Download Oxidative Metabolism - Plant Energy Biology

Control of mitochondrial gene expression
Nuclear encoded mitochondrial gene expression
Mitochondrial encoded genes
Must be coordinated
All of the enzymes required for DNA replication
And transcription are encoded in nucleus
Retrograde regulation General term for mitochondrial signalling
Defined as cellular responses to changes in functional
state of mitochondria
Anterograde regulation
Defined as transfer of information and material
from the nucleus (via cytoplasm) to mitochondria
yeast carbon metabolism
Aerobic metabolism:
(oxidative metabolism)
Glycolysis TCA cycle oxidative phosphorylation
Anaerobic metabolism:
(fermentation)
Glucose  Acetyl CoA  Ethanol
Diauxic shift:
Metabolic change as fermentable carbon source is
used up from…
Glucose Fermentative
(Glycolysis  Ethanol)
to…
Oxidative Metabolism
(Ethanol  TCA cycle)
Outline of Tricarboxylic Acid Cycle
3-Carbons
One pyruvate molecule is
completely oxidised to CO2
CO2
4-Carbons
6-Carbons
NADH
NADH + CO2
FADH
NADH
+ CO2
The NADH and FADH
produced are oxidised
by the respiratory
electron transport chain
metabolic remodelling
Metabolic pathways were altered in respiratory deficient cells
Intermediates of TCA cycle needed for synthesis of amino acids and nucleotides
Metabolic pathways altered:
 OAA and Acetyl-CoA supply
 Acetyl-CoA hydrolase
 in enzymes involved in flux
and conversion of metabolites made
by fatty acid oxidation to TCA and
glyoxylate cycle intermediates.
 in nutrient and metabolite
transporters.
 in enzymes for reoxidation of NADH
reconfigure metabolism by
recruiting peroxisomal activities,
small molecule transport systems and
lipid, sugar and amino acid turnover
to get more OAA and Acetyl-CoA.
CIT2 gene expression upregulated 50 fold
Positive regulators =Rtg1, Rtg2 and Rtg3
Negative regulators = Mks1, Lst8p, Bmh1p, Bmh2p
Rtg1 an Rtg3 - basic helix-loop-helix-leucine zipper
(bhlh-Zip) transcription factors that
heterodimerise and activate transcription
of genes that contain an R Box -GTCAC
Rtg2 acts upstream of Rtg1 and 3
sensor of mitochondrial dysfunction
transducer of signals
Off state
- Rtg1 and 3 sequestered in cytoplasm
- Rtg 3 phosphorylated at multiple sites
On state
- Rtg3 becomes partially dephosphorylayed
- enters nucleus (with Rtg1) to activate
transcription of genes with an R box
Thus it is the location that controls activity
Note in mutants lacking Rtg2 the retrograde response
is completely lost indicating that Rtg2 plays a crucial
role in activating Rtg1 and 3
Regulators of Rtg2
Genetic screens to identify
Mks1p
- negative regulator
- Absence means CIT2 expression
constitutively high
- Phosphoprotein
Retrograde pathway on - Mks1p is largely dephosphorylated
In a complex with Rtg2.
