Publications by authors named "Rosemary A Stuart"

24 Publications

  • Page 1 of 1

Distinct Roles of Mitochondrial HIGD1A and HIGD2A in Respiratory Complex and Supercomplex Biogenesis.

Cell Rep 2020 05;31(5):107607

Department of Neurology, University of Miami Miller School of Medicine, Miami, FL 33136, USA. Electronic address:

The mitochondrial respiratory chain enzymes are organized as individual complexes and supercomplexes, whose biogenesis remains to be fully understood. To disclose the role of the human Hypoxia Inducible Gene Domain family proteins HIGD1A and HIGD2A in these processes, we generate and characterize HIGD-knockout (KO) cell lines. We show that HIGD2A controls and coordinates the modular assembly of isolated and supercomplexed complex IV (CIV) by acting on the COX3 assembly module. In contrast, HIGD1A regulates CIII and CIII-containing supercomplex biogenesis by supporting the incorporation of UQCRFS1. HIGD1A also clusters with COX4-1 and COX5A CIV subunits and, when overexpressed, suppresses the CIV biogenesis defect of HIGD2A-KO cells. We conclude that HIGD1A and HIGD2A have both independent and overlapping functions in the biogenesis of respiratory complexes and supercomplexes. Our data illuminate the existence of multiple pathways to assemble these structures by dynamic HIGD-mediated CIV biogenesis, potentially to adapt to changing environmental and nutritional conditions.
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http://dx.doi.org/10.1016/j.celrep.2020.107607DOI Listing
May 2020

Hypoxia-inducible gene domain 1 proteins in yeast mitochondria protect against proton leak through complex IV.

J Biol Chem 2019 11 7;294(46):17669-17677. Epub 2019 Oct 7.

Department of Cell and Molecular Biology, University of Mississippi Medical Center, Jackson, Mississippi 39216

Hypoxia-inducible gene domain 1 (HIGD1) proteins are small integral membrane proteins, conserved from bacteria to humans, that associate with oxidative phosphorylation supercomplexes. Using yeast as a model organism, we have shown previously that its two HIGD1 proteins, Rcf1 and Rcf2, are required for the generation and maintenance of a normal membrane potential (ΔΨ) across the inner mitochondrial membrane (IMM). We postulated that the lower ΔΨ observed in the absence of the HIGD1 proteins may be due to decreased proton pumping by complex IV (CIV) or enhanced leak of protons across the IMM. Here we measured the ΔΨ generated by complex III (CIII) to discriminate between these possibilities. First, we found that the decreased ΔΨ observed in the absence of the HIGD1 proteins cannot be due to decreased proton pumping by CIV because CIII, operating alone, also exhibited a decreased ΔΨ when HIGD1 proteins were absent. Because CIII can neither lower its pumping stoichiometry nor transfer protons completely across the IMM, this result indicates that HIGD1 protein ablation enhances proton leak across the IMM. Second, we demonstrate that this proton leak occurs through CIV because ΔΨ generation by CIII is restored when CIV is removed from the cell. Third, the proton leak appeared to take place through an inactive population of CIV that accumulates when HIGD1 proteins are absent. We conclude that HIGD1 proteins in yeast prevent CIV inactivation, likely by preventing the loss of lipids bound within the Cox3 protein of CIV.
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http://dx.doi.org/10.1074/jbc.RA119.010317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873203PMC
November 2019

The yeast mitochondrial proteins Rcf1 and Rcf2 support the enzymology of the cytochrome oxidase complex and generation of the proton motive force.

J Biol Chem 2019 03 25;294(13):4867-4877. Epub 2019 Jan 25.

