SLIDE 1
Protein Co-Import of Yeast Catalase A into Peroxisomes & Mitochondria
SLIDE 2 Protein Co-Import of Yeast Catalase A into Peroxisomes & Mitochondria
- catalase A contains two Peroxisomal-Targeting-Signals (PTS):
(1) PTS1: carboyterminal SKF motif (2) internal PTS within the N-terminal part of Cta1p
- Yeast cells possess two types of catalase:
(1) catalase T (cytosolic, Ctt1p) (2) catalase A (peroxisomal, Cta1p)
2 H2O2 2 H2O + O2 catalase
homotetrameric catalase
SLIDE 3 Cta1p is Responsible for Mitochondrial Catalase Activity
2 4 6 8 Catalase Activity (U/mg)
4.7 6.4 cta1 ctt1 cta1 ctt1 CTA1 CTT1
Expression of a Cta1p/GFP fusion protein in a cta1 mutant PTS 1 GFP Catalase A PTS N-
anti-Cox3p anti-Pgk1p Cyt Mit Cyt Mit Cox3p Pgk1p
cta1 [pCTA1-GFP]
fully restores catalase activity in yeast cta1 mitochondria
5.6
SLIDE 4
mt DsRed Cta1p GFP +
Cta1p/GFP mt-DsRed Merged
Co-expression of Cta1p/GFP and mt-DsRed / DsRedSKL
Cta1p/GFP DsRedSKL Merged
DsRedSKL Cta1p GFP +
SLIDE 5
2.0 4.0 6.0 8.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Fractions D-AAO [U/mg x 10-2 ] GDH [U/mg] 4.0 2.0 3.0 1.0
Mitochondria Peroxisomes
Protein [mg/ml] Cta1p-GFP
D-AAO (D-amino acid oxidase, peroxisomal) GDH (glutamate dehydrogenase, mitochondrial)
Peroxisomal & mitochondrial cotargeting of Cta1p
Nycodenz gradient fractions
SLIDE 6
Cell physiology & Cta1p cotransport to different organelles
Cta1p/GFP predominantly targets peroxisomes when cultivated on oleate Cta1p/GFP predominantly targets mitochondria when cultivated on raffinose Oleate Raffinose Glucose
peroxisomal mitochondrial fractions fractions
SLIDE 7
Cells lacking mitochondrial catalase accumulate reactive oxygen species (ROS)
8000 7000 6000 5000 4000 3000 2000 1000
wild-type cta1
10 5 2.5 1.25
Relative ROS content (arbitrary units)
Antimycin-A blocks electron transport in the mitochondrial inner membrane and thus induces oxygen stress in mitochondria
Antimycin A (µg/ml)
SLIDE 8
- Acp1p - Acyl Carrier Protein (essential yeast protein in mitochondria)
- component of mitochondrial fatty acid synthase
- deletion: reduced amount of fatty acids
respiratory defect, unable for growth on lactate
lactate
acp1 ACP1
glucose lactate
Cta1p can drive mitochondrial import of Acp1p
SLIDE 9
Acp1p GFP mt-signal
Expression of ACP variants in a yeast acp1 mutant
Acp1p GFP
Acp1p GFP Cta1p Cta1p targets Acp1p to mitochondria
SLIDE 10 Cta1p contains a mitochondrial targeting signal
Cta1p targets Acp1p to mitochondria allowing aerobic growth of a acp1 mutant on lactate
ACP GFP CTA 1
Acp1p GFP Cta1p positive control
ACP GFP CTA
Acp1p GFP Cta1p
ACP GFP Mitoleader ACP GFP Mitoleader
Acp1p GFP mt-signal
Mitoleader ACP
Acp1p mt-signal negative control
SLIDE 11 Model of the Erv1–Mia40 disulfide relay. Schematic representation of the reactions that mediate redox-driven protein import into the IMS of mitochondria. The sulfhydryl oxidase Erv1 is a dimeric FAD-binding protein that maintains an oxidized state by the use of molecular oxygen as a final electron acceptor. Erv1 directly interacts with Mia40, which functions as a redox-activated import receptor. The oxidized active state of Mia40 can interact with newly imported precursor proteins by intermolecular disulfide
- bonds. It has been proposed that reshuffling of the disulfide bonds releases the substrates from Mia40 in a stably folded oxidized
- state. Because these folded proteins cannot traverse the protein-conducting channel of the TOM complex, they remain trapped in
the IMS. Alternatively, Erv1 might directly interact with some incoming substrates and pass them on to Mia40, which might function as a protein disulfide isomerase. In both cases, the Erv1–Mia40 system constitutes a folding trap that is designed to mediate the unidirectional import of proteins into the IMS of mitochondria. Reduced and oxidized thiol groups are indicated by SH and SS.
SLIDE 12
Yeast Cta1/Erv1p and Erv1/Cta1p expression constructs
SLIDE 13 Experimental strategy and set-up for Cta1/Erv1p expression
no cell growth cell growth
5‘-FOA selection (ura-)
Transformation plasmid shuffling
SLIDE 14 The signal for peroxisomal / mitochondrial cotargeting of catalase is localized within the N-terminal part of Cta1p
PTS1
N- MTS / PTS
Catalase A
SLIDE 15
Summary & Relevance of Mitochondrial Catalase Targeting
Catalase A cotargeting to yeast peroxisomes and mitochondria depends on the physiological status of the cell: Cta1p targets peroxisomes during growth on oleate Cta1p targets mitochondria under aerobic growth conditions (raffinose) Mitochondrial catalase level is significantly increased under oxygen stress (antimycin A) Cta1p might be an essential component in oxidative stress response by maintaining mitochondrial cell functions Protein cotargeting seems a general principle in eukaryotic cell biology that has recently been designated „eclipsed distribution“
Kindly supported by a grant from the Deutsche Forschungsgemeinschaft (DFG)
