Loss of Num1-mediated cortical dynein anchoring negatively impacts respiratory growth.

Num1 is a multifunctional protein that both tethers mitochondria to the plasma membrane and anchors dynein to the cell cortex during nuclear inheritance. Previous work has examined the impact loss of Num1-based mitochondrial tethering has on dynein function in Saccharomyces cerevisiae; here, we elucidate its impact on mitochondrial function. We find that like mitochondria, Num1 is regulated by changes in metabolic state, with the protein levels and cortical distribution of Num1 differing between fermentative and respiratory growth conditions. In cells lacking Num1, we observe a reproducible respiratory growth defect, suggesting a role for Num1 in not only maintaining mitochondrial morphology, but also function. A structure-function approach revealed that, unexpectedly, Num1-mediated cortical dynein anchoring is important for normal growth under respiratory conditions. The severe respiratory growth defect in Δnum1 cells is not specifically due to the canonical functions of dynein in nuclear migration but is dependent on the presence of dynein, as deletion of DYN1 in Δnum1 cells partially rescues respiratory growth. We hypothesize that misregulated dynein present in cells that lack Num1 negatively impacts mitochondrial function resulting in defects in respiratory growth.

Evidence that the catalytic mechanism of heme a synthase involves the formation of a carbocation stabilized by a conserved glutamate.

In eukaryotes and many aerobic prokaryotes, the final step of aerobic respiration is catalyzed by an aa3-type cytochrome c oxidase, which requires a modified heme cofactor, heme a. The conversion of heme b, the prototypical cellular heme, to heme o and ultimately to heme a requires two modifications, the latter of which is conversion of a methyl group to an aldehyde, catalyzed by heme a synthase (HAS). The N- and C-terminal halves of HAS share homology, and each half contains a heme-binding site. Previous reports indicate that the C-terminal site is occupied by a heme b cofactor. The N-terminal site may function as the substrate (heme o) binding site, although this has not been confirmed experimentally. Here, we assess the role of conserved residues from the N- and C-terminal heme-binding sites in HAS from prokaryotic (Shewanella oneidensis) and eukaryotic (Saccharomyces cerevisiae) species - SoHAS/CtaA and ScHAS/Cox15, respectively. A glutamate within the N-terminal site is found to be critical for activity in both types of HAS, consistent with the hypothesis that a carbocation forms transiently during catalysis. In contrast, the residue occupying the analogous C-terminal position is dispensable for enzyme activity. In SoHAS, the C-terminal heme ligands are critical for stability, while in ScHAS, substitutions in either heme-binding site have little effect on global structure. In both species, in vivo accumulation of heme o requires the presence of an inactive HAS variant, highlighting a potential regulatory role for HAS in heme o biosynthesis.

The AAA ATPase Afg1 preserves mitochondrial fidelity and cellular health by maintaining mitochondrial matrix proteostasis.

Mitochondrial functions are critical for cellular physiology; therefore, several conserved mechanisms are in place to maintain the functional integrity of mitochondria. However, many of the molecular details and components involved in ensuring mitochondrial fidelity remain obscure. Here, we identify a novel role for the conserved mitochondrial AAA ATPase Afg1 in mediating mitochondrial protein homeostasis during aging and in response to various cellular challenges. Saccharomyces cerevisiae cells lacking functional Afg1 are hypersensitive to oxidative insults, unable to tolerate protein misfolding in the matrix compartment and exhibit progressive mitochondrial failure as they age. Loss of the Afg1 ortholog LACE-1 in Caenorhabditis elegans is associated with reduced lifespan, impeded oxidative stress tolerance, impaired mitochondrial proteostasis in the motor neuron circuitry and altered behavioral plasticity. Our results indicate that Afg1 is a novel protein quality control factor, which plays an important evolutionarily conserved role in mitochondrial surveillance, and cellular and organismal health.

The Assembly Factor Pet117 Couples Heme a Synthase Activity to Cytochrome Oxidase Assembly.