From the Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233 and

The yeast mitochondrial proteins Rcf1 and Rcf2 are associated with a subpopulation of the cytochrome -cytochrome oxidase supercomplex and have been proposed to play a role in the assembly and/or modulation of the activity of the cytochrome oxidase (complex IV, CIV). Yeast mutants deficient in either Rcf1 or Rcf2 proteins can use aerobic respiration-based metabolism for growth, but the absence of both proteins results in a strong growth defect. In this study, using assorted biochemical and biophysical analyses of Rcf1/Rcf2 single and double null-mutant yeast cells and mitochondria, we further explored how Rcf1 and Rcf2 support aerobic respiration and growth. We show that the absence of Rcf1 physically reduces the levels of CIV and diminishes the ability of the CIV that is present to maintain a normal mitochondrial proton motive force (PMF). Although the absence of Rcf2 did not noticeably affect the physical content of CIV, the PMF generated by CIV was also lower than normal. Our results indicate that the detrimental effects of the absence of Rcf1 and Rcf2 proteins on the CIV complex are distinct in terms of CIV assembly/accumulation and additive in terms of the ability of CIV to generate PMF. Thus, the combined absence of Rcf1 and Rcf2 alters both CIV physiology and assembly. We conclude that the slow aerobic growth of the Rcf1/Rcf2 double null mutant results from diminished generation of mitochondrial PMF by CIV and limits the level of CIV activity required for maintenance of the PMF and growth under aerobic conditions.
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http://dx.doi.org/10.1074/jbc.RA118.006888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442066PMC
March 2019

MrpL35, a mitospecific component of mitoribosomes, plays a key role in cytochrome oxidase assembly.

Mol Biol Cell 2017 Nov 20;28(24):3489-3499. Epub 2017 Sep 20.

Department of Biological Sciences, Marquette University, Milwaukee, WI 53233

Mitoribosomes perform the synthesis of the core components of the oxidative phosphorylation (OXPHOS) system encoded by the mitochondrial genome. We provide evidence that MrpL35 (mL38), a mitospecific component of the yeast mitoribosomal central protuberance, assembles into a subcomplex with MrpL7 (uL5), Mrp7 (bL27), and MrpL36 (bL31) and mitospecific proteins MrpL17 (mL46) and MrpL28 (mL40). We isolated respiratory defective mutant yeast strains, which do not display an overall inhibition in mitochondrial protein synthesis but rather have a problem in cytochrome oxidase complex (COX) assembly. Our findings indicate that MrpL35, with its partner Mrp7, play a key role in coordinating the synthesis of the Cox1 subunit with its assembly into the COX enzyme and in a manner that involves the Cox14 and Coa3 proteins. We propose that MrpL35 and Mrp7 are regulatory subunits of the mitoribosome acting to coordinate protein synthesis and OXPHOS assembly events and thus the bioenergetic capacity of the mitochondria.
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http://dx.doi.org/10.1091/mbc.E17-04-0239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5683760PMC
November 2017

Mutational Analysis of the QRRQ Motif in the Yeast Hig1 Type 2 Protein Rcf1 Reveals a Regulatory Role for the Cytochrome Oxidase Complex.

J Biol Chem 2017 03 6;292(13):5216-5226. Epub 2017 Feb 6.

From the Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233 and

The yeast Rcf1 protein is a member of the conserved family of proteins termed the hypoxia-induced gene (domain) 1 (Hig1 or HIGD1) family. Rcf1 interacts with components of the mitochondrial oxidative phosphorylation system, in particular the cytochrome (complex III)-cytochrome oxidase (complex IV) supercomplex (termed III-IV) and the ADP/ATP carrier proteins. Rcf1 plays a role in the assembly and modulation of the activity of complex IV; however, the molecular basis for how Rcf1 influences the activity of complex IV is currently unknown. Hig1 type 2 isoforms, which include the Rcf1 protein, are characterized in part by the presence of a conserved motif, (Q/I)(R/H)RQ, termed here the QRRQ motif. We show that mutation of conserved residues within the Rcf1 QRRQ motif alters the interactions between Rcf1 and partner proteins and results in the destabilization of complex IV and alteration of its enzymatic properties. Our findings indicate that Rcf1 does not serve as a stoichiometric component, as a subunit of complex IV, to support its activity. Rather, we propose that Rcf1 serves to dynamically interact with complex IV during its assembly process and, in doing so, regulates a late maturation step of complex IV. We speculate that the Rcf1/Hig1 proteins play a role in the incorporation and/or remodeling of lipids, in particular cardiolipin, into complex IV and. possibly, other mitochondrial proteins such as ADP/ATP carrier proteins.
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http://dx.doi.org/10.1074/jbc.M116.758045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392669PMC
March 2017

Supercomplex-associated Cox26 protein binds to cytochrome c oxidase.

Biochim Biophys Acta 2016 Jul 16;1863(7 Pt A):1643-52. Epub 2016 Apr 16.

