Stomatin-Like Protein 2 Is Required for In Vivo Mitochondrial Respiratory Chain Supercomplex Formation and Optimal Cell Function

Stomatin-like protein 2 (SLP-2) is a mainly mitochondrial protein that is widely expressed and is highly conserved across evolution. We have previously shown that SLP-2 binds the mitochondrial lipid cardiolipin and interacts with prohibitin-1 and -2 to form specialized membrane microdomains in the mitochondrial inner membrane, which are associated with optimal mitochondrial respiration. To determine how SLP-2 functions, we performed bioenergetic analysis of primary T cells from T cell-selective Slp-2 knockout mice under conditions that forced energy production to come almost exclusively from oxidative phosphorylation. These cells had a phenotype characterized by increased uncoupled mitochondrial respiration and decreased mitochondrial membrane potential. Since formation of mitochondrial respiratory chain supercomplexes (RCS) may correlate with more efficient electron transfer during oxidative phosphorylation, we hypothesized that the defect in mitochondrial respiration in SLP-2-deficient T cells was due to deficient RCS formation. We found that in the absence of SLP-2, T cells had decreased levels and activities of complex I-III₂ and I-III₂-IV_1-3 RCS but no defects in assembly of individual respiratory complexes. Impaired RCS formation in SLP-2-deficient T cells correlated with significantly delayed T cell proliferation in response to activation under conditions of limiting glycolysis. Altogether, our findings identify SLP-2 as a key regulator of the formation of RCS in vivo and show that these supercomplexes are required for optimal cell function.     

The Mechanism of Pre-transfer Editing in Yeast Mitochondrial Threonyl-tRNA Synthetase

Accurate translation of mRNA into protein is a fundamental biological process critical for maintaining normal cellular functions. To ensure translational fidelity, aminoacyl-tRNA synthetases (aaRSs) employ pre-transfer and post-transfer editing activities to hydrolyze misactivated and mischarged amino acids, respectively. Whereas post-transfer editing, which requires either a specialized domain in aaRS or a trans-protein factor, is well described, the mechanism of pre-transfer editing is less understood. Here, we show that yeast mitochondrial threonyl-tRNA synthetase (MST1), which lacks an editing domain, utilizes pre-transfer editing to discriminate against serine. MST1 misactivates serine and edits seryl adenylate (Ser-AMP) in a tRNA-independent manner. MST1 hydrolyzes 80% of misactivated Ser-AMP at a rate 4-fold higher than that for the cognate threonyl adenylate (Thr-AMP) while releasing 20% of Ser-AMP into the solution. To understand the mechanism of pre-transfer editing, we solved the crystal structure of MST1 complexed with an analog of Ser-AMP. The binding of the Ser-AMP analog to MST1 induces conformational changes in the aminoacylation active site, and it positions a potential hydrolytic water molecule more favorably for nucleophilic attack. In addition, inhibition results reveal that the Ser-AMP analog binds the active site 100-fold less tightly than the Thr-AMP analog. In conclusion, we propose that the plasticity of the aminoacylation site in MST1 allows binding of Ser-AMP and the appropriate positioning of the hydrolytic water molecule.     

Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNA_f^Met). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNA_f^Met, as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNA_f^Met to translation initiation factor 2 (IF2). We hypothesize the existence of an accessory factor that assists mitochondrial IF2 (mIF2) in utilizing unformylated Met-tRNA_f^Met. This accessory factor might be unnecessary when formylated Met-tRNA_f^Met is present but becomes essential when only the unformylated species are available. Using a synthetic petite genetic screen in yeast, we identified a mutation in the AEP3 gene that caused a synthetic respiratory-defective phenotype together with Δfmt1. The same aep3 mutation also caused a synthetic respiratory defect in cells lacking formylated Met-tRNA_f^Met due to loss of the MIS1 gene that encodes the mitochondrial C₁-tetrahydrofolate synthase. The AEP3 gene encodes a peripheral mitochondrial inner membrane protein that stabilizes mitochondrially encoded ATP6/8 mRNA. Here we show that the AEP3 protein (Aep3p) physically interacts with yeast mIF2 both in vitro and in vivo and promotes the binding of unformylated initiator tRNA to yeast mIF2. We propose that Aep3p functions as an accessory initiation factor in mitochondrial protein synthesis.     

