MITOCHONDRIA:

 BRIDGE  NOTES.  
 
A  short  synopsis  of  the  interesting  aspects  of  mitochondrial  biology  that  
bridges  the  information  in  standard  textbooks  and  that  obtained  from  
reading  the  research  papers.  
 
GENERAL  STRUCTURE  OF  MITOCHONDRIA.  
 
Mitochondria  are  often  called  the  powerhouses  of  the  cell  because  of  
their  role  in  ATP  production,  but  they  do  much  more  as  will  be  
discussed  later.  The  classic  picture  of  mitochondria  has  been  that  of  a  
bean  shaped  organelle  with  an  outer  membrane  and  much  invaginated  
inner  membrane  as  shown  in  figure  1,  and  in  the  schematic  section  
through  a  cell  in  Figure  2.

FIGURE  1.  Classic  view  of    a  

mitochondrion  as  a  bean  
shaped  organelle.  

 
 
FIGURE  2.  Mitochondria    
shown  as  bean  shaped  
organelles  in  a  cell    
morphology  diagram.  

 
  
 
 
 
Work  over  the  last  20  or  so  years  now  describes  mitochondria  
significantly  differently.    The  picture  above,  still  used  in  several  
textbooks,  was  based  largely  on  electron  micrographs  of  thin-­‐sectioned  
tissue  as  shown  in  figure  3.    With  the  advent  of  higher  resolution  
electron  microscopes,  tilting  of  specimens  to  allow  reconstruction  of  3D  
images,  and  cryopreservation  of  samples,  the  picture  has  changed.  
 
 
1) The  internal  structure  of  mitochondria.  Frey  TG,  Mannella  CA.  Trends  Biochem  Sci.  
2000  Jul;25(7):319-­‐24  
 

FIGURE  3.  Negatively  stained  electron  micrographs  

of  sperm  mitochondria  (left)  and  human  heart  
mitochondria  (right)  

 
 
   
It  is  now  fairly  clear  that  the  inner  membrane  is  made  up  of  an  inner  
boundary  membrane  that  largely  follows  the  contour  of  the  outer  
membrane,  and  a  series  of  cristae  membranes  extending  from  the  
boundary  membrane  partly  or  fully  across  the  mitochondrion  interior,  
containing  a  fluid  space  called  the  intercristal  space.    Each  so-­‐called  
cristae  is  attached  to  the  inner  boundary  membrane  by  a  specific  
protein  complex.    Thus  a  mitochondrion  contains  three  spaces  each  with  
different  protein  content,  the  intermembrane  space,  the  intercristal  
space  and  the  matrix  space  The  protein  composition  of  the  outer  
membrane,  boundary  membrane  and  cristae  membrane  are  very  
different.  Importantly,  the  cristae  membrane  is  the  seat  of  the  OXPHOS  
complexes.  
Protein  analysis  has  identified  the  complex  that  links  the  outer  
membrane,  inner  boundary  membrane  and  cristae  membranes  and  this  
is  now  called  MINOS  
 
1)      The  mitochondrial  inner  membrane  protein  mitofilin  exists  as  a  complex  with  SAM50,  
metaxins  1  and  2,  coiled-­‐coil-­‐helix  coiled-­‐coil-­‐helix  domain-­‐containing  protein  3  and  6  and  
DnaJC11.  Xie  J,  Marusich  MF,  Souda  P,  Whitelegge  J,  Capaldi  RA.  FEBS  Lett.  2007;58:3545-­‐9.  
2)      Mitofilin  complexes:  conserved  organizers  of  mitochondrial  membrane  architecture.  
Zerbes  RM1,  van  der  Klei  IJ,  Veenhuis  M,  Pfanner  N,  van  der  Laan  M,  Bohnert  M.  Biol  Chem.  
2012;393:1247-­‐61..  
3)        STED  super-­‐resolution  microscopy  reveals  an  array  of  MINOS  clusters  along  human  
mitochondria.  Jans  DC,  Wurm  CA,  Riedel  D,  Wenzel  D,  Stagge  F,  Deckers  M,  Rehling  P,  Jakobs  
S.  Proc  Natl  Acad  Sci  U  S  A.  2013  ;110:8936-­‐41  
 
 
As  the  resolution  and  staining  of  electron  microscopy  specimens  
improved,  it  became  evident  that  the  bean  shaped  organelles  previously  
reported  were  in  fact  often  a  set  of  slices  through  an  undulating  long  
tube  or  reticulum    (Figure  4)  and  this  has  been  confirmed  by  confocal  
fluorescence  microscopy  as  shown  in  Figure  5.  
 

FIGURE  4.    (A)Clear  

evidence  from  EM  that  
mitochondria  are  reticular  
in  glucose  medium  .  (B)  In  
Rho0  cells  which  are  
missing  mtDNA  
mitochondria  are  differently  
shaped  and  fragmented,  (C)  
but  still  retained  cristae.  

 
 
 
More  recently,  it  has  become  clear  that  mitochondria  can  exist  in  two  
extreme  states,  i.e.  they  can  be  small  and  bean  shaped,  now  called  the  
fragmented  state,  or  they  can  form  the  reticulum  shown  in  figure  5.    
What  decides  their  morphology,  and  the  dynamics  of  the  change  in  form  
of  the  organelle,  is  now  the  subject  of  intense  research.    Several  
important  studies  have  established  that  mitochondrial  dynamics  is  a  
manifestation  of  cell  state,  responsive  to  changes  in  cellular  
bioenergetics,  and  is  altered  in  many  diseases.  
 
 

FIGURE  5.  A    3D  

reconstruction  of  
confocal  microscopy  
images  of  a  cell  
(osteosarcoma)  in  
which  green  
fluorescent  protein  has  
been  engineered  to  
locate  in  the  matrix  
space.    

 
 
1)      Cell  cycle  dependent  morphology  changes  and  associated  mitochondrial  DNA  
redistribution  in  mitochondria  of  human  cell  lines.  Margineantu  DH1,  Gregory  Cox  W,  Sundell  
L,  Sherwood  SW,  Beechem  JM,  Capaldi  RA    Mitochondrion.  2002;1:425-­‐35.  
2)      A  hyperfused  mitochondrial  state  achieved  at  G1-­‐S  regulates  cyclin  E  buildup  and  entry  
into  S  phase.  Mitra  K1,  Wunder  C,  Roysam  B,  Lin  G,  Lippincott-­‐Schwartz  J.  Proc  Natl  Acad  Sci  
U  S  A.  2009;106:11960-­‐5  
3)      Shaping  the  dynamic  mitochondrial  network.  Lackner  LL.  
BMC  Biol.  2014;12:12-­‐35  
The  morphology  is  cell  cycle  dependent,  being  reticular  in  the  G1  phase  
but  then  converting  to  the  punctate  form  in  G0  before  returning  to  a  
punctate  state  in  the  S  phase  (Figure  6).  
G1 S G2M
FIGURE  6.    A  time  
course  of  the  
mitochondrial  
morphology  after  
releasing  
osteosarcoma  cells  
from  a  starved  state  to  
synchronize  them.  

 
 
 
Besides  fission  and  fusion,  mitochondria  are  able  to  move  within  cells  in  
response  to  various  stimuli  as  most  evident  in  neurons  because  of  their  
length.  This  movement  has  the  effect  of  concentrating  ATP-­‐producing  
power  to  where  it  is  most  needed  at  any  time  in  the  cell,  for  example  at  
synapses  in  neurons  or  at  the  nucleus  in  times  of  cell  division.  This  
movement  is  assisted  by  interactions  with  microfilaments  and  
microtubules.  
 
There  are  other  morphological  changes  in  mitochondria  that  have  
physiological  relevance.    Thus  the  relative  volume  of  the  intercristal  
space  and  matrix  space  changes  with  organelle  functioning  (Figure  7).  
Under  conditions  of  OXPHOS  the  volume  of  the  intercristal  space  is  
greatly  increased  as  shown  in  the  figure  to  generate  what  has  been  
called  the  orthodox  state  of  the  mitochondrion.    With  reduced  levels  of  
OXPHOS  as  when  cells  are  growing  in  high  glucose  and  using  glycolysis  
for  the  bulk  of  their  ATP  there  cristae  are  much  thinner,  more  regular  
and  this  is  called  the  condensed  state.  
 Figure  7.  These  images  show  the  
fluorescence  microscopy  and  EM    
of    osteosarcoma  and  HeLa  cells  
respectively  cells    grown  in  
glucose  (A-­‐D  and  I-­‐L)  and  
galactose  (E-­‐H  and  M-­‐P)  

 
 
In  considering  the  morphology  of  mitochondria  it  is  important  to  
understand  that  the  organelle  is  linked  in  to  the  rest  of  the  cell  not  only  
functionally  but  also  structurally.    It  forms  tight  complexes  with  the  
endoplasmic  reticulum  through  defined  protein  complexes  called  
ERMES  that  move  Ca++  between  the  two  organelles.    There  is  also  
evidence  of  direct  physical  association  with  peroxisomes  under  some  
cellular  conditions.  
1)        The  ERMES  complex  and  ER  –mitochondria  connections.    Michel  AH.  Kornmann  B.  
Biochem.  Soc.  Trans.  (2012);  40:  445–450  

 
THE  MECHANISM  OF  FISSION  AND  FUSION  OF  MITOCHONDRIA:  KEY  
PLAYERS.  
 