Molekulare Bioenergetik, Zentrum der Biologischen Chemie, Cluster of Excellence Frankfurt Macromolecular Complexes Goethe-Universität Frankfurt, D-60590 Frankfurt, Germany; Functional Proteomics, Institute of Biochemistry I, Faculty of Medicine, Goethe-University of Frankfurt, D-60590 Frankfurt, Germany. Electronic address:

Here we identified a hydrophobic 6.4kDa protein, Cox26, as a novel component of yeast mitochondrial supercomplex comprising respiratory complexes III and IV. Multi-dimensional native and denaturing electrophoretic techniques were used to identify proteins interacting with Cox26. The majority of the Cox26 protein was found non-covalently bound to the complex IV moiety of the III-IV supercomplexes. A population of Cox26 was observed to exist in a disulfide bond partnership with the Cox2 subunit of complex IV. No pronounced growth phenotype for Cox26 deficiency was observed, indicating that Cox26 may not play a critical role in the COX enzymology, and we speculate that Cox26 may serve to regulate or support the Cox2 protein. Respiratory supercomplexes are assembled in the absence of the Cox26 protein, however their pattern slightly differs to the wild type III-IV supercomplex appearance. The catalytic activities of complexes III and IV were observed to be normal and respiration was comparable to wild type as long as cells were cultivated under normal growth conditions. Stress conditions, such as elevated temperatures resulted in mild decrease of respiration in non-fermentative media when the Cox26 protein was absent.
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http://dx.doi.org/10.1016/j.bbamcr.2016.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140176PMC
July 2016

Rcf1 and Rcf2, members of the hypoxia-induced gene 1 protein family, are critical components of the mitochondrial cytochrome bc1-cytochrome c oxidase supercomplex.

Mol Cell Biol 2012 Apr 6;32(8):1363-73. Epub 2012 Feb 6.

Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin, USA.

We report that Rcf1 (formerly Aim31), a member of the conserved hypoxia-induced gene 1 (Hig1) protein family, represents a novel component of the yeast cytochrome bc(1)-cytochrome c oxidase (COX) supercomplex. Rcf1 (respiratory supercomplex factor 1) partitions with the COX complex, and evidence that it may act as a bridge to the cytochrome bc(1) complex is presented. Rcf1 interacts with the Cox3 subunit and can do so prior to their assembly into the COX complex. A close proximity of Rcf1 and members of the ADP/ATP carrier (AAC) family was also established. Rcf1 displays overlapping function with another Hig1-related protein, Rcf2 (formerly Aim38), and their joint presence is required for optimal COX enzyme activity and the correct assembly of the cytochrome bc(1)-COX supercomplex. Rcf1 and Rcf2 can independently associate with the cytochrome bc(1)-COX supercomplex, indicating that at least two forms of this supercomplex exist within mitochondria. We provide evidence that the association with the cytochrome bc(1)-COX supercomplex and regulation of the COX complex are a conserved feature of Hig1 family members. Based on our findings, we propose a model where the Hig1 proteins regulate the COX enzyme activity through Cox3 and associated Cox12 protein, in a manner that may be influenced by the neighboring AAC proteins.
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http://dx.doi.org/10.1128/MCB.06369-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318584PMC
April 2012

Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria.

EMBO Rep 2011 Sep 1;12(9):950-5. Epub 2011 Sep 1.

Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233, USA.

Mitochondrial ribosomal protein 20 (Mrp20) is a component of the yeast mitochondrial large (54S) ribosomal subunit and is homologous to the bacterial L23 protein, located at the ribosomal tunnel exit site. The carboxy-terminal mitochondrial-specific domain of Mrp20 was found to have a crucial role in the assembly of the ribosomes. A new, membrane-bound, ribosomal-assembly subcomplex composed of known tunnel-exit-site proteins, an uncharacterized ribosomal protein, MrpL25, and the mitochondrial peroxiredoxin (Prx), Prx1, accumulates in an mrp20ΔC yeast mutant. Finally, data supporting the idea that the inner mitochondrial membrane acts as a platform for the ribosome assembly process are discussed.
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http://dx.doi.org/10.1038/embor.2011.133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3166459PMC
September 2011

Mapping of the Saccharomyces cerevisiae Oxa1-mitochondrial ribosome interface and identification of MrpL40, a ribosomal protein in close proximity to Oxa1 and critical for oxidative phosphorylation complex assembly.

