LETM1-Mediated K+ and Na+ Homeostasis Regulates Mitochondrial Ca2+ Efflux

HIGHLIGHTS \u2022 Monovalent cation homeostasis is dysregulated upon LETM1 depletion \u2022 K+/H+ exchange activity is decreased in LETM1 knockdown cells \u2022 LETM1 depletion results in K+ accumulation in the mitochondrial matrix \u2022 LETM1 knockdown does not affect expression of major mitochondrial Ca2+ transport modulators \u2022 LETM1-regulated mitochondrial Ca2+ fluxes are dependent on Na+ Ca2+ transport across the inner membrane of mitochondria (IMM) is of major importance for their functions in bioenergetics, cell death and signaling. It is therefore tightly regulated. It has been recently proposed that LETM1\u2014an IMM protein with a crucial role in mitochondrial K+/H+ exchange and volume homeostasis\u2014also acts as a Ca2+/H+ exchanger. Here we show for the first time that lowering LETM1 gene expression by shRNA hampers mitochondrial K+/H+ and Na+/H+ exchange. Decreased exchange activity resulted in matrix K+ accumulation in these mitochondria. Furthermore, LETM1 depletion selectively decreased Na+/Ca2+ exchange mediated by NCLX, as observed in the presence of ruthenium red, a blocker of the Mitochondrial Ca2+ Uniporter (MCU). These data confirm a key role of LETM1 in monovalent cation homeostasis, and suggest that the effects of its modulation on mitochondrial transmembrane Ca2+ fluxes may reflect those on Na+/H+ exchange activity

Multi-level suppression of receptor-PI3K-mTORC1 by fatty acid synthase inhibitors is crucial for their efficacy against ovarian cancer cells

Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions–from FASN towards receptor-PI3K-mTORC1–are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2–EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors

The molecular mechanism of mitochondria autophagy in yeast

Mitochondria are critical for supplying energy to the cell, but during catabolism this organelle also produces reactive oxygen species that can cause oxidative damage. Accordingly, quality control of mitochondria is important to maintain cellular homeostasis. It has been assumed that autophagy is the pathway for mitochondrial recycling, and that the selective degradation of mitochondria via autophagy (mitophagy) is the primary mechanism for mitochondrial quality control, although there is little experimental evidence to support this idea. Recent studies in yeast identified several mitophagy-related genes and have uncovered components involved in the molecular mechanism and regulation of mitophagy. Similarly, studies of Parkinson disease and reticulocyte maturation reveal that Parkin and Nix, respectively, are required for mitophagy in mammalian cells, and these analyses have revealed important physiological roles for mitophagy. Here, we review the current knowledge on mitophagy, in particular on the molecular mechanism and regulation of mitophagy in yeast. We also discuss some of the differences between yeast and mammalian mitophagy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78656/1/j.1365-2958.2009.07035.x.pd

LETM1 haploinsufficiency causes mitochondrial defects in cells from humans with Wolf-Hirschhorn syndrome: implications for dissecting the underlying pathomechanisms in this condition

Wolf-Hirschhorn syndrome (WHS) represents an archetypical example of a contiguous gene deletion disorder – a condition comprising a complex set of developmental phenotypes with a multigenic origin. Epileptic seizures, intellectual disability, growth restriction, motor delay and hypotonia are major co-morbidities in WHS. Haploinsufficiency of LETM1, which encodes a mitochondrial inner-membrane protein functioning in ion transport, has been proposed as an underlying pathomechanism, principally for seizures but also for other core features of WHS, including growth and motor delay. Growing evidence derived from several model organisms suggests that reduced LETM1 expression is associated with some element of mitochondrial dysfunction. Surprisingly, LETM1-dependent mitochondrial functional deficits have not previously been described in cells from individuals with WHS. Here, using a unique panel of WHS-patient-derived cell lines with deletions of differing sizes,incorporating LETM1 or not, we show, for the first time, that LETM1 expression is reduced in mitochondria isolated from WHS-patient cells. Furthermore, we show that this is associated with distinct mitochondrial phenotypes, including altered intracellular [Ca2+] levels, dysfunctional mitochondrial transition-pore opening, hyperpolarizationand superoxide leakage from resting mitochondria. Interestingly, we find that these phenotypes segregate with seizures in our WHScohort. Our findings identify novel cellular phenotypes in WHSattributable to a 50% reduction in LETM1 expression level; thesephenotypes could underlie and/or contribute to some of the core clinical features of this condition

Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

The Parkin ubiquitin ligase marks the mitofusins Mfn1 and Mfn2 for proteasome-dependent degradation, promoting disposal of damaged mitochondria by preventing their fusion with healthy organelles

cAMP/PKA signaling balances respiratory activity with mitochondria dependent apoptosis via transcriptional regulation

Background Appropriate control of mitochondrial function, morphology and biogenesis are crucial determinants of the general health of eukaryotic cells. It is therefore imperative that we understand the mechanisms that co-ordinate mitochondrial function with environmental signaling systems. The regulation of yeast mitochondrial function in response to nutritional change can be modulated by PKA activity. Unregulated PKA activity can lead to the production of mitochondria that are prone to the production of ROS, and an apoptotic form of cell death. Results We present evidence that mitochondria are sensitive to the level of cAMP/PKA signaling and can respond by modulating levels of respiratory activity or committing to self execution. The inappropriate activation of one of the yeast PKA catalytic subunits, Tpk3p, is sufficient to commit cells to an apoptotic death through transcriptional changes that promote the production of dysfunctional, ROS producing mitochondria. Our data implies that cAMP/PKA regulation of mitochondrial function that promotes apoptosis engages the function of multiple transcription factors, including HAP4, SOK2 and SCO1. Conclusions We propose that in yeast, as is the case in mammalian cells, mitochondrial function and biogenesis are controlled in response to environmental change by the concerted regulation of multiple transcription factors. The visualization of cAMP/TPK3 induced cell death within yeast colonies supports a model that PKA regulation plays a physiological role in coordinating respiratory function and cell death with nutritional status in budding yeast

Automatic Morphological Subtyping Reveals New Roles of Caspases in Mitochondrial Dynamics

Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO) cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology

Potassium and Sodium Transport in Yeast

[EN] As the proper maintenance of intracellular potassium and sodium concentrations is vital for cell growth, all living organisms have developed a cohort of strategies to maintain proper monovalent cation homeostasis. In the model yeast Saccharomyces cerevisiae, potassium is accumulated to relatively high concentrations and is required for many aspects of cellular function, whereas high intracellular sodium/potassium ratios are detrimental to cell growth and survival. The fact that S. cerevisiae cells can grow in the presence of a broad range of concentrations of external potassium (10 M–2.5 M) and sodium (up to 1.5 M) indicates the existence of robust mechanisms that have evolved to maintain intracellular concentrations of these cations within appropriate limits. In this review, current knowledge regarding potassium and sodium transporters and their regulation will be summarized. The cellular responses to high sodium and potassium and potassium starvation will also be discussed, as well as applications of this knowledge to diverse fields, including antifungal treatments, bioethanol production and human disease.L.Y. is funded by grant BFU2011-30197-C03-03 from the Spanish Ministry of Science and Innovation (Madrid, Spain) and EUI2009-04147 [Systems Biology of Microorganisms (SysMo2) European Research Area-Network (ERA-NET)].Yenush, L. (2016). Potassium and Sodium Transport in Yeast. Advances in Experimental Medicine and Biology. 892:187-228. https://doi.org/10.1007/978-3-319-25304-6_8S187228892Ahmed A, Sesti F, Ilan N, Shih TM, Sturley SL et al (1999) A molecular target for viral killer toxin: TOK1 potassium channels. Cell 99:283–291Albert A, Yenush L, Gil-Mascarell MR, Rodriguez PL, Patel S et al (2000) X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity. J Mol Biol 295:927–938Albertyn J, Hohmann S, Thevelein JM, Prior BA (1994) GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol Cell Biol 14:4135–4144Alepuz PM, Cunningham KW, Estruch F (1997) Glucose repression affects ion homeostasis in yeast through the regulation of the stress-activated ENA1 gene. Mol Microbiol 26:91–98Ali R, Brett CL, Mukherjee S, Rao R (2004) Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast. J Biol Chem 279:4498–4506Alijo R, Ramos J (1993) Several routes of activation of the potassium uptake system of yeast. Biochim Biophys Acta 1179:224–228Anderson JA, Huprikar SS, Kochian LV, Lucas WJ, Gaber RF (1992) Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 89:3736–3740Anderson JA, Nakamura RL, Gaber RF (1994) Heterologous expression of K+ channels in Saccharomyces cerevisiae: strategies for molecular analysis of structure and function. Symp Soc Exp Biol 48:85–97André B, Scherens B (1995) The yeast YBR235w gene encodes a homolog of the mammalian electroneutral Na(+)-(K+)-C1- cotransporter family. Biochem Biophys Res Commun 217:150–153Andrés MT, Viejo-Díaz M, Fierro JF (2008) Human lactoferrin induces apoptosis-like cell death in Candida albicans: critical role of K+-channel-mediated K+ efflux. Antimicrob Agents Chemother 52:4081–4088Anemaet IG, van Heusden GP (2014) Transcriptional response of Saccharomyces cerevisiae to potassium starvation. BMC Genomics 15:1040Arino J, Ramos J, Sychrova H (2010) Alkali metal cation transport and homeostasis in yeasts. Microbiol Mol Biol Rev 74:95–120Babazadeh R, Furukawa T, Hohmann S, Furukawa K (2014) Rewiring yeast osmostress signalling through the MAPK network reveals essential and non-essential roles of Hog1 in osmoadaptation. Sci Rep 4:4697Baev D, Rivetta A, Li XS, Vylkova S, Bashi E et al (2003) Killing of Candida albicans by human salivary histatin 5 is modulated, but not determined, by the potassium channel TOK1. Infect Immun 71:3251–3260Baev D, Rivetta A, Vylkova S, Sun JN, Zeng GF et al (2004) The TRK1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, Histatin 5. J Biol Chem 279:55060–55072Bagriantsev SN, Ang KH, Gallardo-Godoy A, Clark KA, Arkin MR et al (2013) A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels. ACS Chem Biol 8:1841–1851Bañuelos MA, Sychrová H, Bleykasten-Grosshans C, Souciet JL, Potier S (1998) The Nha1 antiporter of Saccharomyces cerevisiae mediates sodium and potassium efflux. Microbiology 144(Pt 10):2749–2758Bañuelos MA, Ruiz MC, Jiménez A, Souciet JL, Potier S et al (2002) Role of the Nha1 antiporter in regulating K(+) influx in Saccharomyces cerevisiae. Yeast 19:9–15Barnett JA (2008) A history of research on yeasts 13. Active transport and the uptake of various metabolites. Yeast 25:689–731Barreto L, Canadell D, Petrezselyova S, Navarrete C, Maresova L et al (2011) A genomewide screen for tolerance to cationic drugs reveals genes important for potassium homeostasis in Saccharomyces cerevisiae. Eukaryot Cell 10:1241–1250Barreto L, Canadell D, Valverde-Saubí D, Casamayor A, Ariño J (2012) The short-term response of yeast to potassium starvation. Environ Microbiol 14:3026–3042Benito B, Moreno E, Lagunas R (1991) Half-life of the plasma membrane ATPase and its activating system in resting yeast cells. Biochim Biophys Acta 1063:265–268Benito B, Quintero FJ, Rodríguez-Navarro A (1997) Overexpression of the sodium ATPase of Saccharomyces cerevisiae: conditions for phosphorylation from ATP and Pi. Biochim Biophys Acta 1328:214–226Benito B, Garciadeblás B, Rodríguez-Navarro A (2002) Potassium- or sodium-efflux ATPase, a key enzyme in the evolution of fungi. Microbiology 148:933–941Benito B, Garciadeblás B, Schreier P, Rodríguez-Navarro A (2004) Novel p-type ATPases mediate high-affinity potassium or sodium uptake in fungi. Eukaryot Cell 3:359–368Bernardi P (1999) Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 79:1127–1155Bertl A, Slayman CL, Gradmann D (1993) Gating and conductance in an outward-rectifying K+ channel from the plasma membrane of Saccharomyces cerevisiae. J Membr Biol 132:183–199Bertl A, Bihler H, Reid JD, Kettner C, Slayman CL (1998) Physiological characterization of the yeast plasma membrane outward rectifying K+ channel, DUK1 (TOK1), in situ. J Membr Biol 162:67–80Bertl A, Ramos J, Ludwig J, Lichtenberg-Fraté H, Reid J et al (2003) Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations. Mol Microbiol 47:767–780Bihler H, Slayman CL, Bertl A (1998) NSC1: a novel high-current inward rectifier for cations in the plasma membrane of Saccharomyces cerevisiae. FEBS Lett 432:59–64Bihler H, Slayman CL, Bertl A (2002) Low-affinity potassium uptake by Saccharomyces cerevisiae is mediated by NSC1, a calcium-blocked non-specific cation channel. Biochim Biophys Acta 1558:109–118Blomberg A (1995) Global changes in protein synthesis during adaptation of the yeast Saccharomyces cerevisiae to 0.7 M NaCl. J Bacteriol 177:3563–3572Blomberg A (2000) Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions: questions, some answers and a model. FEMS Microbiol Lett 182:1–8Borst-Pauwels GW (1981) Ion transport in yeast. Biochim Biophys Acta 650:88–127Botstein D, Fink GR (2011) Yeast: an experimental organism for 21st Century biology. Genetics 189:695–704Bouillet LE, Cardoso AS, Perovano E, Pereira RR, Ribeiro EM et al (2012) The involvement of calcium carriers and of the vacuole in the glucose-induced calcium signaling and activation of the plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells. Cell Calcium 51:72–81Bowers K, Levi BP, Patel FI, Stevens TH (2000) The sodium/proton exchanger Nhx1p is required for endosomal protein trafficking in the yeast Saccharomyces cerevisiae. Mol Biol Cell 11:4277–4294Breinig F, Tipper DJ, Schmitt MJ (2002) Kre1p, the plasma membrane receptor for the yeast K1 viral toxin. Cell 108:395–405Brett CL, Tukaye DN, Mukherjee S, Rao R (2005) The yeast endosomal Na+K+/H+ exchanger Nhx1 regulates cellular pH to control vesicle trafficking. Mol Biol Cell 16:1396–1405Cagnac O, Leterrier M, Yeager M, Blumwald E (2007) Identification and characterization of Vnx1p, a novel type of vacuolar monovalent cation/H+ antiporter of Saccharomyces cerevisiae. J Biol Chem 282:24284–24293Cagnac O, Aranda-Sicilia MN, Leterrier M, Rodriguez-Rosales MP, Venema K (2010) Vacuolar cation/H+ antiporters of Saccharomyces cerevisiae. J Biol Chem 285:33914–33922Calahorra M, Lozano C, Sánchez NS, Peña A (2011) Ketoconazole and miconazole alter potassium homeostasis in Saccharomyces