The  mitochondrial  fusion-­‐fission  process  is  critical  to  maintaining  
mitochondrial  quality  control.    Fission  allows  separation  of  the  healthy  
mitochondrial  segments  from  the  defective  ones.    

Mitochondrial  fusion  can  be  divided  two  processes,  the  fusion  of  the  
mitochondrial  outer  membrane  followed  by  that  of  the  inner  
membrane.  The  outer  membrane  fusion  requires  proteins  known  as  
mitofusins,  namely  Mfn1  and  Mfn2.    Inner  membrane  fusion  mainly  
involves  the  inner  membrane-­‐localized  protein,  Opa1.      
Mfn1  and  Mfn2,  are  transmembrane  dynamin-­‐related  GTPases  which  
induce  the  joining  of  2  mitochondrial  “fragments”  by  forming  dimers  
across  the  interface.  The  tethering  of  mitochondria  together  is  followed  
by  GTP  hydrolysis,  which  induces  conformation  changes  to  cause  
mitochondrial  fusion  in  a  SNARE-­‐like  mechanism.    Mfn2  shows  tissue  
specific  expression  whereas  Mfn1  shows  ubiquitous  expression.  
Mitofusins  activity  is  regulated  by  ubiquitination,  which  causes  their  
degradation  in  response  to  stress.  The  control  of  this  degradation  
involves  several  proteins  including  PINK1,  Parkin,  E3  ligase  Huwe1,  
MULAN  and  Bcl-­‐2  family  members.    

Opa1,  a  mitochondrial  protein,  is  named  based  on  its  identification  as  a  
mutated  gene  in  optic  atrophy.  It  is  a  dynamin-­‐related  GTPase    that  
interacts  with  cardiolipin,  a  mitochondrial  inner  membrane  lipid.  Opa1  
is  localized  to  mitochondria  and  in  lipid  droplets.  In  the  case  of  
mitochondria,  most  of  the  Opa1  is  found  in  cristae,  consistant  with  its  
role  in  maintaining  cristae  morphology.  

The  mitochondrial  fusion  activity  of  Opa1  involves  the  coordinated  

action  of  a  long  isoform  of  protein  and  its  cleaved  short  isoform.  The  
Opa1  long  isoform  is  located  in  the  mitochondrial  cristae  membrane,  
while  the  short  isoform  is  soluble  in  the  intermembrane  space.  Opa1  
proteolytic  cleavage  is  regulated  by  the  mitochondrial  membrane  
potential,  apoptosis,  ATP  level  and  mtDNA  stability.  

FIGURE  8.    
Schematic  of  the  
fission-­‐fusion  
process.  

 
  
 
Mitochondrial  fission  involves  the  protein  Drp1,  another  GTPase.On  
translocation  from  cytosol  to  mitochondria,  this  protein  oligomerizes  
into  an  X-­‐shaped  dimer  on  mitochondrial  outer  membrane.    The  binding  
sites  for  Drp1  association  include  endoplasmic  reticulum  (ER)-­‐
mitochondria  contact  points.    When  localized  to  the  mitochondrial  outer  
membrane,  Drp1  rims  the  mitochondria  in  multimeric  spirals  at  the  
constriction  site,  with  the  GTPase  domain  pointing  away  from  the  
membrane.  Formation  of  a  complete  spiral  is  thought  to  activate  the  
GTPase  domain  causing  GTP  hydrolysis  leading  to  a  spiral  constriction.    

 In  apoptosis,  Drp1  is  also  involved  in  Bax  oligomerization  on  the  outer  
membrane  and  cytochrome  c  release.  Functioning  of  Drp1  is  controlled  
by  post-­‐translational  modifications  including  phosphorylation  by  GSK3,  
S-­‐nitrosylation,  ubiquitination,  SUMOylation  and  O-­‐linked-­‐N-­‐acetyl-­‐
glucosamine  glycosylation.    

A  second  protein  important  for  mitochondrial  fission  is  human  fission  

protein  1  homologue,  Fis1.  This  protein  binds  to  the  mitochondrial  
outer  membrane  with  the  help  of  C-­‐terminal  transmembrane  domain.  
Unlike  Drp1,  Fis1  is  distributed  evenly  on  outer  mitochondrial  
membrane  surface,  and  appears  as  punctate  complexes.  Functionally  
Fis1  is  involved  in  binding  of  Drp1  (at  least  in  yeast).    Fis1  
overexpression  amplifies  mitochondrial  fragmentation,  suggesting  that  
it  is  a  limiting  factor  for  mitochondrial  fission.  Its  expression  is  
regulated  via  ubiquitination  by  MITOL,  a  mitochondrial  ubiquitin  ligase.  
Note  that  two  of  the  proteins  involved  in  maintaining  the  mitochondrial  
reticulum  are  Pink  1  and  Parkin.    Mutations  of  either  of  these  two  
proteins  causes  early  onset  Parkinsons  disease,  hence  the  great  interest  
in  mitochondrial  dynamics  of  those  interested  in  neurological  diseases.  
1).        The  dynamin  GTPase  Opa1:  more  than  mitochondria?    Belenguer  P.  Pellegrini  L.  
Biochim.Biophys  Acta  2013;  1833:  176-­‐183  

2).        Mitochondrial  fusion  and  fission  in  cell  life  and  death.  Westermann  B.  
Nat.Rev.Mol.Cell.Biol.  2010;  11:872-­‐84  

3).        Mitofusins  and  OPA1  mediate  sequential  steps  in  mitochondrial  membrane  fusion.  
Song  Z.  Ghochani  M.  McCaffrey  JM.  Frey  TG.  Chan  DC.  Mol.  Biol..  Cell  2009;  20:3525-­‐
32  
4).      Mitochondrial  dynamics  in  cell  death  and  neurodegeneration.  Cho  DH.  Nakamura    
T.  Lipton  SA.  Cell  Mol.  Life  Sci.  2010;  67:  3435-­‐47.  

5). Mitochondria: from cell death executioners to regulators of cell differentiation.

Kasahara A. Scorrano L. Trends Cell Biol. 2014 Sep 1
 

     
   
MITOCHONDRIAL  DNA:  origin  and  nature.  
 
Organisms  that  perform  oxidative  phosphorylation  using  molecular  
oxygen  as  the  terminal  electron  acceptor  have  mitochondria.    There  are  
extreme  differences  in  overall  genome  sizes  among  this  array  of  
organisms,  ranging  from  only  16  kb  (in  humans)  to  more  than  500  kb.  
Most  mitochondrial  genomes  contain  between  12  and  20  protein-­‐coding  
genes:    (humans  contain  13  as  shown).  The  mitochondrial  genome  of  
Plasmodium  falciparum  is  one  of  the  simplest,  with  only  two  protein-­‐
coding  genes.  In  contrast,  the  mitochondrial  genome  of  Saccharomyces  
cerevisiae  is  85  779  bp  and  encodes  two  rRNAs,  24  tRNAs  and  30  
proteins.    

Figure  9.    Human  mtDNA  is  

closed  circular  and  encodes  7  
proteins  of  complex  I,  
cytochrome  b  of  complex  III,  3  
subunits  of  cytochrome  c  
oxidase  and  2  proteins  of  the  
ATP  synthase.    In  addition  the  
DNA  encodes  multiple  tRNA  
genes  along  with  12s  and  16s  
rRNAs.  

 
In  all  cases  mitochondrial  DNA  is  closed  and  circular  in  structure  like  
that  the  DNA  of  bacteria,  supporting  the  endosymbiotic  theory  of  
mitochondrial  origin,  and  uses  a  different  genetic  code  than  nuclear  
DNA.    The  DNA  stands  are  not  well  protected  as  by  chromatin  in  the  
nucleus.    Rather,  several  mtDNA  copies  are  bundled  together  in  clusters  
with  several  DNA-­‐binding  proteins  in  so-­‐called  nucleoids.  

There  is  very  limited  repair  of  mtDNA  with  the  result  that  mutations  
readily  accumulate.      Of  considerable  importance,  unlike  the  nuclear  
genome  which  consists  of  a  paternal  and  a  maternal  copy  of  each,  there  
are  anywhere  from  20-­‐  to  several  thousand  copies  of  mtDNA  in  
mammalian  cells,  all  maternal  in  origin.  This  leads  to  the  concept  of  
heteroplasmy,  which  typifies  many  diseases  caused  by  mutations  in  
mtDNA.  

FIGURE  10.  Images  of  the  reticulum  of  osteosarcoma  cells  in  which  mitochondria  
are  labeled  with  mitotracker  red  and  individual  mtDNA  molecules  identified  by  
hybridization  to  green  fluorescently  labeled  DNA  probes.  