Eukaryot Cell 2009 Nov 25;8(11):1792-802. Epub 2009 Sep 25.

Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233, USA.

The Oxa1 protein plays a central role in facilitating the cotranslational insertion of the nascent polypeptide chains into the mitochondrial inner membrane. Mitochondrially encoded proteins are synthesized on matrix-localized ribosomes which are tethered to the inner membrane and in physical association with the Oxa1 protein. In the present study we used a chemical cross-linking approach to map the Saccharomyces cerevisiae Oxa1-ribosome interface, and we demonstrate here a close association of Oxa1 and the large ribosomal subunit protein, MrpL40. Evidence to indicate that a close physical and functional relationship exists between MrpL40 and another large ribosomal protein, the Mrp20/L23 protein, is also provided. MrpL40 shares sequence features with the bacterial ribosomal protein L24, which like Mrp20/L23 is known to be located adjacent to the ribosomal polypeptide exit site. We propose therefore that MrpL40 represents the Saccharomyces cerevisiae L24 homolog. MrpL40, like many mitochondrial ribosomal proteins, contains a C-terminal extension region that bears no similarity to the bacterial counterpart. We show that this C-terminal mitochondria-specific region is important for MrpL40's ability to support the synthesis of the correct complement of mitochondrially encoded proteins and their subsequent assembly into oxidative phosphorylation complexes.
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http://dx.doi.org/10.1128/EC.00219-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2772399PMC
November 2009

Chapter 11 Supercomplex organization of the yeast respiratory chain complexes and the ADP/ATP carrier proteins.

Methods Enzymol 2009 ;456:191-208

Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin, USA.

The enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) are coassembled into higher ordered supercomplexes within the mitochondrial inner membrane. The cytochrome bc(1)-cytochrome c oxidase (COX) supercomplex is formed by the coassociation of the two electron transport chain complexes, the cytochrome bc(1) (cytochrome c reductase) and the COX complex. Recent evidence indicates that a diversity in the populations of the cytochrome bc(1)-COX supercomplexes exists within the mitochondria, because different subpopulations of this supercomplex have been shown to further interact with distinct partner complexes (e.g., the TIM23 machinery and also the Shy1/Cox14 proteins). By use of native gel electrophoresis and affinity purification approaches, the abundant ADP/ATP carrier protein (AAC) isoform in the yeast Saccharomyces cerevisiae, the Aac2 isoform, has recently been found to also exist in physical association with the cytochrome bc(1)-COX supercomplex and its associated TIM23 machinery. The AAC proteins play a central role in cellular metabolism, because they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. The method used to analyze the cytochrome bc(1)-COX-AAC supercomplex and to affinity purify the Aac2 isoform and its associating proteins from S. cerevisiae mitochondria will be outlined in this chapter.
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http://dx.doi.org/10.1016/S0076-6879(08)04411-XDOI Listing
June 2009

Supercomplex organization of the oxidative phosphorylation enzymes in yeast mitochondria.

J Bioenerg Biomembr 2008 Oct 7;40(5):411-7. Epub 2008 Oct 7.

Dept of Biological Sciences, Marquette University, Milwaukee, WI 53233, USA.

Accumulating evidence indicates that the enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) are co-assembled into higher-ordered supercomplexes within the mitochondrial inner membrane. This review will focus largely on the OXPHOS supercomplexes of the yeast Saccharomyces cerevisiae. The recent evidence to indicate that diversity in the populations of the cytochrome bc (1)-COX supercomplexes exist shall be outlined. In addition, the existence of dimeric/oligomeric F(1)F(o)-ATP synthase complexes and their proposed role in establishment of the cristae architecture of the inner mitochondrial membrane shall also be discussed.
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http://dx.doi.org/10.1007/s10863-008-9168-4DOI Listing
October 2008

The yeast Aac2 protein exists in physical association with the cytochrome bc1-COX supercomplex and the TIM23 machinery.

Mol Biol Cell 2008 Sep 9;19(9):3934-43. Epub 2008 Jul 9.

Department of Biological Sciences, Marquette University, Milwaukee, WI 53233, USA.