Glucose Signaling in Yeast Is Partially Mimicked by Galactose and Does Not Require the Tps1 Protein

Glucose produces multiple effects inSaccharomyces cerevisiae,as it controls the expression of many genes and the activity of various enzymes. However, the elements involved in glucose signaling are not well characterized. In this work the capacity of galactose to bring about the same effects than glucose has been assessed. Galactose mimics glucose only partially; it is suggested that it does not interact with a “sensor” in the plasma membrane and that it produces a weaker intracellular signal than glucose. To examine whether trehalose-6P synthase (Tps1) is required to transduce the glucose signal, we have constructed atps1 hxk2/tps1 HXK2strain which, at difference of atps1strain, grows on glucose, and, at difference of atps1 hxk2strain, still possess the Hxk2 protein, possibly involved in glucose repression. From the response of this strain to glucose, we conclude that Tps1 does not play a prominent role in glucose signaling.     

酵母单杂交的原理与应用实例

许多诱导型基因的表达,都受特定的转录因子和顺式元件调控。要阐明各种信号传递途径与基因表达调控的机理,克隆和鉴定转录因子是关键。近年来酵母单杂交方法被广泛应用于克隆和鉴定各种动植物的转录因子,本文以拟南芥DREB转录因子的克隆为例,介绍酵母单杂交方法的原理和具体应用 。     

Insight into Actin Organization and Function in Cytokinesis from Analysis of Fission Yeast Mutants

Actin is a key cytoskeletal protein with multiple roles in cellular processes such as polarized growth, cytokinesis, endocytosis, and cell migration. Actin is present in all eukaryotes as highly dynamic filamentous structures, such as linear cables and branched filaments. Detailed investigation of the molecular role of actin in various processes has been hampered due to the multifunctionality of the protein and the lack of alleles defective in specific processes. The actin cytoskeleton of the fission yeast, Schizosaccharomyces pombe, has been extensively characterized and contains structures analogous to those in other cell types. In this study, primarily with the view to uncover actin function in cytokinesis, we generated a large bank of fission yeast actin mutants that affect the organization of distinct actin structures and/or discrete physiological functions of actin. Our screen identified 17 mutants with specific defects in cytokinesis. Some of these cytokinesis mutants helped in dissecting the function of specific actin structures during ring assembly. Further genetic analysis of some of these actin mutants revealed multiple genetic interactions with mutants previously known to affect the actomyosin ring assembly. We also characterize a mutant allele of actin that is suppressed upon overexpression of Cdc8p-tropomyosin, underscoring the utility of this mutant bank. Another 22 mutant alleles, defective in polarized growth and/or other functions of actin obtained from this screen, are also described in this article. This mutant bank should be a valuable resource to study the physiological and biochemical functions of actin.     

Differential Regulation of Mitochondrial Pyruvate Carrier Genes Modulates Respiratory Capacity and Stress Tolerance in Yeast

Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.     

Channel Formation by Yeast F-ATP Synthase and the Role of Dimerization in the Mitochondrial Permeability Transition

Purified F-ATP synthase dimers of yeast mitochondria display Ca²⁺-dependent channel activity with properties resembling those of the permeability transition pore (PTP) of mammals. After treatment with the Ca²⁺ ionophore ETH129, which allows electrophoretic Ca²⁺ uptake, isolated yeast mitochondria undergo inner membrane permeabilization due to PTP opening. Yeast mutant strains ΔTIM11 and ΔATP20 (lacking the e and g F-ATP synthase subunits, respectively, which are necessary for dimer formation) display a striking resistance to PTP opening. These results show that the yeast PTP originates from F-ATP synthase and indicate that dimerization is required for pore formation in situ.     