   Heteroplasmy  can  arise  from  de-­‐novo  mutations  of  the  mtDNA  but  is  
much  more  often  inherited  by  the  cells  receiving  a  mixture  of  mtDNA  
copies  from  the  egg.    Penetrance  as  it  applies  to  inheritance  of  the  
mitochondrial  genome  thus  depends  on  the  copy  number  of  functioning  
mtDNA  needed  to  make  the  essential  mitochondrially  encoded  proteins  
for  a  particular  cellular  function.  Such  a  threshold  for  functioning  is  
different  in  different  cells  depending  on  their  energetic  need  and  extent  
of  reliance  on  oxidative  phosphorylation.    Note  that  damaged  mtDNA  
copies  can  and  are  removed  as  part  of  the  process  of  mitophagy,  the  
process  in  which  cells  remove  mitochondria  using  the  autophagic  
pathway.  (see  later)  
1) Mitochondrial  threshold  effects.  Rossignol  R,  Faustin  B,  Rocher  C,  Malgat  M,  Mazat  JP,  
Letellier  T.  Biochem  J;370:751-­‐62.  
  
 2).    Mitochondrial  nucleoids  maintain    genetic  autonomy  but  allow  for  functional  
complementation.    Gilkerson  RW.  Schon  EA,    Hernandez  M.  Davidson  MM.    J  Cell  Biol  
2008;181,  1117    

3).      Maternal  inheritance  of  mitochondrial  DNA  by  diverse  mechanisms  to  eliminate  
paternal  mitochondrial  DNA.  Sato  M.  Sato  M.  Bichim.  Biophys.  Acta  2013;1833:  
1979-­‐84  

Mitochondria  contribute  to  intermediary  metabolism  through  

much  more  than  OXPHOS.  

Based  on  the  prior  section  it  follows  that  mitochondria  have  proteins  
contributed  by  2  genomes  i.e  from  mt  DNA  and  from  the  nuclear  genes.  

The  synthesis  of  mtDNA  encoded  proteins  occurs  within  mitochondria  

on  ribosomes  that  have  many  of  the  characteristics  of  those  found  in  
bacteria,  and  different  from  the  cytosolic  ribosomes  that  convert  the  
nuclear  genes  to  proteins.    
1).        Making  proteins  in  the  powerhouse.    Haiilberg  BM.  Larsson  NG.  Cell  Metab.  2014;  
20:  226-­‐240  

Cells  derive  their  energy  predominantly  from  glucose  or  other  sugars  
depending  on  species.    When  glucose  levels  are  low,  they  can  use  
degradation  of  fatty  acids  and  even  amino  acids  as  a  source  for  energy  
production.    Mitochondria  are  the  center  of  this  energy  production  and  
play  a  key  role  in  generating  carbons  for  cell  growth,  repair  and  
reproduction  i.e  in  so-­‐called  intermediary  metabolism.  This  central  role  
is  shown  in  Figure  11  with  pathways  involving  mitochondria  shown  in  
yellow).      

The  extensive  way  that  the  organelle  contributes  to  cell  homeostasis,  
and  overall  viability,  is  clearly  demonstrated  by  proteomic  analyses  of  
mitochondria  from  various  sources.  Lists  of  those  proteins  identified  to  
date  as  having  a  mitochondrial  location  are  presented  in  several  recent  
publications.    
1).        Characterization  of  the  human  heart  mitochondrial  proteome.  Taylor  SW1,  Fahy  E,  
Zhang  B,  Glenn  GM,  Warnock  DE,  Wiley  S,  Murphy  AN,  Gaucher  SP,  Capaldi  RA,  Gibson  BW,  
Ghosh  SS.  Nat  Biotechnol.  2003;  21:281-­‐6.    
2).        The  mitochondrial  proteome  database:  MitoP2  Elstner  M1,  Andreoli  C,  Klopstock  T,  
Meitinger  T,  Prokisch  HMethods  Enzymol.  2009;457:3-­‐20.    
3).        The  proteome  of  Saccharomyces  cerevisiae  mitochondria.  Sickmann  A,  Reinders  J,  
Wagner  Y,  Joppich  C,  Zahedi  R,  Meyer  HE,  Schönfisch  B,  Perschil  I,  Chacinska  A,  Guiard  B,  
Rehling  P,  Pfanner  N,  Meisinger  C.  Proc  Natl  Acad  Sci  U  S  A.  2003  
 

Pentose
Glycogen
Glycolysis Phosphate
Storage
Pathway

Gluconeogenisis N&C
Amino Acid Urea Cycle
Catabolism
pyruvate amino acids

PDH
peroxisomal metabolism
! Oxidation
acetyl CoA FIGURE  11  
Krebs Cycle
Schematic  of  
mitochondrial metabolism
Intermediary  
oxidative
phosphorylation
Metabolism  
Ketogenesis

Triacylglycerol

 
Synthesis

   
 
 Of  note,  a  significant  number  of  mitochondrial  proteins  have  
counterparts  in  other  parts  of  the  cell,  while  many  are  transient  visitors  
as  a  part  of  signaling  pathways  that  tie  mitochondrial  functioning  to  
other  cellular  processes  such  as  autophagy  and  mitophagy.      

Some  of  the  proteins  earlier  claimed  to  be  exclusive  to  mitochondria  
also  turn  out  to  have  different  cellular  locations.    Examples  are  the  ATP  
synthase  that  has  been  shown  to  also  reside  on  the  plasma  membrane  of  
several  different  human  cells  and  in  particular  in  transformed  cells.    
Also  pyruvate  dehydrogenase  has  been  found  in  the  nucleus  where  it  is  
thought  to  provide  acetyl  CoA  moieties  for  histone  modification.    

 The  mechanism  of  oxidative  phosphorylation,  the  workings  of  the  

Krebs  cycle,  conversion  of  lipids  to  acetyl  CoA  and  the  urea  cycle  
pathway  are  all  well  covered  in  standard  textbooks.    Each  pathway  is  
integrated  into  concerted  metabolism  by  signaling  through  
concentrations  of  metabolites  and  by  a  set  of  signaling  pathways  that  
change  the  levels  of  specific  proteins  and/or  post-­‐translational  
modifications  in  response  to  cell  stress.    This  stress  can  take  the  form  of  
substrate  depletion  e.g.  glucose  levels  which  are  monitored  by  the  
insulin  pathway,  and  by  signaling  to  start  mitophagy  or  apoptosis.    

Of  particular  interest  is  the  change  in  metabolism  that  accompanies  

transformation  of  cells  in  cancer.  The  altered  metabolism  of  cancer  cells  
was  first  noted  by  Warburg  and  is  now  called  the  Warburg  Effect.    
Cancer  cells  make  the  energy  for  their  growth  and  replication  by  
glycolysis  rather  than  oxidative  phosphorylation  even  when  oxygen  is  
plentiful.  They  source  their  carbons  for  building  new  proteins  and  fats  
mainly  from  amino  acids,  particularly  glutamine  and  serine  in  a  process  
called  anapleurosis.  

   

FIGURE  12.    Use  

of  amino  acids  
and  fats  as    
carbon  source.    
Proteins  in  
green  are  
mitochondrial.  

 
1).      The  diverse  functions  of  glutamine  in  metabolism,  cell  biology  and  cancer.  
DeBerardinis  RJ.    Cheng  T.  Oncogene.  2010  Jan  21;29(3):313-­‐24  
2).          How  does  the  metabolism  of  tumour  cells  differ  from  that  of  normal  cells.  
Amoêdo  ND,  Valencia  JP,  Rodrigues  MF,  Galina  A,  Rumjanek  FD.  Biosci  Rep.  2013;  
33(6).  456-­‐73  
3).          Defects  in  mitochondrial  metabolism  and  cancer.  Gaude  E.  Frezza  C.  Cancer  metab.2014;  
2.  10-­‐20  

Mitochondria  as  the  key  generator  of  free  radicals  in  the  cell.  
It  has  been  known  for  many  years  that  oxidative  phosphorylation  
generates  more  than  energy  and  bi-­‐products  water  and  carbon  dioxide.  
In  an  oxygen  environment,  reduction  of  O2  is  not  entirely  efficient  with  
the  result  that  both  oxygen  free  radicals  and  nitrogen  free  radicals  are  
formed.    Electron  transfer  through  complexes  I  and  III  of  the  electron  
transfer  chain  are  considered  to  produce  the  majority  of  free  radicals,  
but  other  enzymes  including  alpha  ketoglutarate  dehydrogenase  are  
thought  to  contribute.    

   

FIGURE  13  
Different  forms  of  
ROS  generated  by  
mitochondria  

 
Generation  of  ROS  is  both  good  and  bad.  H2O2  is  a  signaling  molecule  
within  the  cell  (see  text  box)  but  it,  along  with  other  reactive  oxygen  and  
nitrative  species,  are  damaging  to  the  organelle  and  the  cell  more  
broadly.    They  modify  proteins,  DNA  and  lipids  both  inside  
mitochondria  and  in  the  cytosol.    

Mitochondria  and  the  cytosol  contain  free  radical  scavengers  including  

both  small  molecules  such  as  glutathione  and  proteins  such  as  
superoxide  dismutases,  thioredoxins  and  peroxiredoxins.  However  with  
high  rates  of  ROS  production  these  can  be  overwhelmed  and  damage  
induced.    
1) Mitochondrial  production  of  reactive  oxygen  species.  Grivennikova  VG,  
Vinogradov  AD.Biochemistry  (Mosc).  2013;78:1490-­‐511.    
2) Unearthing  the  secrets  of  mitochondrial  ROS  and  glutathione  in  bioenergetics.  
Mailloux  RJ,  McBride  SL,  Harper  ME.  Trends  Biochem  Sci.  2013;38:592-­‐602.  
3) The  Yin  and  Yang  of  redox  regulation.Olsen  LF,  Issinger  OG,  Guerra  B.Redox  Rep.  
2013;18:245-­‐52.  
 
