolutionary genetics analysis (MEGA) for macOS. Mol. Biol. Evol. 37, 1237–1239 (2020).31904846
29. Robert X Gouet P Deciphering key features in protein structures with the new ENDscript server Nucleic Acids Res. 2014 42 W320 W324 10.1093/nar/gku316 24753421
Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).24753421
30. Fukasawa Y MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites Mol. Cell Proteom. 2015 14 1113 1126 10.1074/mcp.M114.043083
Fukasawa, Y. et al. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites. Mol. Cell Proteom. 14, 1113–1126 (2015).
31. Meisinger C Pfanner N Truscott KN Isolation of yeast mitochondria Methods Mol. Biol. 2006 313 33 39 16118422
Meisinger, C., Pfanner, N. & Truscott, K. N. Isolation of yeast mitochondria. Methods Mol. Biol. 313, 33–39 (2006).16118422
32. Meisinger C Sommer T Pfanner N Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations Anal. Biochem. 2000 287 339 342 10.1006/abio.2000.4868 11112284
Meisinger, C., Sommer, T. & Pfanner, N. Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations. Anal. Biochem. 287, 339–342 (2000).11112284
33. Diekert K de Kroon AI Kispal G Lill R Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae Methods Cell Biol. 2001 65 37 51 10.1016/S0091-679X(01)65003-9 11381604
Diekert, K., de Kroon, A. I., Kispal, G. & Lill, R. Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae. Methods Cell Biol. 65, 37–51 (2001).11381604
34. Lemaire C Dujardin G Preparation of respiratory chain complexes from Saccharomyces cerevisiae wild-type and mutant mitochondria : Activity measurement and subunit composition analysis Methods Mol. Biol. 2008 432 65 81 10.1007/978-1-59745-028-7_5 18370011
Lemaire, C. & Dujardin, G. Preparation of respiratory chain complexes from Saccharomyces cerevisiae wild-type and mutant mitochondria : Activity measurement and subunit composition analysis. Methods Mol. Biol. 432, 65–81 (2008).18370011
35. Matsuo Y Asakawa K Toda T Katayama S A rapid method for protein extraction from fission yeast Biosci. Biotechnol. Biochem. 2006 70 1992 1994 10.1271/bbb.60087 16926515
Matsuo, Y., Asakawa, K., Toda, T. & Katayama, S. A rapid method for protein extraction from fission yeast. Biosci. Biotechnol. Biochem. 70, 1992–1994 (2006).16926515
36. Luo Y Schizosaccharomyces pombe Mti2 and Mti3 act in conjunction during mitochondrial translation initiation FEBS J. 2019 286 4542 4553 10.1111/febs.15021 31350787
Luo, Y. et al. Schizosaccharomyces pombe Mti2 and Mti3 act in conjunction during mitochondrial translation initiation. FEBS J. 286, 4542–4553 (2019).31350787
37. Brown RM Brown GK Complementation analysis of systemic cytochrome oxidase deficiency presenting as Leigh syndrome J. Inherit. Metab. Dis. 1996 19 752 760 10.1007/BF01799168 8982948
Brown, R. M. & Brown, G. K. Complementation analysis of systemic cytochrome oxidase deficiency presenting as Leigh syndrome. J. Inherit. Metab. Dis. 19, 752–760 (1996).8982948
38. Fontanesi F Jin C Tzagoloff A Barrientos A Transcriptional activators HAP/NF-Y rescue a cytochrome c oxidase defect in yeast and human cells Hum. Mol Genet. 2008 17 775 788 10.1093/hmg/ddm349 18045776
Fontanesi, F., Jin, C., Tzagoloff, A. & Barrientos, A. Transcriptional activators HAP/NF-Y rescue a cytochrome c oxidase defect in yeast and human cells. Hum. Mol Genet. 17, 775–788 (2008).18045776
39. Teraoka M Two novel mutations of SURF1 in Leigh syndrome with cytochrome c oxidase deficiency Hum. Genet. 1999 105 560 563 10.1007/s004399900191 10647889
Teraoka, M. et al. Two novel mutations of SURF1 in Leigh syndrome with cytochrome c oxidase deficiency. Hum. Genet. 105, 560–563 (1999).10647889
40. Barrientos A Zambrano A Tzagoloff A Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae EMBO J. 2004 23 3472 3482 10.1038/sj.emboj.7600358 15306853
Barrientos, A., Zambrano, A. & Tzagoloff, A. Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae. EMBO J. 23, 3472–3482 (2004).15306853
41. Perez-Martinez X Broadley SA Fox TD Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p EMBO J. 2003 22 5951 5961 10.1093/emboj/cdg566 14592991
Perez-Martinez, X., Broadley, S. A. & Fox, T. D. Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p. EMBO J. 22, 5951–5961 (2003).14592991