The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (Saccharomyces cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome c reductase (cytochrome bc(1))-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity-purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc(1)-COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc(1)-COX supercomplex is perturbed, whereby a decrease in the III(2)-IV(2) assembly state relative to the III(2)-IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc(1)-COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc(1)-COX-TIM23 supercomplex are also discussed.
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http://dx.doi.org/10.1091/mbc.e08-04-0402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2526699PMC
September 2008

In vitro analysis of yeast mitochondrial protein import.

Curr Protoc Cell Biol 2007 Mar;Chapter 11:Unit 11.19

Marquette University, Milwaukee, Wisconsin, USA.

This unit describes methods for importing in vitro-translated or recombinant proteins into isolated yeast mitochondria and for exporting mitochondrial proteins translated in the yeast mitochondrial matrix into the inner mitochondrial membrane. The methods use mitochondria isolated from yeast cells and mitochondrial protein precursors derived from an in vitro transcription/translation reaction or purified from an E. coli recombinant protein expression system. The described translocation assays can be used to determine whether a protein is targeted to mitochondria and its location within the mitochondrion. It can also be used to study the import mechanism and to investigate mitochondrial matrix translation of proteins and their export.
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http://dx.doi.org/10.1002/0471143030.cb1119s34DOI Listing
March 2007

Structural characterization of the transmembrane domain from subunit e of yeast F1Fo-ATP synthase: a helical GXXXG motif located just under the micelle surface.

Biochemistry 2008 Feb 26;47(7):1910-7. Epub 2008 Jan 26.

Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, Wisconsin 53201-1881, USA.

F1Fo-ATP synthase is a large multiprotein complex, including at least 10 subunits in the membrane-bound Fo-sector. One of these Fo proteins is subunit e (Su e), involved in the stable dimerization of F1Fo-ATP synthase, and required for the establishment of normal cristae membrane architecture. As a step toward enabling structure-function studies of the Fo-sector, the Su e transmembrane region was structurally characterized in micelles. Based on a series of NMR and CD (circular dichroism) studies, a structural model of the Su e/micelle complex was constructed, indicating Su e is largely helical, and emerges from the micelle with Arg20 near the phosphate head groups. Su e only adopts this folded conformation in the context of the micelle, and is essentially disordered in DMSO, water or trifluoroethanol/water. Within the micelle the C-terminal Ala10-Arg20 stretch is helical, while the region N-terminal may be transiently helical, based on negative CSI (chemical shift index) values. The Ala10-Arg20 helix contains the G14XXXG18 motif, which has been proposed to play an important role in dimer formation with another protein from the Fo-sector. The Gly on the C-terminal end of this motif (Gly18) is slightly more mobile than the more buried Gly14, based on NMR order parameter measurements (Gly14 S2 = 0.950; Gly18 S2 = 0.895). Only one Su e transmembrane peptide is bound per micelle, and micelles are 22-23 A in diameter, composed of 51 +/- 4 dodecylphosphocholine detergent molecules. Although there is no evidence for Su e homodimerization via the transmembrane domain, potentially synergistic roles for N-terminal (membrane) and C-terminal (soluble) domain interactions may still occur. Furthermore, the presence of a buried charged residue (Arg7) suggests there may be interactions with other Fo-sector protein(s) that stabilize this charge, and possibly drive the folding of the N-terminal 9 residues of the transmembrane domain.
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http://dx.doi.org/10.1021/bi7015475DOI Listing
February 2008

The F1F0-ATP synthase complex influences the assembly state of the cytochrome bc1-cytochrome oxidase supercomplex and its association with the TIM23 machinery.

J Biol Chem 2008 Mar 10;283(11):6677-86. Epub 2008 Jan 10.

Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233, USA.

The enzyme complexes involved in mitochondrial oxidative phosphorylation are organized into higher ordered assemblies termed supercomplexes. Subunits e and g (Su e and Su g, respectively) are catalytically nonessential subunits of the F1F0-ATP synthase whose presence is required to directly support the stable dimerization of the ATP synthase complex. We report here that Su g and Su e are also important for securing the correct organizational state of the cytochrome bc1-cytochrome oxidase (COX) supercomplex. Mitochondria isolated from the Delta su e and Delta su g null mutant strains exhibit decreased levels of COX enzyme activity but appear to have normal COX subunit protein levels. An altered stoichiometry of the cytochrome bc1-COX supercomplex was observed in mitochondria deficient in Su e and/or Su g, and a perturbation in the association of Cox4, a catalytically important subunit of the COX complex, was also detected. In addition, an increase in the level of the TIM23 translocase associated with the cytochrome bc1-COX supercomplex is observed in the absence of Su e and Su g. Together, our data highlight that a further level of complexity exists between the oxidative phosphorylation supercomplexes, whereby the organizational state of one complex, i.e. the ATP synthase, may influence that of another supercomplex, namely the cytochrome bc1-COX complex.
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http://dx.doi.org/10.1074/jbc.M708440200DOI Listing
March 2008