Retrograde  signaling  from  mitochondria  to  the  rest  of  the  cell.  

The  level  of  mitochondria  in  a  cell  is  a  balance  between  biogenesis  of  
new,  and  destruction  of  damaged,  organelle.  The  biogenesis  of  
mitochondria  is  controlled  by  specific  cell  signaling  pathways  involving  
PGC1,  PPARs  and  other  nuclear  transcription  factors    

Dysfunctional  mitochondria  result  from  any  of  several  cellular  events.  

Cells  must  continually  respond  to  stress  events  whether  induced  
externally  e.g.  lack  of  oxygen  or  other  essential  substrates,  or  internally  
e.g.  ROS  production,  failure  of  proteins  to  fold  properly  etc.  Any  of  these  
conditions  will  transiently  or  permanently  adversely  affect  
mitochondrial  functioning.    

Among  the  signals  from  the  organelle,  the  altered  ratio  of  ATP  to  ADP,  
Ca++  accumulation  inside  the  mitochondria,  excess  ROS  production,  
altered  metabolite  shuttling  e.g.  of  citrate,  malate  etc.  and  loss  of  
membrane  potential  are  all  recognized  by  the  interconnected  signaling  
pathways  in  the  cell.    The  signaling  from  mitochondria  to  alter  
metabolism  more  globally  is  called  the  retrograde  signaling  response.  

7:"78B7!%:(9=B)!<=)B(G78&(&="8?*8)%7=!'(

!"#$!%#$!&#'(
HI,/,(!"#( FIGURE  14.  Different  
/,JC@/,K(L3/(
4/01A23/4( -4>#7'( retrograde  signals  from  
mitochondria  to  the  
&:"!;8<=":(
9*>""<:9( cytosol,  ER  and  nucleus.  
?@4/04,(
&0604,( )!%*$)!%+(
!A20/404,( )!%#*$)!%#+'(
B6C40-04,( 789$7)9'(
#D/CE04,(
!F,4D6(?3!( ?06F@C-(
(
&,-./01,( #78":=)(=&#87"$()N0220(;O(
HI,/,(-,-./01,( 234,1506'( !%*:7:)?:(;?6PO(#=)QPO&?6P(
234,1506$2/3431( ,4F(!)%(7:<:!9:'(
M/0K@,14(K/@E,A(4/01A23/4( !=GO?D43FI/3-,(F(,4F'(
(  
The  response  to  mitochondrial  dysfunction  can  take  the  form  of  
organelle  repair  such  as  when  there  is  a  build  up  of  unfolded  proteins,  in  
the  same  way  as  when  there  is  an  accumulation  of  unfolded  proteins  in  
the  ER.  The  key  protein  in  the  mitochondrial  UPR  is  the  molecular  
chaperone  HSP60,  the  levels  of  which  signal  the  JNK  pathway  and  PKR.    

FIGURE  15.  Key  

e  
players   in  the  
mitochondria  
unfolded  protein  
response  (yellow)  
and  ER  UPR  (blue)  

 
Alternatively  there  can  be  destruction  of  the  dysfunctional  
mitochondria  by  a  process  now  called  mitophagy  if  full  repair  is  not  
possible.  This  is  shown  in  the  figure  for  build  up  and  damage  by  ROS.  

FIGURE  16.  Free  

radicals  elicit  a  
retrograde  singaling  
response  thst  leads  
to  an  attempt  to  
repair  the  damage  
(autophagy,  
mitophagy)  or  to  cell  
death(apoptosis)  

 
1).      Mitochondrial  retrograde  signaling  at  the  crossroads  of  tumor  bioenergetics,  genetics  and  
epigenetics.  Guha  M,  Avadhani  NG.  
Mitochondrion.  2013;13:577-­‐91.    
  
2).      Mitochondrial  quality  control  and  communications  with  the  nucleus  are  important  in  
maintaining  mitochondrial  function  and  cell  health.  Kotiadis  VN,  Duchen  MR,  Osellame  LD.  
Biochim  Biophys  Acta.  2014;1840:1254-­‐65.  
 
3).      Evaluating  and  responding  to  mitochondrial  dysfunction:  the  mitochondrial  unfolded-­‐
protein  response  and  beyond.  Haynes  CM,  Fiorese  CJ,  Lin  YF.Trends  Cell  Biol.  2013;23:311-­‐8.  
 
4). Mitochondrial stress signaling in longevity: A new role for mitochondrial function in aging. Hill
S. Van Remmen H. Redox Biol. 2014 Jul 27;2:936-944
 
 

Protein  post-­‐translational  modifications  in  the  control  of  

metabolism:  phosphorylation  and  acetylation  and  more.  

Many  proteins  in  mitochondria  are  subject  to  phosphorylation/  

dephosphorylation  reactions  at  Thr,  Ser  and  Tyr  residues  via  
mitochondrial  kinases  and  phosphatases.    The  phosphoproteome  of  the  
organelle  in  humans  has  been  extensively  characterized.    Such  
modifications  are  particularly  important  in  the  control  of  oxidative  
phosphorylation  as  well  as  in  lipid  metabolism.  
1)      Phosphoproteome  analysis  reveals  regulatory  sites  in  major  pathways  of  cardiac  
mitochondria.  Deng  N,  Zhang  J,  Zong  C,  Wang  Y,  Lu  H,  Yang  P,  Wang  W,  Young  GW,  
Wang  Y,  Korge  P,  Lotz  C,  Doran  P,  Liem  DA,  Apweiler  R,  Weiss  JN,  Duan  H,  Ping  P.  
Mol  Cell  Proteomics.  2011  Feb;10(2):M110.000117  
2)      A  quantitative  map  of  the  liver  mitochondrial  phosphoproteome  reveals  
posttranslational  control  of  ketogenesis.  Grimsrud  PA,  Carson  JJ,  Hebert  AS,  Hubler  
SL,  Niemi  NM,  Bailey  DJ,  Jochem  A,  Stapleton  DS,  Keller  MP,  Westphall  MS,  Yandell  
BS,  Attie  AD,  Coon  JJ,  Pagliarini  DJ.  Cell  Metab.  2012  Nov  7;16(5):672-­‐83.  
 

The  acetylation  of  lysine  residues  by  several  acetylases  in  the  organelle  
and  deacetylation  by  a  set  of  proteins,  collectively  called  sirtuins,  also  
regulates  oxidative  phosphorylation,  as  well  as  controlling  the  enzymes  
of  the  urea  cycle,  fatty  acid  oxidation  and  antioxidant  proteins.  At  least  3  
of  the  7  well-­‐defined  sirtuins,  i.e.  3,  4  and  5,  are  mitochondrial  in  
location.  These  deacetylases  has  drawn  widespread  attention  because  of  
suggestions  that  their  modulation  may  alter  longevity.  For  example  
there  is  considerable  work  on  the  natural  product  resveratrol,  an  
inhibitor  of  sirtuins,  as  an  anti-­‐aging  compound.  
1)      Mitochondrial  sirtuins  in  the  regulation  of  mitochondrial  activity  and  metabolic  
adaptation.  Lombard  DB,  Tishkoff  DX,  Bao  J.  Handb  Exp  Pharmacol.  2011;206:163-­‐88  
2)      Mitochondrial  protein  acetylation  as  a  cell-­‐intrinsic,  evolutionary  driver  of  fat  storage:  
chemical  and  metabolic  logic  of  acetyl-­‐  lysine  modifications.  Ghanta  S.  Grossman  RE,  Brenner  
C.  Crit  Rev  Biochem  Mol  Biol.  2013  ;  48:  561–574.  

3)      SIRT3  regulates  mitochondrial  fatty-­‐acid  oxidation  by  reversible  enzyme  deacetylation.  

Hirschey  MD1,  Shimazu  T,  Goetzman  E,  Jing  E,  Schwer  B,  Lombard  DB,  Grueter  CA,  Harris  C,  
Biddinger  S,  Ilkayeva  OR,  Stevens  RD,  Li  Y,  Saha  AK,  Ruderman  NB,  Bain  JR,  Newgard  CB,  
Farese  RV  Jr,  Alt  FW,  Kahn  CR,  Verdin  E.  Nature.  2010;  464:121-­‐5  

4)      SIRT5  regulates  the  mitochondrial  lysine  succinylome  and  metabolic  networks.  Rardin  
MJ1,  He  W,  Nishida  Y,  Newman  JC,  Carrico  C,  Danielson  SR,  Guo  A,  Gut  P,  Sahu  AK,  Li  B,  
Uppala  R,  Fitch  M,  Riiff  T,  Zhu  L,  Zhou  J,  Mulhern  D,  Stevens  RD,  Ilkayeva  OR,  Newgard  CB,  
Jacobson  MP,  Hellerstein  M,  Goetzman  ES,  Gibson  BW,  Verdin  E.  Cell  Metab.  2013;18:920-­‐33  
 
 
Mitophagy:  the  removal  of  damaged  mitochondria.  

