Chaperone mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels Diego De Stefani
Department of Experimental and Diagnostic Medicine
Section of General Pathology
University of Ferrara
Chaperone mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels Diego De Stefani
Department of Experimental and Diagnostic Medicine
Section of General Pathology
University of Ferrara
Title
The following experimental data aim to demonstrate the existence of a protein complex (formed by IP3R, VDAC and GRP75) at the interface between endoplasmic reticulum and mitochondria which mediate efficient calcium transfer from one organelle to the other.
ER-Mitochondria contact sites
This high rate in mitochondrial calcium uptake is mainly explained by the existence of close contact sites between ER, which rapresents the main intracellular calcium store, and mitochondria. These contact sites can be seen both by high resolution fluorescence microscopy, as shown in this figure, and by electron microscopy. In the figure you can see mitochondria surface rendered in green and red spots on it rapresent ER contact sites.
Close contacts between ER and mitochondria are not only a morphological feature but play an active role in calcium transfer from one organelle to the other. Indeed, IP3 induced calcium release from ER creates a local microdomain with high calcium concentration just near the release channels. In this way mitochondria that are strategically positioned at the mouth of this release sites can sense a local environment of high calcium levels, much higher than that reached in the bulk cytosol. This means that mitochondria can efficiently uptake a large quantity of this ion. Mitochondrial calcium signaling is involved in several relevant (patho)physiological processes such as regulation of aerobic metabolism, release of caspase cofactors and feedback control of calcium homeostasis.
In spite of the well known functional aspects of mitochondrial calcium signaling, the molecular determinants of this cellular process is far from being understood. The molecular nature of the systems that mediate accumulation (namely the MCU) and extrusion (mNCX and mHCX) of calcium in and from the mitochondrial matrix are not yet identified, in spite of the great effort spent in this way.
Rapizzi E. et al., J Cell Biol. 2002
One of the main goal in calcium signalling is trying to decode what are, if any, the molecular determinants of this crosstalk between the two organelles. The main components of this functional interaction are IP3R (which releases calcium from ER), and the OMM protein VDAC which transfers the locally established calcium microdomain to the so called yet unidentified Mitochondrial Calcium Uniporter (MCU) of te IMM.
We have previously demonstrated that VDAC is a key component involved in this processe, since VDAC overexpressing cells shows an higher uptake in mitochondrial calcium. So we used VDAC as a starting point for our proteomic anaysis.
Yeast two-hybrid screen using VDAC as bait
Schwarzer et. al. IJBCB, 2002
We performed a yeast two-hybrid screen of human liver and kidney cDNA libraries using rat VDAC as bait. Among the several interacting proteins, one of them, known as GRP75, can exert an effect on mitochondrial calcium uptake. This protein resides mainly inside mitochondrial matrix, but many experimental evidences have shown also an extra mitochondrial localisation of this protein. Moreover, a paper by Schwarzer et al has previously demonstrated the interaction between VDAC and GRP75 and also has shown that the voltage/conductance proprerties of VADC channel in lipid bilayers can be modified by the presence of GRP75.
HeLa Liver WB: apan-IP3R agrp-75 aVDAC-1 C P Mito Mic MAM MAM 250 75 35 30 (kD) 30 aCOX-II agrp-75 aVDAC-1 1st dimension BN 250 75 35 30 (kD) Mito P MAM 2nd dimension SDS-PAGE 75 agrp-75 270 500 600 750 1200 (kD) 2nd dimension
SDS-PAGE (kD) WB: WB: apan-IP3R
Could mitochondrial Ca2+ uptake machinery be regulated by IP3 receptor?
Structure of the IP3R
NH2 COOH Inhibitory
domain IP3-binding
domain Modulatory and transduction
domain Channel
domain “Pore” 225 576 IP3R-structure two or three conserved armadillo domains
involved in intra- and intermolecular interactions The structure of the IP3R ligand binding domain
(Bosanac, Nature 2002)
Constructs containing IP3R-LBD
Local effect
mtTracker IP3R-LBD-mRFP1 overlay OMM- targeting
mtTracker IP3R-LBD-mRFP1 overlay ER-targeting
B A IP3R-LBD increases mitochondrial Ca2+ uptake
The effect on [Ca2+]mt depends on the specific protein-protein interaction
Then we performed several experiments to demonstrate that this effect on mitochondrial calcium uptake depends on a specific protein-protein interaction. First we used a LBD mutant (K508A), where a lysine in a critical position for IP3 binding has been replaced by an alanine: thus, this mutant is unable to bind IP3 as you can see by looking at the cytosolic response which is unchanged (because there is no inhibition in IP3 induced calcium release). Anyway this mutant can still increase mitochondrial calcium uptake, which means that this effect does not depend on IP3 binding. Next to demonstrate that this effect is dependent on a specific protein protein interaction, we developed a similar construct containing a PH domain from a PLC-like protein (p130) instead of IP3R-LDB and we could see a parallel inhibition on both cytosolic and mitochondrial calcium response (blue bars). This means that a construct having the same IP3 binding properties but which is structurally uncorrelated to IP3R-LBD causes a general inhibition of calcium response due only to its IP3 buffering effect. Finally to demonstrate that the stimulatory effect on mitochondrial calcium uptake is independent on the source of calcium we measured the so called capacitative calcium entry. We induced calcium stores depletion by inhibiting SERCA pumps with tBHQ: this treatment triggers the entry of calcium from the extracellular environment through the opening of plasmamembrane calcium channels. From the right panel ...
Coupling of ER and mitochondrial Ca2+ channels depends on the presence of GRP75
Conclusions
VDAC-1, IP3R and GRP75 reside in a common macromolecular complex
IP3R-LBD enhances mitochondrial Ca2+ uptake
This effect is independent of IP3 binding and channel function of the IP3R
The molecular chaperone GRP75 is required to stabilize this interaction
Thanks to
Prof. Rosario Rizzuto
Dott. Gyorgy Szabadkai
Dott. Katiuscia Bianchi
Dott. Paolo Pinton Dott. Sara Leo
Dott. Paola Aguiari
Dott. Erika Zecchini
Dott. Angela Bononi
Dott. Anna Maria Simoni
Dott. Valeria Guaran
Dott. Alessandro Rimessi
Dott. Anna Romagnoli
Dott. Saverio Marchi
Dott. Massimo Bonora
Dott. Claudio Baldini
Dott. Mariusz Wieckowski
Dott. Roberta Siviero Dott. Peter Varnai
Dott. Tamas Balla
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