Mitochondrial biogenesis: is an old dog still teaching us new tricks? Meeting on the Assembly of the Mitochondrial Respiratory Chain.

EMBO Rep 2008 Jan 7;9(1):33-8. Epub 2007 Dec 7.

Department of Biological Sciences, Marquette University, 530 N. 15th Street, Milwaukee, WI 53233, USA.

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http://dx.doi.org/10.1038/sj.embor.7401136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2246626PMC
January 2008

Oxa1 directly interacts with Atp9 and mediates its assembly into the mitochondrial F1Fo-ATP synthase complex.

Mol Biol Cell 2007 May 7;18(5):1897-908. Epub 2007 Mar 7.

Department of Biological Sciences, Marquette University, Milwaukee, WI 53233, USA.

The yeast Oxa1 protein is involved in the biogenesis of the mitochondrial oxidative phosphorylation (OXPHOS) machinery. The involvement of Oxa1 in the assembly of the cytochrome oxidase (COX) complex, where it facilitates the cotranslational membrane insertion of mitochondrially encoded COX subunits, is well documented. In this study we have addressed the role of Oxa1, and its sequence-related protein Cox18/Oxa2, in the biogenesis of the F(1)F(o)-ATP synthase complex. We demonstrate that Oxa1, but not Cox18/Oxa2, directly supports the assembly of the membrane embedded F(o)-sector of the ATP synthase. Oxa1 was found to physically interact with newly synthesized mitochondrially encoded Atp9 protein in a posttranslational manner and in a manner that is not dependent on the C-terminal, matrix-localized region of Oxa1. The stable manner of the Atp9-Oxa1 interaction is in contrast to the cotranslational and transient interaction previously observed for the mitochondrially encoded COX subunits with Oxa1. In the absence of Oxa1, Atp9 was observed to assemble into an oligomeric complex containing F(1)-subunits, but its further assembly with subunit 6 (Atp6) of the F(o)-sector was perturbed. We propose that by directly interacting with newly synthesized Atp9 in a posttranslational manner, Oxa1 is required to maintain the assembly competence of the Atp9-F(1)-subcomplex for its association with Atp6.
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http://dx.doi.org/10.1091/mbc.e06-10-0925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855041PMC
May 2007

Characterization of Mmp37p, a Saccharomyces cerevisiae mitochondrial matrix protein with a role in mitochondrial protein import.

Mol Biol Cell 2006 Sep 21;17(9):4051-62. Epub 2006 Jun 21.

Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.
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http://dx.doi.org/10.1091/mbc.e06-04-0366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1556384PMC
September 2006

The yeast F(1)F(0)-ATP synthase: analysis of the molecular organization of subunit g and the importance of a conserved GXXXG motif.

J Biol Chem 2005 Jul 9;280(26):24435-42. Epub 2005 May 9.

Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233, USA.

The F(1)F(0)-ATP synthase enzyme is located in the inner mitochondrial membrane, where it forms dimeric complexes. Dimerization of the ATP synthase involves the physical association of the neighboring membrane-embedded F(0)-sectors. In yeast, the F(0)-sector subunits g and e (Su g and Su e, respectively) play a key role in supporting the formation of ATP synthase dimers. In this study we have focused on Su g to gain a better understanding of the function and the molecular organization of this subunit within the ATP synthase complex. Su g proteins contain a GXXXG motif (G is glycine, and X is any amino acid) in their single transmembrane segment. GXXXG can be a dimerization motif that supports helix-helix interactions between neighboring transmembrane segments. We demonstrate here that the GXXXG motif is important for the function and in particular for the stability of Su g within the ATP synthase. Using site-directed mutagenesis and cross-linking approaches, we demonstrate that Su g and Su e interact, and our findings emphasize the importance of the membrane anchor regions of these proteins for their interaction. Su e also contains a conserved GXXXG motif in its membrane anchor. However, data presented here would suggest that an intact GXXXG motif in Su g is not essential for the Su g-Su e interaction. We suggest that the GXXXG motif may not be the sole basis for a Su g-Su e interaction, and possibly these dimerization motifs may enable both Su g and Su e to interact with another mitochondrial protein.
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http://dx.doi.org/10.1074/jbc.M502804200DOI Listing
July 2005

