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Claire Lemaire

UMR8221, CNRS, CEA, Université Paris-SudCEA Saclay/ iBiTec-S/SB2SM/LPM

Mitochondrial phosphoproteome

Mai 2014 1

Mai 2014 C. Lemaire 2

Importance of the regulation by phosphorylation

� A large proportion of the proteome is subjected to post-translational 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

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 phosphoproteinsthan normal cells (Krause, D.S. and R.A. Van Etten. 2005).


Mitochondria


� Providing energy to the cell: synthesis of ATP

Outer membrane

Inner membrane

Intermembranary space

Matrix

Cristae

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


Regulation of mitochondrial functions by phosphorylation


Kinase

Phosphatase

Phosphoproteins

SER, THR,TYR

Steadily increasing number of mitochondrial phosphoproteins, kinases andphosphatases suggests that reversible protein phosphorylation could be an important

level of regulation in mitochondria

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 proteinphosphorylation following an internal or external stimulus


Kinases and mitochondria

� Kinases are translocated to the mitochondria and are present in mitochondria particularly in the intermembrane space and innermembrane ( MAPKs: mitogen-activated protein kinases; Akt: proteinkinase 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)


Regulation of mitochondrial enzymatic content during growth by the kinase Tpk3p in S. cerevisiae


Ras/ cAMP/ PKA pathway

C.Chevtzoff et al., 2005

Decrease in carbon source

Ras/ cAMP cascade

Tpk3p

Mitochondria adaptationto maintain optimal growth

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 limitedset of proteins


Identification of mitochondrial phosphoproteins


MITOCHONDRIA PURIFICATION

MS analysis

Identification of mitochondrial phosphoproteins

� Reinders et al, 2007S. cerevisiae

� Feng et al, 2008 Mouse heart

� Deng et al, 2010 Mouse heart

� Zhao et al, 2010Human skeletal muscle


80 mitochondrial phosphoproteins

14 OXPHOS subunits


21 OXPHOS subunits


7 OXPHOS subunits


23 OXPHOS subunits

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)


Schematic representation of the respiratory chain in Saccharomyces cerevisiae


NADH

ATP

O2

cyt c

III

H+

succinate

V

External NADHase

IV

ADP+ PiH2O

NAD+

fumarate

Internal NADHase

NADHNAD+

Subunit number: 3 4 10 11 14

H+

H+

H+

Qe-IIe-

Matrix

Intermembranary space

Inner Membrane

Outer Membrane

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)

Few data on mitochondrial proteins� isolated mitochondria:

� 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

Quantitative variations of the proteome and phosproteome of the yeast Saccharomyces cerevisiae during a metabolic change


Michel ZIVY

Ludovic BONHOMME

Marlène DAVANTURE

Benoit VALOT

Thierry BAILLEAU

PhD thesis: Margaux Renvoisé

Renvoisé et al., J. of Proteomics, 2014

Quantitative variations of the proteome and phosproteome of the yeast Saccharomyces cerevisiae during a metabolic change


Growth in fermentation and respiration conditions� Glucose, Galactose

� Lactate

Isolation and purification of mitochondria

3 different isotopic labelling of peptides

Enrichment of phosphopeptides by SCX-IMAC

A B C

A B C

mix

Protein accumulation and phosphorylation level are quantified in the 3 growth conditions


� Proteome

� 544 mitochondrial proteins quantified� 176 significantly vary

� Phosphoproteome

� 287 phosphorylation sites quantified159 sites seen for the first time !� 90 significantly vary

368

176

0

100

200

300

400

500

600

197

90

0

50

100

150

200

250

300

350


OXPHOS proteins are regulated during a change of metabolism

C II (CIII)2 (CIV)2CV

Proteome� 32 proteins more abundant in

lactate

Phosphoproteome

� 12 phosphorylation sites vary

INH1matrix

Intermembranaryspace

Innermembrane

Ndi1p

Nde1p

Acknowledgements


The team « Regulation of

mitochondrial energy-

transducing complexes »

UMR 8821 - Saclay

Francis Haraux

Margaux Renvoisé Mehdi Lembrouk

Aurélie StanislasAlix de PaliminyDeborah BoubouneAurore DavoustErwan Durocher

Tiona AndrianaivomananjaonaQian Wu

Ohio State UniversityColumbus, U.S.A.

Patrice Hamel

I.B.G.C. ,Bordeaux

Marie-France GiraudDaniel BrèthesIsabelle Larrieu

Anne DevinMichel Rigoulet

Collaborations

SBIGEM, Saclay

Gwenaëlle Le RouxChristophe Carles

Financial support and encouragements: Bruno Robert

Thank you for your attention

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