Mitochondrial  quality  control  is  essential  for  the  health  of  a  cell.    
Mitochondrial  functioning  is  constantly  being  evaluated.  As  discussed  
above  in  the  event  of  a  build  up  of  unfolded  proteins,  a  response  is  
mounted  in  which  protein  synthesis  is  temporarily  suspended  and  the  
unfolded  proteins  are  removed  by  mitochondrial  AAA  proteases  
including  Lon.    A  second  cleansing  mechanism  involves  vesicular  
transport  of  defective  mitochondrial  proteins  to  the  lysosome  for  
degradation.  Finally,  whole  mitochondria  (fragmented  form)  can  be  
removed  by  mitophagy..

 
An  example  of  particular  importance  is  in  the  inheritance  pattern  of  
mitochondria.  As  discussed  before,  on  fertilization  of  an  egg,  the  male  
sperm  mitochondria  are  all  destroyed.  Specifically  they  are  
programmed  for  destruction  by  mitophagy  by  prior  widespread  
ubiquitinylation  of  outer  membrane  proteinsAs  would  be  expected,  the  
trigger  for  mitophagy  is  dysfunction  of  mitochondria.    The  key  signal  is  
loss  of  membrane  potential,  which  induces  fragmentation  of  the  
organelle  and  initiates  association  of  proteins  involved  in  the  mitophagy  
process.    The  best-­‐characterized  form  of  mitophagy  is  that  initiated  by  
the  pink1/parkin  reaction.  In  healthy  mitochondria,  PINK1  is  imported  
to  the  inner  mitochondrial  membrane,  presumably  through  the  
TOM/TIM  complex.  The  TIM  complex-­‐associated  protease,  
mitochondrial  MPP,  cleaves  PINK1  mitochondrial  targeting  sequence  
(MTS).  PINK1  is  also  cleaved  by  the  inner  membrane  presenilin-­‐
associated  rhomboid-­‐like  protease  PARL  and  ultimately  proteolytically  
degraded.  Loss  of  membrane  potential  in  damaged  mitochondria  
prevents  the  import  of  PINK1  leading  to  the  accumulation  of  
unprocessed  PINK1  on  the  outer  membrane  surface  where  it  associates  
with  the  TOM  complex,  and  recruits  cytosolic  Parkin  and  promotes  
mitophagy  of  damaged  mitochondria  in  two  major  ways.  (i)  In  one  
mechanism,  Parkin,  presumably  through  its  ubiquitin–  ligase  activity,  
causes  the  degradation  of  its  substrates  such  as  Miro  and  Mitofusin.      

Also  involved  are  BCL-­‐2  and  BCL-­‐XL  two  anti-­‐apoptotic  proteins  that  
bind  the  essential  autophagy  protein  BECLIN-­‐1  to  prevent  its  activation,  
and  disruption  of  BCL-­‐2  dissociation  also  allows  BECLIN-­‐1  activation  by  
AMBRA1.  In  order  to  facilitate  phagophore  formation,  AMBRA1  
translocates  to  the  mitochondria  and  ER  after  initiation  of  autophagy  

Other  proteins  involved  in  regulating  mitophagy  are  BNIP3  and  

BNIP3L/NIX,  two  pro-­‐apoptotic  BH3-­‐only  proteins  that  cause  
permeabilization  of  the  mitochondrial  membrane  via  opening  of  the  
mitochondrial  permeability  transition  pore  or  activation  of  BAX/BAK.    
BNIP3  resides  at  mitochondria,  and  appears  to  have  distinct  functions  in  
mitophagy/autophagy  and  in  apoptosis.    Both  BNIP3  and  NIX  interact  
directly  with  LC3  on  the  phagophore  to  tether  mitochondria  to  forming  
autophagosomes.  Mitochondrial  fragmentation  and  arrest  of  motility,  
through  loss  of  Mitofusin  and  Miro,  quarantine  damaged  mitochondria  
and  promote  their  autophagosomal  engulfment.  (ii)  Alternatively  or  in  
addition,  Parkin-­‐  mediated  hyper-­‐ubiquitination  of  the  mitochondrial  
outer  membrane  is  recognized  by  ubiquitin-­‐binding  adaptors,  such  as  
p62,  HDAC6,  and  unknown  others,  that  may  recruit  damaged  
mitochondria  to  the  isolation  membrane  through  their  interaction  with  
the  autophagosomal  protein  L3.  
1) Mitochondrial quality control in the myocardium: Cooperation between protein
degradation and mitophagy. Hammerling BC. Gustafsson AB. J Mol Cell Cardiol.
2014 Jul 30;75C:122-130.
2) Mitochondrial quality control and communications with the nucleus are important in
maintaining mitochondrial function and cell health. Kotiadis VN, Duchen MR, Osellame
LD. Biochim Biophys Acta. 2014;1840:1254-65.
3). Parkin and PINK1: much more than mitophagy. Scarffe LA, Stevens DA,
Dawson VL3 Dawson TM. Trends Neurosci. 2014;37:315-24
4) Mitochondrial dismissal in mammals, from protein degradation to mitophagy.
Campello S. Strappazzon F. Cecconi F. Biochim Biophys Acta. 2014;1837:451-60.
 

FIGURE  17.  Key  players  

in  mitophagy.    The  upper  
image  shows  the  
different  roles  of  BNIP3  
in  cell  death  versus  
  autophagy/mitophagy.  
The  lower  image  shows  
  the  parkin/pink1  
  pathway  of  mitophagy.  

 
Mitochondrially-­‐directed  apoptosis.  
 
Apoptosis  can  be  signaled  from  outside  the  cell  by  signaling  molecules  
and  growth  factors  as  in  development  of  tissues  and  organs  (extrinsic),  
or  from  within  the  cell  in  response  to  various  cell  stress  events  
(intrinsic).  Intrinsic  apoptosis  can  be  a  response  to  ER  stress  (as  shown  
in  the  figure)  or  from  mitochondrial  dysfunction,  DNA  modifications,  
loss  of  energy  or  other  substrate  molecules.  
J*>5+ 1<55+F<6GH+
J*>+
J6FF+ 15<6I<F+
<=+>#)?>>+ B6=B+

1*>7*>?+@+
7)$%*>7*>?;+
12?*E?(+
0"(+ 1*>7;+
BL!*+
7;@A6BC+
0%24.5+ 0"!+ A129+

0123+ !"#$%&$'()"*+ %*>7*>?D+

6KJ+
7:;+ M$.*+ 0*(+
!" ,*.+
#$% ,*-+ %/#%+
&'& 67#$>$!?+
() 67*849+
$
B)$%*>7+;+
 

FIGURE  18.  The  key  players  in  apoptosis.  MCl1  and  BCl-­‐xl  are  anti-­‐apoptotic  
proteins  on  the  mitochondrial  outer  membrane.  Noxa,  Bcl2,bax,  bak  bid  bad  and  
puma  are  proapoptotic  proteins  that  respond  to  cell  signals  by  interacting  with  
the  organelle.  

Whether  initiated  externally  or  internally,  mitochondria  play  an  

important  role  in  the  cell  death  process  and  are  often  called  the  central  
executioner.  

The  key  is  that  the  mitochondrial  outer  membrane  contains  or  has  
attached  a  set  of  anti-­‐apoptotic  proteins  that  protect  the  cell  from  death.  
During  apoptosis  these  are  neutralized  by  altered  interactions  and/or  
proteolytic  digestion.  The  outcome  of  this  is  release  of  molecules  from  
the  mitochondria,  specifically  cytochrome  c,  AIF,  endonuclease  G,  Smac  
and  OMI/HtrA2.  

The  release  of  cytochrome  c  induces  formation  of  the  aptosome  which  
in  turn  activates  a  caspase  cascade  that  leads  to  cleavage  of  proteins  and  
DNA  and  further  degradation  of  these  fragments  by  uptake  into  
macrophages  and  other  inflammatory  cells.    

As  shown  above,  during  apoptosis  the  outer  membrane,  and  as  a  result  
the  mitochondrial  permeability  transition,  is  kept  in  check  by    a  set  of  
proteins  of  the  Bcl-­‐2  family.  The  prototype  member  of  this  family,  Bcl-­‐2  
itself,  was  initially  identified  in  a  common  form  of  B-­‐cell  lymphoma,  
where  a  chromosome  translocation  causes  overproduction  of  the  Bcl-­‐2  
protein.  The  high  levels  of  Bcl-­‐2  promote  cancer  by  inhibiting  apoptosis,  
thereby  prolonging  cell  survival.    