Functional analysis of subunit e of the F1Fo-ATP synthase of the yeast Saccharomyces cerevisiae: importance of the N-terminal membrane anchor region.

Eukaryot Cell 2005 Feb;4(2):346-55

Department of Biological Sciences, Marquette University, Milwaukee, WI 53233, USA.

Mitochondrial F1Fo-ATP synthase complexes do not exist as physically independent entities but rather form dimeric and possibly oligomeric complexes in the inner mitochondrial membrane. Stable dimerization of two F1Fo-monomeric complexes involves the physical association of two membrane-embedded Fo-sectors. Previously, formation of the ATP synthase dimeric-oligomeric network was demonstrated to play a critical role in modulating the morphology of the mitochondrial inner membrane. In Saccharomyces cerevisiae, subunit e (Su e) of the Fo-sector plays a central role in supporting ATP synthase dimerization. The Su e protein is anchored to the inner membrane via a hydrophobic region located at its N-terminal end. The hydrophilic C-terminal region of Su e resides in the intermembrane space and contains a conserved coiled-coil motif. In the present study, we focused on characterizing the importance of these regions for the function of Su e. We created a number of C-terminal-truncated derivatives of the Su e protein and expressed them in the Su e null yeast mutant. Mitochondria were isolated from the resulting transformant strains, and a number of functions of Su e were analyzed. Our results indicate that the N-terminal hydrophobic region plays important roles in the Su e-dependent processes of mitochondrial DNA maintenance, modulation of mitochondrial morphology, and stabilization of the dimer-specific Fo subunits, subunits g and k. Furthermore, we show that the C-terminal coiled-coil region of Su e functions to stabilize the dimeric form of detergent-solubilized ATP synthase complexes. Finally, we propose a model to explain how Su e supports the assembly of the ATP synthase dimers-oligomers in the mitochondrial membrane.
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http://dx.doi.org/10.1128/EC.4.2.346-355.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC549337PMC
February 2005

Yeast Oxa1 interacts with mitochondrial ribosomes: the importance of the C-terminal region of Oxa1.

EMBO J 2003 Dec;22(24):6438-47

Department of Biological Sciences, Marquette University, 530 N. 15th Street, Milwaukee, WI 53233, USA.

The yeast mitochondrial Oxa1 protein is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1 mediates the insertion of proteins (nuclearly and mitochondrially encoded) into the inner membrane. The mitochondrially encoded substrates interact directly with Oxa1 during their synthesis as nascent chains and in a manner that is supported by the associated ribosome. We have investigated if the Oxa1 complex interacts with the mitochondrial ribosome. Evidence to support a physical association between Oxa1 and the large ribosomal subunit is presented. Our data indicate that the matrix-exposed C-terminal region of Oxa1 plays an important role supporting the ribosomal-Oxa1 interaction. Truncation of this C-terminal segment compromises the ability of Oxa1 to support insertion of substrate proteins into the inner membrane. Oxa1 can be cross-linked to Mrp20, a component of the large ribosomal subunit. Mrp20 is homologous to L23, a subunit located next to the peptide exit tunnel of the ribosome. We propose that the interaction of Oxa1 with the ribosome serves to enhance a coupling of translation and membrane insertion events.
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http://dx.doi.org/10.1093/emboj/cdg624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC291819PMC
December 2003

Cardiolipin stabilizes respiratory chain supercomplexes.

J Biol Chem 2003 Dec 15;278(52):52873-80. Epub 2003 Oct 15.

Zentrum der Biologischen Chemie, Universitätsklinikum Frankfurt, D-60590 Frankfurt, Germany.

