Mitochondrial phosphoproteome

Transcription

1 Mitochondrial phosphoproteome Claire Lemaire UMR8221, CNRS, CEA, Université Paris-Sud CEA Saclay/ ibitec-s/sb2sm/lpm Mai

2 Importance of the regulation by phosphorylation A large proportion of the proteome is subjected to posttranslational modifications (PTMs), often associated with vital processes like: cell cycle regulation, stress response, apoptosis. Among the PTMs, phosphorylation is of key importance (at least 500 protein kinases and more than 100 phosphatases are predicted in the human genome). It is a fast and reversible process Phosphorylation has indeed been shown to modify different proteins in their functional properties, cellular localization, interactions with partners Mai 2014 C. Lemaire 2

3 Implications of phosphorylation in diseases It is also known that abnormal phosphorylation events are associated with neurodegenerative diseases (Alzheimer s, Parkinson s or Huntington s disease) ( Cohen, P. 2002). Identification of phosphoproteins has been done in patients with type II diabetes (Bridges, A.J. 2005). In cancers, transformed cells have higher levels of phosphoproteins than normal cells (Krause, D.S. and R.A. Van Etten. 2005). Mai 2014 C. Lemaire 3

4 Mitochondria Providing energy to the cell: synthesis of ATP Inner membrane Outer membrane Cristae Matrix Intermembranary space Mai 2014 C. Lemaire 4

5 Mitochondria and diseases In humans, deregulation of mitochondrial functions, in particular of the respiratory chain, is associated with several pathologies, including neurodegenerative diseases neuromuscular diseases liver dysfunction type II diabetes cancer cardiovascular diseases ageing Mai 2014 C. Lemaire 5

6 Regulation of mitochondrial functions by phosphorylation Phosphoproteins Kinase Phosphatase SER, THR,TYR Steadily increasing number of mitochondrial phosphoproteins, kinases and phosphatases suggests that reversible protein phosphorylation could be an important level of regulation in mitochondria Mai 2014 C. Lemaire 6

7 Kinases The kinome constitutes about 2% of human genes Protein kinases recognize specific sequences Phosphorylate their target proteins in few milliseconds Responses to stimuli are often transduced through a cascade of several kinases - several seconds or minutes to observe protein phosphorylation following an internal or external stimulus Mai 2014 C. Lemaire 7

8 Kinases and mitochondria Kinases are translocated to the mitochondria and are present in mitochondria particularly in the intermembrane space and inner membrane ( MAPKs: mitogen-activated protein kinases; Akt: protein kinase B; PKA: protein kinase A; PKC: protein kinase C Kinases regulate mitochondrial activities such as phosphorylation of respiratory proteins (cytochrome oxidase: Lee, 2002; complex I: Chen, 2004) The TOM complex is essential for the import of most mitochondrial proteins from the cytosol. Its biogenesis and function has been shown to be regulate by two cytosolic kinases CK2 (casein kinase 2) and PKA ( protein kinase A). ( Schmidt, 2011) Mai 2014 C. Lemaire 8

9 Regulation of mitochondrial enzymatic content during growth by the kinase Tpk3p in S. cerevisiae Ras/ camp/ PKA pathway Decrease in carbon source Ras/ camp cascade Tpk3p C.Chevtzoff et al., 2005 Mitochondria adaptation to maintain optimal growth Mai 2014 C. Lemaire 9

10 Phosphoproteome Advances in phosphoprotein/peptide enrichment methods and in mass spectrometry techniques have allowed high-throughput and large-scale mapping of in vivo phosphosites for a wide variety of organisms such as human mouse yeast fly bacteria plants Functional consequences of phosphorylation investigated for a limited set of proteins Mai 2014 C. Lemaire 10