More  than  20  members  of  the  Bcl-­‐2  family  have  been  identified,  all  
defined  by  the  presence  of  one  to  four  Bcl-­‐2  homology  (BH)  domains.    
The  proapoptotic  Bcl-­‐2  proteins  can  be  further  subdivided  into  two  
subfamilies  based  on  the  sharing  of  BH  domains.  BH  multidomain  
proteins,  such  as  Bax  and  Bak,  are  the  triggers  of  apoptosis,  most  likely  
as  a  result  of  their  ability  to  form  pores  in  the  outer  mitochondrial  
membrane.  The  other  subfamily,  the  BH3-­‐only  proteins  shown  in  the  
figure,  which  contain  only  the  BH3  domain,  act  as  upstream  regulators  
by  controlling  the  allosteric  activation  of  the  gatekeepers  Bax  and  Bak.  
In  healthy  cells,  BH3-­‐only  proteins  are  either  not  expressed  or  are  
inactive,  until  rapidly  activated  following  exposure  to  cellular  stresses.  
Different  types  of  stresses  activate  distinct  sets  of  BH3-­‐only  proteins,  
suggesting  that  BH3-­‐only  proteins  act  as  essential  sensors  of  different  
death  stimuli.    

The  modulation  of  the  Bcl2  family  proteins  is  through  the  
Ub/proteasome  system.  Under  normal  growth  conditions  the  half-­‐  life  of  
Mcl1  has  been  estimated  to  be  in  the  range  of  ~40–60  min.  With  
induction  of  apoptosis,  Mcl1  is  rapidly  degraded  in  a  Ub  and  
proteasome-­‐dependent  manner.  The  apoptotic  degradation  of  Mcl1,  as  
well  as  its  turnover  in  non-­‐apoptotic  cells,  is  regulated  by  the  
counteracting  activities  of  the  Ub  ligase  ARF-­‐BP1/Mule  and  the  
deubiquitinase  Usp9x.  Expression  levels  of  ARF-­‐  BP1/Mule  and  Usp9x  
appears  to  be  critical  for  the  maintenance  of  proper  cellular  balance  of  
anti-­‐  and  pro-­‐apoptotic  proteins,  and  contributes  to  cell  sensitivity  to  
apoptosis  and  is  linked  to  tumor.  Turnover  of  other  mitochondria-­‐
associated  Bcl-­‐2  family  proteins,  including  Bax  and  Bcl-­‐2  is  also  under  
Ub/proteasome  control.  Bax,  a  pro-­‐  apoptotic  Bcl-­‐2  family  protein,  is  
mainly  localized  in  the  cytosol  in  an  apoptotically  inactive  form  which  
moves  to  mitochondria  upon  pro-­‐apoptotic  trigger-­‐  induced  change  in  
its  conformation.    Proteasome-­‐dependent  degradation  of  Bax  occurs  
specifically  on    mitochondria,    suggesting  that  the  apoptotic  
conformation  of  Bax  might  be  recognized  by  the  Ub  conjugation  
machinery,  and  serve  as  a  degradation  signal  preventing  the  
accumulation  of  potentially  dangerous  apoptotically-­‐active  Bax  in  
healthy  cell  mitochondria.  Baxβ,  a  24-­‐kD  splice  variant  of  Bax  that  has  
shorter  half-­‐life,  and  is  a  more  efficient  pro-­‐apoptotic  protein  than  the  
more  abundant  21-­‐kD.    Baxα,  is  continuously  degraded  in  a  proteasome-­‐
dependent  manner  in  non-­‐  apoptotic  cells.  In  addition  to  Mcl-­‐1  and  Bax,  
ubiquitination  of  other  Bcl-­‐2  family  proteins,  including  Bcl-­‐2,  and  a  
truncated  form  of  Bid,  regulates  their  expression  and  activity.    

   

The  pro  –apoptotic  Bcl-­‐2  proteins  Smac  and  the  serine  protease  
Omi/HtrA2,  once  released  from  mitochondria  along  with  cytochrome  c,  
promote  apoptosis  by  counteracting  the  inhibitor-­‐of-­‐apoptosis  proteins  
(IAPs),  which  comprise  a  family  of  endogenous  caspase  inhibitors.    
Other  proteins  released  from  mitochondria,  i.e.  apoptosis-­‐inducing  
factor  (AIF)  and  endonuclease  G  promote  cell  death  in  a  caspase-­‐
independent  manner  by  inducing  chromatin  condensation  and  DNA  
degradation.  Thus,  if  for  some  reason  cells  do  not  activate  caspases  after  
MOMP,  these  mediators  might  still  ensure  that  cell  death  proceeds.  
Phagocytic  uptake  of  apoptotic  cells,  the  last  step  of  apoptosis  is  
identified  by  a  phospholipid  asymmetry  and  externalization  of  
phosphatidylserine  on  the  surface  of  apoptotic  cells.    

1). Regulating Mitochondrial Outer Membrane Proteins by Ubiquitination and

Proteasomal Degradation. Mariusz Karbowski. Richard J. Youle. Curr Opin Cell
Biol. 2011; 23: 476–482.

2). Mitochondria and cell death: outer membrane permeabilization and beyond.
Tait SW. Green DR. Nat Rev Mol Cell Biol. 2010;11:621-32.

3) How do Bax and Bak lead to permeabilization of the outer mitochondrial

membrane? Antignani A. Youle RJ. Curr Opin Cell Biol. 2006;18:685-9.

NADH  ubiquinone  oxidoreductase:  the  most  complex  electron  

transfer  complex.

NADH  ubiquinone  oxidoreductase,  also  called  Complex  I,  in  mammals  is  
a  complex  of  45  different  subunits  in  mammals,  present  in  unit  
stoichiometry  with  a  total  MW  of  over  900K.    Seven  of  these  subunits  
ND1-­‐ND6  and  ND4L  are  encoded  on  mtDNA,  the  rest  in  the  nucleus.  The  
enzyme  complex  contains  a  flavin  and  multiple  non-­‐heme  iron  centers  
as  prosthetic  group.    Seven  of  the  nDNA-­‐encoded  subunits,  NDUFV1,  
NDUFV2,  NDUFS1,  NDUFS2,  NDUFS3,  NDUFS7  and  NDUFS8,  represent  
the  “core  subunits”  in  the  peripheral  part  of  the  complex,  catalyzing  the  
oxidation  of  NADH  and  electron  transfer.  The  Q  binding  site,  responsible  
for  the  electron  transfer  to  ubiquinone,  includes  at  a  minimum  the  
NDUFS2,  NDUFS3,  NDUFS7  and  NDUFS8  subunits.    Electron  transfer  
from  NADH  to  ubiquinone  through  complex  I  is  coupled  to  proton  
translocation.

FIGURE  19.  structure  of  

Complex  I  from  T.  
thermophilus.  This  
bacterial  form  of  the  
complex  is  the  core  
element  of  the  mammalian  
enzyme.  

 
3D structure of complex I from T> thermophiles (a536kDa complex with flavin and 9 non-heme iron
centers. (from Baradaran R. Berrisford JM. Minhas GS. Sazanov LA. Nature 2013;494: 443-448

Recently, good progress has been made in understanding how such a large
complex assembles given that it contains both mitochondrially and nuclear
encoded subunits. Not surprisingly, assembly occurs in stages with the
different additions of subunits each involving assembly factors i.e. molecular
chaperones that are not components of the final complex. One of these is
AIF, apoptosis inducing factor has a dual function as it is released from the
organelle to function in apoptosis. Other assembly factors including
NDUFAF, MidA, Ind1(NUBPL) and C20orf7 were mostly identified by
genetic analysis of human mutations that led to only partially assembled
complex I
1). Understanding mitochondrial complex I assembly in health and disease.Mimaki M.
Wang X. McKenzie M. Thorburn DM. RyanMT., Biochimica et Biophysica Acta (BBA) -
Bioenergetics.2012;1817: 851‒862

A second aspect of complex I biology that is receiving widespread attention

is the sensitivity of its functioning to many environmental chemicals (see
table 1).
It has been hypothesized that inhibition of complex I activity, leading to
increased oxidative and nitrative damage of the complex, is an important
factor in several neurodegenerative diseases including Parkinsons,
Alzheimers, epilepsy and even bipolar disorder.
Several studies have identified “hot spots” for oxidative and nitrative
damage in the complex. In one set of experiments it was shown that both
oxidative damage and partial disassembly of the complex occurs in
Parkinsons disease brain. Another study has shown oxidative modification
and loss of complex I function in the brain of patients with epilepsy.
1). Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is
functionally impaired and misassembled. Keeney PM, Xie J, Capaldi RA, Bennett JP Jr. J
Neurosci. 2006;26:5256-64.
2). Nitrosylation and nitration of mitochondrial complex I in Parkinson's disease. Chinta SJ,
Andersen JK. Free Radic Res. 2011;45:53-8.
3). Post-Translational Oxidative Modification and Inactivation of Mitochondrial Complex I in
Epileptogenesis Ryan K.Baccus S. Regan. Patel P. J Neurosci. ,2012; 32: 11250–11258.

4). Mitochondrial Complex I Activity and Oxidative Damage to Mitochondrial Proteins in the
Prefrontal Cortex of Patients With Bipolar Disorder Andreazza AC. Shao L. Wang JF.Young LT.
Arch Gen Psychiatry. 2010;67:360-368

Mitochondrial deficiencies, involving mutations in the mtDNA encoded or

nuclear encoded genes of the 5 OXPHOS complexes are relatively common.
They have proved difficult to diagnose because they can occur at any age, in
any tissue, with any penetrance, and with almost any symptoms!
A simple antibody screen for altered assembly of each of the OXPHOS
complexes (the majority of cases) has been developed and validated.
However it has not been widely adopted by pathologists as it is extremely
cheap as well as easy to administer.