42. Zhao Z Su W Yuan S Huang Y Functional conservation of tRNase ZL among Saccharomyces cerevisiae,Schizosaccharomyces pombe and humans Biochem. J. 2009 422 483 492 10.1042/BJ20090743 19555350
Zhao, Z., Su, W., Yuan, S. & Huang, Y. Functional conservation of tRNase ZL among Saccharomyces cerevisiae, Schizosaccharomyces pombe and humans. Biochem. J. 422, 483–492 (2009).19555350
43. Zuin A Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast PLoS ONE 2008 3 e2842 10.1371/journal.pone.0002842 18665268
Zuin, A. et al. Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast. PLoS ONE 3, e2842 (2008).18665268
44. Wang Y The Schizosaccharomyces pombe PPR protein Ppr10 associates with a novel protein Mpa1 and acts as a mitochondrial translational activator Nucleic Acids Res. 2017 45 3323 3340 10.1093/nar/gkx127 28334955
Wang, Y. et al. The Schizosaccharomyces pombe PPR protein Ppr10 associates with a novel protein Mpa1 and acts as a mitochondrial translational activator. Nucleic Acids Res. 45, 3323–3340 (2017).28334955
45. Mick DU Coa3 and Cox14 are essential for negative feedback regulation of COX1 translation in mitochondria J. Cell Biol. 2010 191 141 154 10.1083/jcb.201007026 20876281
Mick, D. U. et al. Coa3 and Cox14 are essential for negative feedback regulation of COX1 translation in mitochondria. J. Cell Biol. 191, 141–154 (2010).20876281
46. Mick DU Shy1 couples Cox1 translational regulation to cytochrome c oxidase assembly EMBO J. 2007 26 4347 4358 10.1038/sj.emboj.7601862 17882259
Mick, D. U. et al. Shy1 couples Cox1 translational regulation to cytochrome c oxidase assembly. EMBO J. 26, 4347–4358 (2007).17882259
47. Pagliarini DJ Rutter J Hallmarks of a new era in mitochondrial biochemistry Genes Dev. 2013 27 2615 2627 10.1101/gad.229724.113 24352419
Pagliarini, D. J. & Rutter, J. Hallmarks of a new era in mitochondrial biochemistry. Genes Dev. 27, 2615–2627 (2013).24352419
48. Watson SA McStay GP Functions of cytochrome c oxidase assembly factors Int. J. Mol. Sci. 2020 2020 21
Watson, S. A. & McStay, G. P. Functions of cytochrome c oxidase assembly factors. Int. J. Mol. Sci. 2020, 21 (2020).
49. Dell’agnello C Increased longevity and refractoriness to Ca(2+)-dependent neurodegeneration in Surf1 knockout mice Hum. Mol. Genet. 2007 16 431 444 10.1093/hmg/ddl477 17210671
Dell’agnello, C. et al. Increased longevity and refractoriness to Ca(2+)-dependent neurodegeneration in Surf1 knockout mice. Hum. Mol. Genet. 16, 431–444 (2007).17210671
50. Da-Rè C Leigh syndrome in Drosophila melanogaster: Morphological and biochemical characterization of Surf1 post-transcriptional silencing J. Biol. Chem. 2014 289 29235 29246 10.1074/jbc.M114.602938 25164807
Da-Rè, C. et al. Leigh syndrome in Drosophila melanogaster: Morphological and biochemical characterization of Surf1 post-transcriptional silencing. J. Biol. Chem. 289, 29235–29246 (2014).25164807
51. Kühn K Complete mitochondrial complex I deficiency induces an up-regulation of respiratory fluxes that is abolished by traces of functional complex I Plant Physiol. 2015 168 1537 1549 10.1104/pp.15.00589 26134164
Kühn, K. et al. Complete mitochondrial complex I deficiency induces an up-regulation of respiratory fluxes that is abolished by traces of functional complex I. Plant Physiol. 168, 1537–1549 (2015).26134164
52. Acín-Pérez R Respiratory complex III is required to maintain complex I in mammalian mitochondria Mol. Cell. 2004 13 805 815 10.1016/S1097-2765(04)00124-8 15053874
Acín-Pérez, R. et al. Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol. Cell. 13, 805–815 (2004).15053874
53. Lee IC Chiang KL Clinical diagnosis and treatment of leigh syndrome based on SURF1: Genotype and phenotype Antioxidants Basel 2021 2021 10
Lee, I. C. & Chiang, K. L. Clinical diagnosis and treatment of leigh syndrome based on SURF1: Genotype and phenotype. Antioxidants Basel 2021, 10 (2021).
54. Rahman S Leigh syndrome: Clinical features and biochemical and DNA abnormalities Ann. Neurol. 1996 39 343 351 10.1002/ana.410390311 8602753
Rahman, S. et al. Leigh syndrome: Clinical features and biochemical and DNA abnormalities. Ann. Neurol. 39, 343–351 (1996).8602753
55. Kumar S Stecher G Li M Knyaz C Tamura K MEGA X: Molecular evolutionary genetics analysis across computing platforms Mol. Biol. Evol. 2018 35 1547 1549 10.1093/molbev/msy096 29722887
Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549 (2018).29722887