Determination of the Order of Gene Function in the Yeast Nuclear Division Pathway Using cs and ts Mutants

Cold-sensitive (cs) and heat-sensitive (ts) conditional-lethal mutations that affect specifically the cell division cycle of budding yeast (Saccharomyces cerevisiae) were used to determine the order of gene function. Reciprocal temperature-shift experiments using cs-ts double mutants revealed a detailed order of function among genes whose execution points and mutant phenotypes are very similar. The data suggest that the nuclear branch of the overall cell-cycle pathway itself contains at least one branch.     

The Yeast Gene, MDM20, Is Necessary for Mitochondrial Inheritance and Organization of the Actin Cytoskeleton

In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.     

The Production of Reactive Oxygen Species in Intact Isolated Nerve Terminals Is Independent of the Mitochondrial Membrane Potential

Dependence on mitochondrial membrane potential (m) of hydrogen peroxide formation of in situ mitochondria in response to inhibition of complex I or III was studied in synaptosomes. Blockage of electron flow through complex I by rotenone or that through complex III by antimycin resulted in an increase in the rate of H₂O₂ generation as measured with the Amplex red assay. Membrane potential of mitochondria was dissipated by either FCCP (250nM) or DNP (50mM) and then the rate of H₂O₂ production was followed. Neither of the uncouplers had a significant effect on the rate of H₂O₂ production induced by rotenone or antimycin. Inhibition of the F₀F₁-ATPase by oligomycin, which also eliminates m in the presence of rotenone and antimycin, respectively, was also without effect on the ROS formation induced by rotenone and only slightly reduced the antimycin-induced H₂O₂ production. These results indicate that ROS generation of in situ mitochondria in nerve terminals in response to inhibition of complex I or complex III is independent of m. In addition, we detected a significant antimycin-induced H₂O₂ production when the flow of electrons through complex I was inhibited by rotenone, indicating that the respiratory chain of in situ mitochondria in synaptosomes has a substantial electron influx distal from the rotenone site, which could contribute to ROS generation when the complex III is inhibited.     

Responses of the Mitochondrial Respiratory System to Low Temperature in Plants

Low-temperature (LT) stress induces significant changes to plant cells including perturbations of various physio-biochemical and metabolic processes, which impact primary metabolism, respiratory rate, and the ATP production for biosynthesis and growth. Mitochondria from LT-tolerant species respond to LT through remodeling their composition that changes the structural and functional properties of the organelles. In this review, we discuss physiological aspects of mitochondrial respiration rate that are affected by LT, as well as, changes in the abundance of respiratory components under LT. The latter includes components of the phosphorylating and non-phosphorylating pathways and adjustments of mitochondrial membrane composition. Our objective is to provide a detailed overview of the often-contrasting reports of mitochondrial-specific changes and responses to LT and look for consensus themes to explain changes and draw more generally applicable observations about the LT response of plant respiration.     

Phosphatidylethanolamine Is Required for Normal Cell Morphology and Cytokinesis in the Fission Yeast Schizosaccharomyces pombe