Complex I Function is Sensitive to Over Sixty Different Families of Chemicals

Type of Compound, Representative Potent Inhibitor Minimal I50 Assay Relative References
Source, or Main use System Potency
(Rotenone)
a) Synthetic Pesticides Sandoz 547A 2 nM NADH:Q-1 >2 M. Degli Esposti, Biochem. Biophys.
Acta 1364 (1998) 222-235
Acaricides, Insecticides, Pyridaben 2.4 nM NADH:Q-1 2
B. Gassner et al., J. Pharm. Exp. Ther.
Insecticide Synergists, and Fenpyroximate 4.6 nM NADH:Q-1 0.5 281 (1997) 855-860
Antihelmintic Mimics Benzimidazole 3 nM NADH:Q-1 1.4 A. Andreani et al., J. Med. Chem. 38
Cyhalothrin 0.6 µM NADH:DBQ 4 x10-2 (1995) 1090-1097
D.L. Gil, J. Ferreira, Xenobiotica
6-chloro-benzothiadiazole 0.1 mM NADH:O2 1x10-4
10 (1980) 7-15
2-methyl-6-(2-thienyl)imidazo[2,1-b]thiazole 40 µM NADH:Q-1 6x10-3
b) Synthetic Drugs and Toxins Amytal 0.2 mM NADH:Q-1 5x10-5 L. Ernster et al., Exp. Cell. Res. 3
(1955) 1090-1097
Sedatives, Analgesics, Demerol 0.1 mM NADH:Q-1 1x10-4 W. J Nicklas et al., Life Sci. 36 (1986)
Neurotoxins, Neuroleptics, 1-methyl-4-phenyl-pyridinium (MPP+) 0.3 mM NADH:Q-1 3x10-5 2503-2508
Antiseptics, Antihistaminics, Haloperidol 3 µM NADH:Q-1 2x10-2 K. Veitch et al., Mol. Pharmacol. 45
Antianginals, (1994) 158-163
Antihypertensives, Dequalinium chloride 11 µM NADH:O2 3x10-3 K.M. Wyatt et al. Biochem. Pharmacol.
Anesthetics, and Drug Cinnarizine 5-10 µM NADH:Q-1 1x10-3 50 (1995) 1599-1606
metabolites Ranolazine 23 µM NADH:Q-1 2x10-3
c) Phenolic Compounds Vacor 50 µM NADH:Q 5x10-3 M. Degli Esposti et al., Diabetes 45
(1996) 1531-1534
Rodenticides and Pollutants Nonyl-phenol 20 µM NADH:Q 3x10-3
d) Quinone Antagonists 7-chloro-4-octyloxy analogue 0.2 µM NADH:Q-1 5x10-2 W. Oettmeier et al., Biochem. Biophys.
Acta 1099 (1992) 262-266
Ancridones, Quinolones, 2-unidecyl-3-methyl analogue 20 nM NADH:Q-1 <0.1 M. R. Glick et al., J. Bio. Chem. 269
Quinolines, and 4-hydroxy analogue 37 10 nM NADH:O2 ca. 1 (1994) 3167-3174
Phenylpyridines 4䇻-heptyl analogue 1.7 µM NADH:O2 ca. 1
e) Fluorescent Dyes Erythrosin 5䇻-iodoacetamide 20 nM NADH:O2 0.8 W. M. Anderson et al., Biochem.
Biophys. Acta 1230 (1995) 186-193
Fluoresceins, Acridines, and Safranine 17 µM NADH:Q 2x10-3
Carbocyanines diOC6(3) 80 nM NADH:O2 0.2
f) Antibiotics Thiangazole 0.04nmol/mg NADH:Q 2 T. Freidrich et al., Eur. J. Biochem. 219
0.3 nmol/mg (1994) 691-698
Myxobacterial and others Aureothin NADH:Q 0.23
g) Plant Toxins Rotenone 0.07nmol/mg NADH:Q 1 D. J. Horgan et al., J. Biol. Chem. 243
0.03nmol/mg (1968) 5967-5976
Rotenoids, Acetogenins, and Rolliniastatin-1 NADH:Q 13 M. Degli Esposti et al., Biochem J. 301
Vanilloids 20-50 µM
Capsaicin NADH:Q 1x10-3 (1994) 161-167

1). Differential effects of mitochondrial Complex I inhibitors on production of reactive

oxygen species. Fato R. Bergamini C. Bortolus M. Maniero C. Leoni C. Tomoko
Ohnishi T. Lenaz G. Biochim Biophys Acta. 2009; 1787: 384–392.
FIGURE  20.  Evaluation  of  4  patients  with  mitochondrial  disease  
cause  by  a  different  OXPHOS  complex  or  with  PDH  deficiency.  
Fibroblasts  from  each  patient  were  cultured  and  then  stained  with  
mitotracker  red  to  identify  the  organelle  (red)  and  with  an  antibody  
to  each  of  the  five  OXPHOS  complexes  and  PDH.  Clearly  patient  1  has  
a  mutation  in  Complex  I,  patient  2  has  PDH  deficiency  while  patients  
3  and  4  are  cytochrome  c  oxidase  mutants.    
 ES))S;(T;RSO;(UOOSOV(S@()UW<RSXTV)7(
!"#$%&&&'()*+,-.,/01*23(0*4,105146(!5787%(9*+,-.,/01*23(:;<(05=35>,/(
4?/01,954'(
(
!"#"&%&&&'(@2A?(2-*0(,B*02>,/(0*4,10514((
*/-7(950*C9D-.2*/(2-?3DE,<(05.?01,85/245(05F-*5/-?(!"#G&%&&&'(
(
!"#"H%&&&'(I.5/?3J5+,/C1*2(
(
!"#"H%&&&'()5+.?3923,/*-2-*0C1*27()5+.?3923,/?3D(
E,<(9C+245%(-,K2329*/(95+2K,3*49((
((
!"#L&%&&&'(<9*/,2-*0,=2+.*54((
(
!"#H&%&&&'(I51,B*4,923(0*4,105146(5787%(M533N5851(4?/01,95%(/5,/2+23(
2015/,35CJ,0?4+1,=.?%(O5P4C9Q4(0*45245'(
(
!"#"H&%&&&'()2=35(4?1C=(C1*/5(0*45245(!RES<:'(
(

Of these, the altered assembly and or function of complex I is the most

prevalent as shown by the table of mutations causing cardiomyopathy,
Leighs syndrome, MERRF etc.

Disease Tissues/Systems Affected Mutations

Alzheimer䇻s Nervous System (NS)

Alper䇻s Syndrome NS

Barth䇻s Syndrome (X-linked dilated Cardiac and Skeletal Muscle, NS

cardiomyopathy)
Cardiomyopathy & Encephalopathy Cardiac and Skeletal Muscle, NS NDUFS2

Leber䇻s Hereditary Optic Neuropathy NS (visual loss) ND1, ND4, ND6 & tRNA Lys
(LHON)
Leigh䇻s Syndrome NS, Cardiac and Skeletal Muscle ND3, ND4, NDUFV1,
NDUFS7 & NDUFS8
Mitochondrial Encephalomyopathy, NS, Cardiac and Skeletal Muscle tRNA Leu
Lactic Acidosis, Stroke (MELAS)
Myclonic Epilepsy, Red Ragged Fibers NS, Skeletal Muscle ND1, tRNA Leu, tRNA Lys &
(MERRF) tRNA Ser
Myopathy (+ CNS) Skeletal Muscle, NS tRNA Leu, ND1, ND4 &
NDUFS1
Parkinsonism NS
Progressive External Opthalmoplegia NS, Skeletal Muscle tRNA Leu, tRNA Ala, tRNA Glu
(PEO) & tRNA Tyr
ATP SYNTHASE: A ROTARY MOTOR AND TARGET FOR MANY DRUGS.
The ATP synthase is ubiquitous to all organisms. It is in the plasma
membrane of prokaryotes along with respiratory chain proteins. In
eukaryotes it is located predominantly in the mitochondrial crista
membrane (but see later).

FIGURE  21.    Negatively  

stained  EM  image  of  sub-­‐
mitochondrial  particles  
(inverted  vesicles  of  crista  
membranes  showing  
“knobs”  of  ATP  synthase)  

Figure  22.    The  

arrangement  of  
subunits  in  the  
prokaryote  ATP  
synthase  
The prokaryote form of the enzyme, e.g. as isolated from E. coli,
contains 5 different subunits, alpha3,beta3, gamma, delta and epsilon
as shown in Figure 22.The F0 part contains 3 different subunits a, b2
and c 12 respectively. There are 3 catalytic sites per F1part, at the
interfaces of the 3alpha/beta pairs respectively.

The c subunits are arranged as a ring. The F1 part and F0 parts are
connected by two stalks, one (the rotor includes gamma and epsilon,
the other the stator contains the b subunit pair).

The eukaryote ATP synthase is more complex than that of prokaryotes.

The mammalian enzyme has the additional subunits F6, d, e, A6L, f, g
and OSCP with most of these in the so-called stalk region of the
complex.