Heme a is an essential metalloporphyrin cofactor of the mitochondrial respiratory enzyme cytochrome c oxidase (CcO). Its synthesis from heme b requires several enzymes, including the evolutionarily conserved heme a synthase (Cox15). Oligomerization of Cox15 appears to be important for the process of heme a biosynthesis and transfer to maturing CcO. However, the details of this process remain elusive, and the roles of any additional CcO assembly factors that may be involved remain unclear. Here we report the systematic analysis of one such uncharacterized assembly factor, Pet117, and demonstrate in Saccharomyces cerevisiae that this evolutionarily conserved protein is necessary for Cox15 oligomerization and function. Pet117 is shown to reside in the mitochondrial matrix, where it is associated with the inner membrane. Pet117 functions at the later maturation stages of the core CcO subunit Cox1 that precede Cox1 hemylation. Pet117 also physically interacts with Cox15 and specifically mediates the stability of Cox15 oligomeric complexes. This Cox15-Pet117 interaction observed by co-immunoprecipitation persists in the absence of heme a synthase activity, is dependent upon Cox1 synthesis and early maturation steps, and is further dependent upon the presence of the matrix-exposed, unstructured linker region of Cox15 needed for Cox15 oligomerization, suggesting that this region mediates the interaction or that the interaction is lost when Cox15 is unable to oligomerize. Based on these findings, it was concluded that Pet117 mediates coupling of heme a synthesis to the CcO assembly process in eukaryotes.

Analysis of Oligomerization Properties of Heme a Synthase Provides Insights into Its Function in Eukaryotes.

Heme a is an essential cofactor for function of cytochrome c oxidase in the mitochondrial electron transport chain. Several evolutionarily conserved enzymes have been implicated in the biosynthesis of heme a, including the heme a synthase Cox15. However, the structure of Cox15 is unknown, its enzymatic mechanism and the role of active site residues remain debated, and recent discoveries suggest additional chaperone-like roles for this enzyme. Here, we investigated Cox15 in the model eukaryote Saccharomyces cerevisiae via several approaches to examine its oligomeric states and determine the effects of active site and human pathogenic mutations. Our results indicate that Cox15 exhibits homotypic interactions, forming highly stable complexes dependent upon hydrophobic interactions. This multimerization is evolutionarily conserved and independent of heme levels and heme a synthase catalytic activity. Four conserved histidine residues are demonstrated to be critical for eukaryotic heme a synthase activity and cannot be substituted with other heme-ligating amino acids. The 20-residue linker region connecting the two conserved domains of Cox15 is also important; removal of this linker impairs both Cox15 multimerization and enzymatic activity. Mutations of COX15 causing single amino acid conversions associated with fatal infantile hypertrophic cardiomyopathy and the neurological disorder Leigh syndrome result in impaired stability (S344P) or catalytic function (R217W), and the latter mutation affects oligomeric properties of the enzyme. Structural modeling of Cox15 suggests these two mutations affect protein folding and heme binding, respectively. We conclude that Cox15 multimerization is important for heme a biosynthesis and/or transfer to maturing cytochrome c oxidase.

The eukaryotic enzyme Bds1 is an alkyl but not an aryl sulfohydrolase.

The eukaryotic enzyme Bds1 in Saccharomyces cerevisiae is a metallo-ß-lactamase-related enzyme evolutionarily originating from bacterial horizontal gene transfer that serves an unknown biological role. Previously, Bds1 was reported to be an alkyl and aryl sulfatase. However, we demonstrate here that Bds1 acts on primary alkyl sulfates (of 6-12 carbon atoms) but not the aryl sulfates p-nitrophenyl sulfate and p-nitrocatechol sulfate. The apparent catalytic rate constant for hydrolysis of the substrate 1-hexyl sulfate by Bds1 is over 100 times lower than that of the reaction catalyzed by its bacterial homolog SdsA1. We show that Bds1 shares a catalytic mechanism with SdsA1 in which the carbon atom of the sulfate ester is the subject of nucleophilic attack, rather than the sulfur atom, resulting in C-O bond lysis. In contrast to SdsA1 and another bacterial homolog with selectivity for secondary alkyl sulfates named Pisa1, Bds1 does not show any substantial activity towards secondary alkyl sulfates. Neither Bds1 nor SdsA1 have any significant activity towards a branched primary alkyl sulfate, primary and secondary steroid sulfates, or phosphate diesters. Therefore, the enzymes homologous to SdsA1 that have been identified and characterized thus far vary in their selectivity towards primary and secondary alkyl sulfates but do not exhibit aryl sulfatase activity.

Modulation of the respiratory supercomplexes in yeast: enhanced formation of cytochrome oxidase increases the stability and abundance of respiratory supercomplexes.