To investigate the contributions of phosphatidylethanolamine to the growth and morphogenesis of the fission yeast Schizosaccharomyces pombe, we have characterized three predicted genes in this organism, designated psd1, psd2, and psd3, encoding phosphatidylserine decarboxylases, which catalyze the conversion of phosphatidylserine to phosphatidylethanolamine in both eukaryotic and prokaryotic organisms. S. pombe mutants carrying deletions in any one or two psd genes are viable in complex rich medium and synthetic defined minimal medium. However, mutants carrying deletions in all three psd genes (psd1-3Δ mutants) grow slowly in rich medium and are inviable in minimal medium, indicating that the psd1 to psd3 gene products share overlapping essential cellular functions. Supplementation of growth media with ethanolamine, which can be converted to phosphatidylethanolamine by the Kennedy pathway, restores growth to psd1-3Δ cells in minimal medium, indicating that phosphatidylethanolamine is essential for S. pombe cell growth. psd1-3Δ cells produce lower levels of phosphatidylethanolamine than wild-type cells, even in medium supplemented with ethanolamine, indicating that the Kennedy pathway can only partially compensate for the loss of phosphatidylserine decarboxylase activity in S. pombe. psd1-3Δ cells appear morphologically indistinguishable from wild-type S. pombe cells in medium supplemented with ethanolamine, but when cultured in nonsupplemented medium, they produce high frequencies of abnormally shaped cells as well as cells exhibiting severe septation defects, including multiple, mispositioned, deformed, and misoriented septa. Our results demonstrate that phosphatidylethanolamine is essential for cell growth and for normal cytokinesis and cellular morphogenesis in S. pombe, and they illustrate the usefulness of this model eukaryote for investigating potentially conserved biological and molecular functions of phosphatidylethanolamine.Phosphatidylethanolamine (PE) is a major phospholipid component of cell membranes in both prokaryotic and eukaryotic organisms (34, 35). There are three distinct pathways for PE synthesis in eukaryotic cells: (i) decarboxylation of phosphatidylserine (PS) via reactions catalyzed by PS decarboxylase (PSD) enzymes; (ii) the CDP-ethanolamine branch of the Kennedy pathway, which converts ethanolamine to PE (34); and (iii) acylation of lysophosphatidylethanolamine (21, 29), a reaction that in the budding yeast Saccharomyces cerevisiae is catalyzed by the enzyme Ale1 (22). Genetic studies have demonstrated that PE is essential for cell viability in S. cerevisiae, although the minimal threshold of PE required for cell growth in this organism can apparently be provided by any of the routes of PE synthesis listed above (22). In contrast, the results of mouse knockout experiments indicate that both PSD- and Kennedy pathway-catalyzed pathways for PE synthesis are essential for embryonic development (9, 28, 35).While PE is present in most, if not all, eukaryotic cell membranes, it is particularly enriched in the membranes of mitochondria (32, 35, 37). Indeed, S. cerevisiae mutants carrying a null mutation in the PSD1 gene, which encodes a mitochondrially localized PSD, exhibit phenotypes indicative of mitochondrial dysfunction, as do cells derived from mouse embryos carrying a disruption of the Psid gene, which encodes a protein highly homologous in structure to S. cerevisiae Psd1 (28, 32). A second PSD enzyme in S. cerevisiae, encoded by the PSD2 gene, is localized to Golgi and vacuolar membranes (33, 37). Consistent with a role in vacuolar function, PE has been implicated in the process of autophagy by genetic studies utilizing S. cerevisiae vacuolar targeting mutants and by studies showing that Atg8, a ubiquitin-like protein required for yeast autophagy, is conjugated to PE, as are several related mammalian proteins (19, 20, 27).Interestingly, studies utilizing a streptavidin-conjugated form of the PE-binding peptide cinnamycin demonstrated that PE is enriched at cell division sites in S. cerevisiae, the fission yeast Schizosaccharomyces pombe, and mammalian cells (6, 11). Moreover, streptavidin-conjugated cinnamycin was shown to inhibit the disassembly of the contractile ring and the completion of cytokinesis in cultures of Chinese hamster ovary cells, and a PE-deficient cell line from the same species was found to arrest growth in cytokinesis with an intact contractile ring (7). PE has also been shown to be enriched at the growing ends of interphase S. pombe cells and at the emerging bud cortex in dividing cells of S. cerevisiae, findings that implicate PE in processes controlling polarized cell growth (11).Although S. pombe mutants defective in enzymes that directly catalyze PE synthesis have not been described previously, we recently showed that mutants carrying a null mutation in the PS synthase gene pps1 are ethanolamine auxotrophs that exhibit severe morphology- and cytokinesis-defective phenotypes under ethanolamine-limited growth conditions (17). These findings implicated PE in the regulation of cellular morphogenesis and cytokinesis in S. pombe. To investigate the biological functions of PE in S. pombe, in particular its contributions to the control of cell morphology and cytokinesis, we have in the present study generated and characterized mutants carrying null mutations in three open reading frames predicted to encode PSD enzymes in this organism. In this paper, we describe the phenotypes of S. pombe PSD-null mutants, which demonstrate central roles for PE in the regulation of cell morphology and cytokinesis in this model eukaryote.