The full X ray structure of the yeast ATP synthase has been obtained,
while the full structure of all of the segments of the ATP synthase of
mammals have been determined and modeled into the unit complex.

The structure of the ATP synthase is an important clue to the workings

of this complex as first recognized by Boyer in his alternating site
hypothesis, and made clearer as the detailed X-ray data emerged.

It is a rotary motor, as shown most convincingly in the videos listed

below. Real time visualization of the rotation of the gamma subunit
against the alpha3beta3 ring has been presented, also seen in the
vidoes.

1) The ATP synthase--a splendid molecular machine.Boyer PD.

Annu Rev Biochem. 1997;66:717-49.

2) The structure of bovine F1-ATPase in complex with its regulatory protein

IF1.Cabezón E, Montgomery MG, Leslie AG, Walker JE. Nat Struct Biol.
2003;10:744-50.
3) Molecular architecture of the rotary motor in ATP synthase. Stock D,
Leslie AG, Walker JE. Science. 1999;286:1700–1705.
4) The structure of the membrane extrinsic region of bovine ATP synthase.
Rees DM, Leslie AG, Walker JE. Proc Natl Acad Sci USA.
2009;106:21597–21601
5) Structure of the c(10) ring of the yeast mitochondrial ATP synthase in the
open conformation. Symersky J. Pagadala V, Osowski D, Krah A, Meier T,
Faraldo-Gómez JD, Mueller DM. . Nat Struct Mol Biol. 2012;19:485-91
6) Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the
mitochondrial cristae. Davies KM. Anselmi C, Wittig I, Faraldo-Gómez JD,
Kühlbrandt W. Proc Natl Acad Sci U S A. 2012;109:13602-7

Three aspects of the ATP synthase are particularly significant in terms

of the working of this enzyme and control of energy metabolism. The
first is the presence of the ATP synthase on the plasma membrane
of cells in some tissues e.g. hepatocytes and endothelial cells, (shown
in Figure 24) along with a great increase in levels of the complex on
the plasma membrane of cancer cells

FIGURE  23.  Image  of  a  

cancer  cell  in  which  the  
mitochondria  have  been  
labeled  with  green  
fluorescent  protein  and  the  
surface  ATP  synthase  
labeled  by  a  monoclonal  
antibody  (red)  reacted  with  
the  intact  cell.  

Initial claims that the “mitochondrial” ATP synthase is present on the

plasma membrane of some cells was met with considerable skepticism
but now the evidence is overwhelming. It raises the interesting, and as
yet unanswered question, of how it gets there. Assembly of a
functional complex requires subunits encoded on mtDNA and
synthesized in the mitochondria. As recent evidence suggests that
other OXPHOS complexes are found under some conditions on the
plasma membrane, the most likely way is that there is fusion of
mitochondria with the cell membrane and some re-sorting of proteins
during this process.

The plasma membrane enzyme, also called ectopic ATP synthase, has
been shown to have several interesting functions that go beyond its
enzymatic activity to control of ATP and ADP in the cellular milieu.

1) It is the receptor for HDL and induces uptake of these particles

into liver, an important role in cholesterol control.
Ecto-F -ATPase: a moonlighting protein complex and an unexpected apoA-I
receptor. Vantourout P1, Radojkovic C, Lichtenstein L, Pons V, Champagne E,
Martinez LO. World J Gastroenterol. 2010;16:5925-35.

2) It acts to control pH in the cytosol allowing cancer cells which

have high levels of the enzyme on the plasma membrane to
survive as the milieu gets more acid.
Cell surface F1/FO ATP synthase contributes to interstitial flow-mediated
development of the acidic microenvironment in tumor tissues. Kawai Y, Kaidoh
M, Yokoyama Y, Ohhashi T. Am J Physiol Cell Physiol. 2013 305(11):C1139-
50.

3) It is involved in blood pressure control. One of the subunits, CF1

is released by sheer stress to affect prostaglandin functioning.

Coupling factor 6-induced activation of ecto-F1F(o) complex induces insulin

resistance, mild glucose intolerance and elevated blood pressure in mice. Osanai
T, Tanaka M, Magota K, Tomita H, Okumura K. Diabetologia. 2012;55:520-9.

4) Evidence has been presented that the ectopic ATP synthase

binds beta amyloid protein.
Amyloid precursor protein and amyloid beta-peptide bind to ATP synthase and
regulate its activity at the surface of neural cells. Schmidt C, Lepsverdize E, Chi
SL, Das AM, Pizzo SV, Dityatev A, Schachner M. Mol Psychiatry. 2008;10:953-
69.

5) There is data to indicate that it is a receptor for HIV virus.

Ectopic ATP synthase facilitates transfer of HIV-1 from antigen-presenting cellsto
CD4target cells. Yavlovich A. Viard M. Zhou M. Veenstra TD. Wang JM Gong
Heldman M. Blumenthal R. RavivY. Blood 2012;120:1246-54
Second is the presence of an inhibitor protein, IF1, which controls
whether the enzyme acts as an ATP synthase or an ATP hydrolase.
IF1 binds to the F1 part of the ATP synthase. It is a small,
predominantly alpha helical, polypeptide that interacts at the interface
of an alpha and a beta subunit by associations with 5 subunits of the
enzyme in total. IF1 is a dimer at pH below 6.5 when it binds to the
ATP synthase to inhibit ATP hydrolysis. At higher pH the inhibitor
progressively aggregates into a tetramer, which can no longer bind to
the enzyme. In this way IF1 responds to the electron transfer-derived
proton gradient that turns the matrix space acidic when OXPHOS is
favored, thereby preventing wasteful ATP hydrolysis and indirectly
promoting glycolysis. Interestingly, IF1 is highly expressed in cancer
cells, a feature that is now considered a part of the Warburg effect. In
forming a dimer, IF1 helps to stabilize the ATP synthase dimer in the
cristae membranes. The dimer enzyme is now considered to act as
the mitochondrial permeability pore. Thus, it is not surprising that
down regulation of IF1 levels by RNAi induces increased apoptosis in
response to cell stressors.

1) Assessing actual contribution of IF1, inhibitor of mitochondrial FoF1, to ATP

homeostasis, cell growth, mitochondrial morphology, and cell viability.
Fujikawa M. Imamura H, Nakamura J, Yoshida M. J Biol Chem. 2012;287:18781-7.

Third there is accumulating evidence that the ATP synthase by itself

forms the mitochondrial permeability transition (MPT) pore. At
one time the MPT was considered to be complex of the VDAC,
adenylate kinase in the intermembrane space, the peripheral
benzodiazepine receptor, cyclophilin D and the adenine nucleotide
translocator. More recently it has been claimed that the MPT is the
dimeric form of the ATP synthase. The evidence comes from
reconstitution experiments with purified ATP synthase (free of the
other components above). In lipid bilayers and on addition of a
benzodiazepine derivative known to induce MPT, the enzyme dimer
forms a channel with many of the same features seen on MPT opening
in vitro. Further the yeast ATP synthase when incorporated into
liposomes likewise forms a channel in response to Ca++ as seen in
yeast mitochondria. Additionally, it has been shown that the c subunit
ring of the complex forms a channel that is Ca++ sensitive and partly
blocked by the beta subunit of the F1 part. This involvement in MPT
and thus in cell death is another reason why interest in the ATP
synthase as a drug-able target is now so strong.

1). Dimers of mitochondrial ATP synthase form the permeability transition pore.
Valentina Giorgio, Sophia von Stockum, Manuela Antoniel, Astrid Fabbro, Federico
Fogolari, Michael Forte, Gary D. Glick, Valeria Petronilli, Mario Zoratti, Ildikó Szabó,
Giovanna Lippe, Paolo Bernardi Proc Natl Acad Sci U S A. 2013; 110: 5887–5892.

Mitochondria: movies

http://biovisions.mcb.harvard.edu/anim_mitochondria.html

ATP Synthase: http://www.mrc-mbu.cam.ac.uk/node/448

ATP Synthase: quicktime cell movies\14.3-ATP_synthase.mov

ATP Synthase From Above: http://www.mrc-mbu.cam.ac.uk/node/447

ATP Formation: http://www.mrc-mbu.cam.ac.uk/node/450

Mitochondrial Transport:
http://www.youtube.com/watch?v=LfDYGanMi6Q

Mitochondrial Import of Proteins:

http://www.dnatube.com/video/4183/Mitochondrial-Protein-Import

Mitochondria, TOM/TIM:
http://www.youtube.com/watch?v=4OgCMzvjaws

Additional useful web-sites/reviews

http://themedicalbiochemistrypage.org/index.php  
 
Cell  Signalling  Biology  Michael  J.  Berridge    Module  2    Cell  Signaling  
Pathways  

Toward  a  systems-­‐level  view  of  dynamic  phosphorylation  networks.  

Newman  RH,  Zhang  J,  Zhu  H.  Front  Genet.  2014;5:263.    
 
Mammalian  target  of  rapamycin:  a  central  node  of  complex  signaling  
cascades.  Dobashi  Y,  Watanabe  Y,  Miwa  C,  Suzuki  S,  Koyama  S.  Int  J  Clin  
Exp  Pathol.  2011;4:476-­‐95.