Yeast cells deficient in the Rieske iron-sulfur subunit (Rip1) of ubiquinol-cytochrome c reductase (bc1) accumulate a late core assembly intermediate, which weakly associates with cytochrome oxidase (CcO) in a respiratory supercomplex. Expression of the N-terminal half of Rip1, which lacks the C-terminal FeS-containing globular domain (designated N-Rip1), results in a marked stabilization of trimeric and tetrameric bc1-CcO supercomplexes. Another bc1 mutant (qcr9Δ) stalled at the same assembly intermediate is likewise converted to stable supercomplex species by the expression of N-Rip1, but not by expression of intact Rip1. The N-Rip1-induced stabilization of bc1-CcO supercomplexes is independent of the Bcs1 translocase, which mediates Rip1 translocation during bc1 biogenesis. N-Rip1 induces the stabilization of bc1-CcO supercomplexes through an enhanced formation of CcO. The association of N-Rip1 with the late core bc1 assembly intermediate appears to confer stabilization of a CcO assembly intermediate. This induced stabilization of CcO is dependent on the Rcf1 supercomplex stabilization factor and only partially dependent on the presence of cardiolipin. N-Rip1 exerts a related induction of CcO stabilization in WT yeast, resulting in enhanced respiration. Additionally, the impact of CcO stabilization on supercomplexes was observed by means other than expression of N-Rip1 (via overexpression of CcO subunits Cox4 and Cox5a), demonstrating that this is a general phenomenon. This study presents the first evidence showing that supercomplexes can be stabilized by the stimulated formation of CcO.

Loss of cardiolipin leads to perturbation of mitochondrial and cellular iron homeostasis.

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.

LYRM7/MZM1L is a UQCRFS1 chaperone involved in the last steps of mitochondrial Complex III assembly in human cells.

The mammalian Complex III (CIII) assembly process is yet to be completely understood. There is still a lack in understanding of how the structural subunits are put together and which additional factors are involved. Here we describe the identification and characterization of LYRM7, a human protein displaying high sequence homology to the Saccharomyces cerevisiae protein Mzm1, which was recently shown as an assembly factor for Rieske Fe-S protein incorporation into the yeast cytochrome bc(1) complex. We conclude that human LYRM7, which we propose to be renamed MZM1L (MZM1-like), works as a human Rieske Fe-S protein (UQCRFS1) chaperone, binding to this subunit within the mitochondrial matrix and stabilizing it prior to its translocation and insertion into the late CIII dimeric intermediate within the mitochondrial inner membrane. Thus, LYRM7/MZM1L is a novel human CIII assembly factor involved in the UQCRFS1 insertion step, which enables formation of the mature and functional CIII enzyme.

The C-terminal propeptide of a plant defensin confers cytoprotective and subcellular targeting functions.

BACKGROUND: Plant defensins are small (45-54 amino acids), basic, cysteine-rich proteins that have a major role in innate immunity in plants. Many defensins are potent antifungal molecules and are being evaluated for their potential to create crop plants with sustainable disease resistance. Defensins are produced as precursor molecules which are directed into the secretory pathway and are divided into two classes based on the absence (class I) or presence (class II) of an acidic C-terminal propeptide (CTPP) of about 33 amino acids. The function of this CTPP had not been defined. RESULTS: By transgenically expressing the class II plant defensin NaD1 with and without its cognate CTPP we have demonstrated that NaD1 is phytotoxic to cotton plants when expressed without its CTPP. Transgenic cotton plants expressing constructs encoding the NaD1 precursor with the CTPP had the same morphology as non-transgenic plants but expression of NaD1 without the CTPP led to plants that were stunted, had crinkled leaves and were less viable. Immunofluorescence microscopy and transient expression of a green fluorescent protein (GFP)-CTPP chimera were used to confirm that the CTPP is sufficient for vacuolar targeting. Finally circular dichroism and NMR spectroscopy were used to show that the CTPP adopts a helical confirmation. CONCLUSIONS: In this report we have described the role of the CTPP on NaD1, a class II defensin from Nicotiana alata flowers. The CTPP of NaD1 is sufficient for vacuolar targeting and plays an important role in detoxification of the defensin as it moves through the plant secretory pathway. This work may have important implications for the use of defensins for disease protection in transgenic crops.

Biogenesis of the cytochrome bc(1) complex and role of assembly factors.

The cytochrome bc(1) complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc(1) complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc(1) complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc(1) complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc(1) complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron-sulfur protein and its role in completing the assembly of functional bc(1) complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Reprint of: Biogenesis of the cytochrome bc(1) complex and role of assembly factors.

The cytochrome bc(1) complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc(1) complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc(1) complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc(1) complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc(1) complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron-sulfur protein and its role in completing the assembly of functional bc(1) complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Down the Iron Path: Mitochondrial Iron Homeostasis and Beyond.