11 Identification of mitochondrial phosphoproteins MITOCHONDRIA PURIFICATION MS analysis Mai 2014 C. Lemaire 11

12 Identification of mitochondrial phosphoproteins Reinders et al, 2007 S. cerevisiae 80 mitochondrial phosphoproteins 14 OXPHOS subunits Feng et al, 2008 Mouse heart Deng et al, 2010 Mouse heart Zhao et al, 2010 Human skeletal muscle 61 mitochondrial phosphoproteins 21 OXPHOS subunits 181 mitochondrial phosphoproteins 7 OXPHOS subunits 86 mitochondrial phosphoproteins 23 OXPHOS subunits Mai 2014 C. Lemaire 12

13 Regulation of mitochondrial proteins by phosphorylation The effect of mitochondrial protein phosphorylation has been first documented in the case of pyruvate dehydrogenase (Linn, 1969; Uhlinger, 1986) Later reports concerned : complex I (Papa et al., 2002) complex IV (Bender, 2000; Lee et al, 2009) complex V (Reinders et al., 2007; Højlund, 2010) complex II (Tomitsuka et al., 2009) ADP/ATP translocase (Feng et al, 2008, 2009) Mai 2014 C. Lemaire 13

14 Schematic representation of the respiratory chain in Saccharomyces cerevisiae Outer Membrane Intermembranary space External NADHase NADH NAD + cyt c H + Inner Membrane e - Q e - II III IV V Internal NADHase Matrix NADH NAD + succinate fumarate O 2 H + H + H 2 O ATP Subunit number: H + ADP + Pi Mai 2014 C. Lemaire 14

15 Proteomic and phosphoproteomic studies on yeast grown on different carbon sources whole cell extracts: Change in protein accumulation (Fendt and Sauer, BMC Syst Biol, 2010) and in phosphorylation (Oliveira et al., Mol Syst Biol, 2012) isolated mitochondria: Few data on mitochondrial proteins Change in protein accumulation (Ohlmeier et al., JBC, 2004) and in phosphorylation (Ohlmeier et al., Electrophoresis, 2010) Only qualitative studies, performed on 2D-gels Lack of significant and quantitative data on mitochondrial proteins Mai 2014 C. Lemaire 15

16 Quantitative variations of the proteome and phosproteome of the yeast Saccharomyces cerevisiae during a metabolic change PhD thesis: Margaux Renvoisé Michel ZIVY Ludovic BONHOMME Marlène DAVANTURE Benoit VALOT Thierry BAILLEAU Renvoisé et al., J. of Proteomics, 2014 Mai 2014 C. Lemaire 16

17 Quantitative variations of the proteome and phosproteome of the yeast Saccharomyces cerevisiae during a metabolic change A B C Growth in fermentation and respiration conditions Glucose, Galactose Lactate Isolation and purification of mitochondria mix 3 different isotopic labelling of peptides A B C Enrichment of phosphopeptides by SCX-IMAC Mai 2014 C. Lemaire 17

18 Protein accumulation and phosphorylation level are quantified in the 3 growth conditions Proteome 544 mitochondrial proteins quantified 176 significantly vary Phosphoproteome 287 phosphorylation sites quantified 159 sites seen for the first time! 90 significantly vary Mai 2014 C. Lemaire 18

19 OXPHOS proteins are regulated during a change of metabolism Intermembranary space Nde1p Inner membrane matrix Ndi1p INH1 C II (CIII) 2 (CIV) 2 CV Proteome 32 proteins more abundant in lactate Phosphoproteome 12 phosphorylation sites vary Mai 2014 C. Lemaire 19

20 Acknowledgements The team «Regulation of mitochondrial energytransducing complexes» UMR Saclay Francis Haraux Margaux Renvoisé Mehdi Lembrouk Aurélie Stanislas Alix de Paliminy Deborah Bouboune Aurore Davoust Erwan Durocher I.B.G.C.,Bordeaux Marie-France Giraud Daniel Brèthes Isabelle Larrieu Anne Devin Michel Rigoulet SBIGEM, Saclay Gwenaëlle Le Roux Christophe Carles Collaborations Ohio State University Columbus, U.S.A. Patrice Hamel Tiona Andrianaivomananjaona Qian Wu Financial support and encouragements: Bruno Robert Thank you for your attention Mai 2014 C. Lemaire 20