cerevisiae. Biochim Biophys Acta 1808:433–445Canadell D, González A, Casado C, Ariño J (2015) Functional interactions between potassium and phosphate homeostasis in Saccharomyces cerevisiae. Mol Microbiol 95:555–572Casado C, Yenush L, Melero C, del Carmen Ruiz M, Serrano R et al (2010) Regulation of Trk-dependent potassium transport by the calcineurin pathway involves the Hal5 kinase. FEBS Lett 584:2415–2420Causton HC, Ren B, Koh SS, Harbison CT, Kanin E et al (2001) Remodeling of yeast genome expression in response to environmental changes. Mol Biol Cell 12:323–337Clotet J, Posas F (2007) Control of cell cycle in response to osmostress: lessons from yeast. Methods Enzymol 428:63–76Cornet M, Gaillardin C (2014) pH signaling in human fungal pathogens: a new target for antifungal strategies. Eukaryot Cell 13:342–352Courchesne WE (2002) Characterization of a novel, broad-based fungicidal activity for the antiarrhythmic drug amiodarone. J Pharmacol Exp Ther 300:195–199Courchesne WE, Ozturk S (2003) Amiodarone induces a caffeine-inhibited, MID1-dependent rise in free cytoplasmic calcium in Saccharomyces cerevisiae. Mol Microbiol 47:223–234Crespo JL, Daicho K, Ushimaru T, Hall MN (2001) The GATA transcription factors GLN3 and GAT1 link TOR to salt stress in Saccharomyces cerevisiae. J Biol Chem 276:34441–34444Cunningham KW, Fink GR (1996) Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae. Mol Cell Biol 16:2226–2237Curto M, Valledor L, Navarrete C, Gutiérrez D, Sychrova H et al (2010) 2-DE based proteomic analysis of Saccharomyces cerevisiae wild and K+ transport-affected mutant (trk1,2) strains at the growth exponential and stationary phases. J Proteomics 73:2316–2335D’Avanzo N, Cheng WW, Xia X, Dong L, Savitsky P et al (2010) Expression and purification of recombinant human inward rectifier K+ (KCNJ) channels in Saccharomyces cerevisiae. Protein Expr Purif 71:115–121Daran-Lapujade P, Daran JM, Luttik MA, Almering MJ, Pronk JT et al (2009) An atypical PMR2 locus is responsible for hypersensitivity to sodium and lithium cations in the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D. FEMS Yeast Res 9:789–792Davis DA (2009) How human pathogenic fungi sense and adapt to pH: the link to virulence. Curr Opin Microbiol 12:365–370de Nadal E, Posas F (2011) Elongating under stress. Genet Res Int 2011:326286de Nadal E, Clotet J, Posas F, Serrano R, Gomez N et al (1998) The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase. Proc Natl Acad Sci U S A 95:7357–7362de Nadal E, Calero F, Ramos J, Ariño J (1999) Biochemical and genetic analyses of the role of yeast casein kinase 2 in salt tolerance. J Bacteriol 181:6456–6462de Nadal E, Alepuz PM, Posas F (2002) Dealing with osmostress through MAP kinase activation. EMBO Rep 3:735–740De Nadal E, Zapater M, Alepuz PM, Sumoy L, Mas G et al (2004) The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes. Nature 427:370–374Dimmer KS, Fritz S, Fuchs F, Messerschmitt M, Weinbach N et al (2002) Genetic basis of mitochondrial function and morphology in Saccharomyces cerevisiae. Mol Biol Cell 13:847–853Durell SR, Guy HR (1999) Structural models of the KtrB, TrkH, and Trk1,2 symporters based on the structure of the KcsA K(+) channel. Biophys J 77:789–807Eide DJ, Clark S, Nair TM, Gehl M, Gribskov M et al (2005) Characterization of the yeast ionome: a genome-wide analysis of nutrient mineral and trace element homeostasis in Saccharomyces cerevisiae. Genome Biol 6:R77Elicharova H, Sychrova H (2014) Fluconazole affects the alkali-metal-cation homeostasis and susceptibility to cationic toxic compounds of Candida glabrata. Microbiology 160:1705–1713Endele S, Fuhry M, Pak SJ, Zabel BU, Winterpacht A (1999) LETM1, a novel gene encoding a putative EF-hand Ca(2+)-binding protein, flanks the Wolf-Hirschhorn syndrome (WHS) critical region and is deleted in most WHS patients. Genomics 60:218–225Eraso P, Mazón MJ, Portillo F (2006) Yeast protein kinase Ptk2 localizes at the plasma membrane and phosphorylates in vitro the C-terminal peptide of the H+-ATPase. Biochim Biophys Acta 1758:164–170Erez O, Kahana C (2002) Deletions of SKY1 or PTK2 in the Saccharomyces cerevisiae