Cellular iron homeostasis and mitochondrial iron homeostasis are interdependent. Mitochondria must import iron to form iron-sulfur clusters and heme, and to incorporate these cofactors along with iron ions into mitochondrial proteins that support essential functions, including cellular respiration. In turn, mitochondria supply the cell with heme and enable the biogenesis of cytosolic and nuclear proteins containing iron-sulfur clusters. Impairment in cellular or mitochondrial iron homeostasis is deleterious and can result in numerous human diseases. Due to its reactivity, iron is stored and trafficked through the body, intracellularly, and within mitochondria via carefully orchestrated processes. Here, we focus on describing the processes of and components involved in mitochondrial iron trafficking and storage, as well as mitochondrial iron-sulfur cluster biogenesis and heme biosynthesis. Recent findings and the most pressing topics for future research are highlighted.

Mitochondrial contact site and cristae organizing system (MICOS) machinery supports heme biosynthesis by enabling optimal performance of ferrochelatase.

Heme is an essential cofactor required for a plethora of cellular processes in eukaryotes. In metazoans the heme biosynthetic pathway is typically partitioned between the cytosol and mitochondria, with the first and final steps taking place in the mitochondrion. The pathway has been extensively studied and its biosynthetic enzymes structurally characterized to varying extents. Nevertheless, understanding of the regulation of heme synthesis and factors that influence this process in metazoans remains incomplete. Therefore, we investigated the molecular organization as well as the physical and genetic interactions of the terminal pathway enzyme, ferrochelatase (Hem15), in the yeast Saccharomyces cerevisiae. Biochemical and genetic analyses revealed dynamic association of Hem15 with Mic60, a core component of the mitochondrial contact site and cristae organizing system (MICOS). Loss of MICOS negatively impacts Hem15 activity, affects the size of the Hem15 high-mass complex, and results in accumulation of reactive and potentially toxic tetrapyrrole precursors that may cause oxidative damage. Restoring intermembrane connectivity in MICOS-deficient cells mitigates these cytotoxic effects. These data provide new insights into how heme biosynthetic machinery is organized and regulated, linking mitochondrial architecture-organizing factors to heme homeostasis.

Late-stage maturation of the Rieske Fe/S protein: Mzm1 stabilizes Rip1 but does not facilitate its translocation by the AAA ATPase Bcs1.

The final step in the assembly of the ubiquinol-cytochrome c reductase or bc(1) complex involves the insertion of the Rieske Fe/S cluster protein, Rip1. Maturation of Rip1 occurs within the mitochondrial matrix prior to its translocation across the inner membrane (IM) in a process mediated by the Bcs1 ATPase and subsequent insertion into the bc(1) complex. Here we show that the matrix protein Mzm1 functions as a Rip1 chaperone, stabilizing Rip1 prior to the translocation step. In the absence of Mzm1, Rip1 is prone to either proteolytic degradation or temperature-induced aggregation. A series of Rip1 truncations were engineered to probe motifs necessary for Mzm1 interaction and Bcs1-mediated translocation of Rip1. The Mzm1 interaction with Rip1 persists in Rip1 variants lacking its transmembrane domain or containing only its C-terminal globular Fe/S domain. Replacement of the globular domain of Rip1 with that of the heterologous folded protein Grx3 abrogated Mzm1 interaction; however, appending the C-terminal 30 residues of Rip1 to the Rip1-Grx3 chimera restored Mzm1 interaction. The Rip1-Grx3 chimera and a Rip1 truncation containing only the N-terminal 92 residues each induced stabilization of the bc(1):cytochrome oxidase supercomplex in a Bcs1-dependent manner. However, the Rip1 variants were not stably associated with the supercomplex. The induced supercomplex stabilization by the Rip1 N terminus was independent of Mzm1.

The LYR protein Mzm1 functions in the insertion of the Rieske Fe/S protein in yeast mitochondria.

The assembly of the cytochrome bc(1) complex in Saccharomyces cerevisiae is shown to be conditionally dependent on a novel factor, Mzm1. Cells lacking Mzm1 exhibit a modest bc(1) defect at 30°C, but the defect is exacerbated at elevated temperatures. Formation of bc(1) is stalled in mzm1Δ cells at a late assembly intermediate lacking the Rieske iron-sulfur protein Rip1. Rip1 levels are markedly attenuated in mzm1Δ cells at elevated temperatures. Respiratory growth can be restored in the mutant cells by the overexpression of the Rip1 subunit. Elevated levels of Mzm1 enhance the stabilization of Rip1 through physical interaction, suggesting that Mzm1 may be an important Rip1 chaperone especially under heat stress. Mzm1 may function primarily to stabilize Rip1 prior to inner membrane (IM) insertion or alternatively to aid in the presentation of Rip1 to the inner membrane translocation complex for extrusion of the folded domain containing the iron-sulfur center.