Cardiolipin stabilized supercomplexes of Saccharomyces cerevisiae respiratory chain complexes III and IV (ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase, respectively), but was not essential for their formation in the inner mitochondrial membrane because they were found also in a cardiolipin-deficient strain. Reconstitution with cardiolipin largely restored wild-type stability. The putative interface of complexes III and IV comprises transmembrane helices of cytochromes b and c1 and tightly bound cardiolipin. Subunits Rip1p, Qcr6p, Qcr9p, Qcr10p, Cox8p, Cox12p, and Cox13p and cytochrome c were not essential for the assembly of supercomplexes; and in the absence of Qcr6p, the formation of supercomplexes was even promoted. An additional marked effect of cardiolipin concerns cytochrome c oxidase. We show that a cardiolipin-deficient strain harbored almost inactive resting cytochrome c oxidase in the membrane. Transition to the fully active pulsed state occurred on a minute time scale.
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http://dx.doi.org/10.1074/jbc.M308366200DOI Listing
December 2003

Su e of the yeast F1Fo-ATP synthase forms homodimers.

J Biol Chem 2002 Dec 10;277(50):48484-9. Epub 2002 Oct 10.

Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233, USA.

The yeast F(1)F(o)-ATP synthase forms a dimeric complex in the mitochondrial inner membrane. Dimerization of two F(1)F(o) monomeric complexes involves the physical association of two membrane-embedded F(o) sectors and in a manner, which is dependent on the F(o) subunit, Su e. Sequence analysis of Su e protein family members indicated the presence of a conserved coiled-coil motif. As this motif is often the basis for protein homodimerization events, it was hypothesized that Su e forms homodimers in the inner membrane and that formation of Su e dimers between two neighboring F(o) complexes would facilitate dimerization of the F(1)F(o)-ATP synthase complex (Arnold, I., Pfeiffer, K., Neupert, W., Stuart, R. A., and Schägger, H. (1998) EMBO J. 17, 7170-7178). Using a histidine-tagged derivative of yeast Su e, Su e-His(12), combined with cross-linking and affinity purification approaches, we have directly demonstrated the ability of the yeast Su e protein to form homodimers. Functionality of the Su e-His(12) derivative was confirmed by its ability to assemble into the ATP synthase complex and to support its dimerization in the Deltasu e null mutant yeast cells. The close association of two neighboring Su e proteins was also demonstrated using cross-linking with Cu(2+), which binds and cross-links a unique Cys residue in neighboring Su e proteins. Finally, we propose a model for the molecular basis of the homodimerization of the Su e proteins.
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http://dx.doi.org/10.1074/jbc.M209382200DOI Listing
December 2002

Formation of the yeast F1F0-ATP synthase dimeric complex does not require the ATPase inhibitor protein, Inh1.

J Biol Chem 2002 Oct 6;277(42):39289-95. Epub 2002 Aug 6.

Department of Biology, Marquette University, Milwaukee, Wisconsin 53233, USA.

The yeast F1F0-ATP synthase forms dimeric complexes in the mitochondrial inner membrane and in a manner that is supported by the F0-sector subunits, Su e and Su g. Furthermore, it has recently been demonstrated that the binding of the F1F0-ATPase natural inhibitor protein to purified bovine F1-sectors can promote their dimerization in solution (Cabezon, E., Arechaga, I., Jonathan P., Butler, G., and Walker J. E. (2000) J. Biol. Chem. 275, 28353-28355). It was unclear until now whether the binding of the inhibitor protein to the F1 domains contributes to the process of F1F0-ATP synthase dimerization in intact mitochondria. Here we have directly addressed the involvement of the yeast inhibitor protein, Inh1, and its known accessory proteins, Stf1 and Stf2, in the formation of the yeast F1F0-ATP synthase dimer. Using mitochondria isolated from null mutants deficient in Inh1, Stf1, and Stf2, we demonstrate that formation of the F(1)F(0)-ATP synthase dimers is not adversely affected by the absence of these proteins. Furthermore, we demonstrate that the F1F0-ATPase monomers present in su e null mutant mitochondria can be as effectively inhibited by Inh1, as its dimeric counterpart in wild-type mitochondria. We conclude that dimerization of the F1F0-ATP synthase complexes involves a physical interaction of the membrane-embedded F0 sectors from two monomeric complexes and in a manner that is independent of inhibitory activity of the Inh1 and accessory proteins.
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http://dx.doi.org/10.1074/jbc.M205720200DOI Listing
October 2002