trk1Deltatrk2Delta mutant cells exert dual effect on ion homeostasis. Biochem Biophys Res Commun 295:1142–1149Estrada E, Agostinis P, Vandenheede JR, Goris J, Merlevede W et al (1996) Phosphorylation of yeast plasma membrane H+-ATPase by casein kinase I. J Biol Chem 271:32064–32072Fairman C, Zhou X, Kung C (1999) Potassium uptake through the TOK1 K+ channel in the budding yeast. J Membr Biol 168:149–157Farnaud S, Evans RW (2003) Lactoferrin – a multifunctional protein with antimicrobial properties. Mol Immunol 40:395–405Fell GL, Munson AM, Croston MA, Rosenwald AG (2011) Identification of yeast genes involved in k homeostasis: loss of membrane traffic genes affects k uptake. G3 (Bethesda) 1:43–56Fernandes AR, Sá-Correia I (2003) Transcription patterns of PMA1 and PMA2 genes and activity of plasma membrane H+-ATPase in Saccharomyces cerevisiae during diauxic growth and stationary phase. Yeast 20:207–219Ferrando A, Kron SJ, Rios G, Fink GR, Serrano R (1995) Regulation of cation transport in Saccharomyces cerevisiae by the salt tolerance gene HAL3. Mol Cell Biol 15:5470–5481Ferrigno P, Posas F, Koepp D, Saito H, Silver PA (1998) Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1. EMBO J 17:5606–5614Flegelova H, Haguenauer-Tsapis R, Sychrova H (2006) Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae. Biochim Biophys Acta 1760:504–516Flis K, Hinzpeter A, Edelman A, Kurlandzka A (2005) The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p. Biochem J 390:655–664Forment J, Mulet JM, Vicente O, Serrano R (2002) The yeast SR protein kinase Sky1p modulates salt tolerance, membrane potential and the Trk1,2 potassium transporter. Biochim Biophys Acta 1565:36–40Froschauer E, Nowikovsky K, Schweyen RJ (2005) Electroneutral K+/H+ exchange in mitochondrial membrane vesicles involves Yol027/Letm1 proteins. Biochim Biophys Acta 1711:41–48Fukuda A, Nakamura A, Tagiri A, Tanaka H, Miyao A et al (2004) Function, intracellular localization and the importance in salt tolerance of a vacuolar Na(+)/H(+) antiporter from rice. Plant Cell Physiol 45:146–159Gaber RF (1992) Molecular genetics of yeast ion transport. Int Rev Cytol 137:299–353Gaber RF, Styles CA, Fink GR (1988) TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae. Mol Cell Biol 8:2848–2859Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper SL et al (1999) The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast. Proc Natl Acad Sci U S A 96:1480–1485Gelis S, Curto M, Valledor L, González A, Ariño J et al (2012) Adaptation to potassium starvation of wild-type and K(+)-transport mutant (trk1,2) of Saccharomyces cerevisiae: 2-dimensional gel electrophoresis-based proteomic approach. Microbiologyopen 1:182–193Gómez MJ, Luyten K, Ramos J (1996) The capacity to transport potassium influences sodium tolerance in Saccharomyces cerevisiae. FEMS Microbiol Lett 135:157–160González A, Casado C, Petrezsélyová S, Ruiz A, Ariño J (2013) Molecular analysis of a conditional hal3 vhs3 yeast mutant links potassium homeostasis with flocculation and invasiveness. Fungal Genet Biol 53:1–9Goossens A, de La Fuente N, Forment J, Serrano R, Portillo F (2000) Regulation of yeast H(+)-ATPase by protein kinases belonging to a family dedicated to activation of plasma membrane transporters. Mol Cell Biol 20:7654–7661Gupta SS, Canessa CM (2000) Heterologous expression of a mammalian epithelial sodium channel in yeast. FEBS Lett 481:77–80Gustin MC, Martinac B, Saimi Y, Culbertson MR, Kung C (1986) Ion channels in yeast. Science 233:1195–1197Haass FA, Jonikas M, Walter P, Weissman JS, Jan YN et al (2007) Identification of yeast proteins necessary for cell-surface function of a potassium channel. Proc Natl Acad Sci U S A 104:18079–18084Haro R, Rodríguez-Navarro A (2002) Molecular analysis of the mechanism of potassium uptake through the TRK1 transporter of Saccharomyces cerevisiae. Biochim Biophys Acta 1564:114–122Haro R, Rodríguez-Navarro A (2003) Functional analysis of the M2(D) helix of the TRK1 potassium transporter of Saccharomyces cerevisiae. Biochim Biophys Acta 1613:1–6Haro R, Garciadeblas B, Rodríguez-Navarro A (1991) A novel P-type ATPase from yeast involved in