Retrograde pathway off - Mks1p is highly phosphoryalyed
and not bound to Rtg2, but to 14-3-3- proteins
Bmh1p and Bmh2p - as yet not known how this complex
negatively regulates
Lst8p - negative regulator of retrograde response
- Acts at two points
upstream of Rtg2
downstream of Rtg2
Shown by the fact two classes of mutants exists for Lst8p
- class I cannot bypass need for Rtg2 = upstream
-class II - no need for Rtg2 = downstream
TOR signalling in yeast (target for rapamycin)
growth control
nutrient signalling
RTG pathway activated when TOR signalling is inactivated
Lst8p is an integral component of Tor1 and 2 complexes
Acts as a positive regulator in these complexes
Details not yet clear on interaction of TOR and RTG
pathway but thought to be indirect as RTG response
still takes place if TOR pathway active
Retrograde response in mammalian cells
Transcription of the mammalian mitochondrial genome
Transcription of mitochondrial encoded genes
polygenic - followed by processing
Light strand - 1 mRNA and 8 tRNA
Heavy Strand - 12 mRNA, 14 tRNA and 2 rRNA
Mitochondrial transcription factor A
(Tfam, mtTF-1, mtTFA)
Stimulates transcription by binding to sequence
specific elements
Transcription of Nuclear encoded mitochondrial genes
Complex and integrated with cellular growth
Nuclear Respiratory Factor 1
Transcription factor that can be phosphorylated
Binds DNA as a Homodimer
Binding domain found in many but not all
nuclear encoded mitochondrial genes
Other factors characterised
NRF2
Sp1
Ets
YY1
It is the combination of these
factors that achieves the
coordination
Coordination of nuclear and mitochondrial gene
expression
NRF-1
Nuclear gene
expression
T-fam
Mitochondrial gene
expression
Respiratory complex
synthesis
Plant Respiratory Chain
ADP + Pi
ATP
MATRIX
NAD+
NAD(P)+
NADH
H+
succinate
NAD(P)H
int
I
O2
fumarate
O2
II
H2O
H+
H+
III
IV
AOX
UQ
ext
V
c
IMS
NAD(P)H
H2O
NAD(P)+
Number of genes in Arabidopsis:
• cytochrome pathway – approx. 150
• Aox – 5
• alternative NAD(P)H DHs - 7
H+
Transgenic tobacco cells expressing Aox or anti-Aox
wt
antisense
overexpressor
ROS
ROS
Maxwell et al 1998
Arabidopsis Suspension Cells
cystine
redox
carotenoids
tetrapyrrols
paraquat
PSII
NUCLEUS
norflurazon
chloroplast
CHLOROPLAST protein synthesis
chloramphenicol
erythromycin
carotenoid
biosynthesis
Treatments indicated at
site of action in RED
Genes studied by quantitative
real-time PCR indicated in blue
(blue box indicates mitochondrially
encoded genes)
keto acids
ROS
C
H+
III
AA
rotenone NDB1,2,3,4
NDC1
EXT
UCP
I
UQ
UCP1,2
H+
H+
Cyt C
H+
IV
AOX1a,1c,2
succinyl
CoA synthase
ANT
BKA
PTP
pyruvate
translocator
fumarate
fumarase
L-malate
succinyl-CoA
MITOCHONDRIA
MDH
IMS
a-ketoglutatate
IDH1,2
OMM
pyruvate
PDH
matrix
IMM
OAA
citrate
synthase
isocitrate aconitase citrate
Acon1,2,3
SA
H2O2
mannitol
ROS
H+
II malonate
succinate
IDH
glucose
oligomycin
SDH
-KDH
pyruvate
F1alpha
V F1beta
Aox
INT
NDA1,2
Signalling pathways
mechanisms indicated in green
sugars
FCCP
acetylCoA
CoA-SH
mitochondrial
protein synthesis
chloramphenicol
cytoplasm
ALTERNATIVE RESPIRATORY PATHWAY
WAS INDUCED
Alternative oxidase in Arabidopsis
•Encoded by five genes
•Aox1a - highest expressed in a variety of tissues
- highly stress inducible
•Aox1c - expressed in a variety of tissues
- not stressed inducible
•Aox2
- restricted expression
-induced by addition of compounds that
affect plastid function
•Aox1b and Aox1d are expressed are very low levels
Alternative oxidase in Arabidopsis
•Encoded by five genes
•Aox1a - highest expressed in a variety of tissues
- highly stress inducible
•Aox1c - expressed in a variety of tissues
- not stressed inducible
•Aox2
- restricted expression
-induced by addition of compounds that
affect plastid function
•Aox1b and Aox1d are expressed are very low levels
Do all genes have same function or role in cells/plants?
Aox1a is induced by a variety of stresses
Identification of regulatory motifs
Different resources used to identify regulatory promoter motifs:
Transcript data from detailed lab studies
Transcript data from past experiments reveals co-expression.
Large transcript data-sets
Clustering of array data over development, biotic/abiotic stresses
in order to discover co-regulation through co-expression.
Comparative genomics: Inter-species promoter comparison
Comparison of promoter sequences of a gene between different
species can identify elements highly conserved since divergence.
Testing functionality
Measure
reporter gene
Predicted motifs in Aox1a
I1
Functional motifs in Aox1a
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I1
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•9 unique motifs identified, 3 overlapping
Elements also functional in NDB2
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•Aox1a and NDB2 are co-regulated