sodium transport. FEBS Lett 291:189–191Hasenbrink G, Schwarzer S, Kolacna L, Ludwig J, Sychrova H et al (2005) Analysis of the mKir2.1 channel activity in potassium influx defective Saccharomyces cerevisiae strains determined as changes in growth characteristics. FEBS Lett 579:1723–1731Herrera R, Álvarez MC, Gelis S, Ramos J (2013) Subcellular potassium and sodium distribution in Saccharomyces cerevisiae wild-type and vacuolar mutants. Biochem J 454:525–532Herrera R, Alvarez MC, Gelis S, Kodedová M, Sychrová H et al (2014) Role of Saccharomyces cerevisiae Trk1 in stabilization of intracellular potassium content upon changes in external potassium levels. Biochim Biophys Acta 1838:127–133Hess DC, Lu W, Rabinowitz JD, Botstein D (2006) Ammonium toxicity and potassium limitation in yeast. PLoS Biol 4:e351Hoeberichts FA, Perez-Valle J, Montesinos C, Mulet JM, Planes MD et al (2010) The role of K+ and H+ transport systems during glucose- and H2O2-induced cell death in Saccharomyces cerevisiae. Yeast 27:713–725Hohmann S (2002) Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66:300–372Hohmann S, Krantz M, Nordlander B (2007) Yeast osmoregulation. Methods Enzymol 428:29–45Idnurm A, Walton FJ, Floyd A, Reedy JL, Heitman J (2009) Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis. Eukaryot Cell 8:315–326Jung KW, Strain AK, Nielsen K, Jung KH, Bahn YS (2012) Two cation transporters Ena1 and Nha1 cooperatively modulate ion homeostasis, antifungal drug resistance, and virulence of Cryptococcus neoformans via the HOG pathway. Fungal Genet Biol 49:332–345Kafadar KA, Cyert MS (2004) Integration of stress responses: modulation of calcineurin signaling in Saccharomyces cerevisiae by protein kinase A. Eukaryot Cell 3:1147–1153Kahm M, Navarrete C, Llopis-Torregrosa V, Herrera R, Barreto L et al (2012) Potassium starvation in yeast: mechanisms of homeostasis revealed by mathematical modeling. PLoS Comput Biol 8:e1002548Kallay LM, Brett CL, Tukaye DN, Wemmer MA, Chyou A et al (2011) Endosomal Na+(K+)/H+ exchanger Nhx1/Vps44 functions independently and downstream of multivesicular body formation. J Biol Chem 286:44067–44077Kane PM (2007) The long physiological reach of the yeast vacuolar H+-ATPase. J Bioenerg Biomembr 39:415–421Kane PM (2012) Targeting reversible disassembly as a mechanism of controlling V-ATPase activity. Curr Protein Pept Sci 13:117–123Ke R, Ingram PJ, Haynes K (2013) An integrative model of ion regulation in yeast. PLoS Comput Biol 9:e1002879Ketchum KA, Joiner WJ, Sellers AJ, Kaczmarek LK, Goldstein SA (1995) A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem. Nature 376:690–695Kinclová O, Ramos J, Potier S, Sychrová H (2001) Functional study of the Saccharomyces cerevisiae Nha1p C-terminus. Mol Microbiol 40:656–668Kinclova-Zimmermannova O, Sychrova H (2006) Functional study of the Nha1p C-terminus: involvement in cell response to changes in external osmolarity. Curr Genet 49:229–236Kinclová-Zimmermannová O, Flegelová H, Sychrová H (2004) Rice Na+/H+-antiporter Nhx1 partially complements the alkali-metal-cation sensitivity of yeast strains lacking three sodium transporters. Folia Microbiol (Praha) 49:519–525Kinclova-Zimmermannova O, Gaskova D, Sychrova H (2006) The Na+, K+/H+ -antiporter Nha1 influences the plasma membrane potential of Saccharomyces cerevisiae. FEMS Yeast Res 6:792–800Klee CB, Draetta GF, Hubbard MJ (1988) Calcineurin. Adv Enzymol Relat Areas Mol Biol 61:149–200Klipp E, Nordlander B, Krüger R, Gennemark P, Hohmann S (2005) Integrative model of the response of yeast to osmotic shock. Nat Biotechnol 23:975–982Ko CH, Gaber RF (1991) TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae. Mol Cell Biol 11:4266–4273Ko CH, Buckley AM, Gaber RF (1990) TRK2 is required for low affinity K+ transport in Saccharomyces cerevisiae. Genetics 125:305–312Ko CH, Liang H, Gaber RF (1993) Roles of multiple glucose transporters in Saccharomyces cerevisiae. Mol Cell Biol 13:638–648Kojima A, To

Mitochondrial Alterations in PINK1 Deficient Cells Are Influenced by Calcineurin-Dependent Dephosphorylation of Dynamin-Related Protein 1

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential