Lecture III Flashcards
What are the 2 classes of dynamin GTPases?
mitofusins (MFN1 & MFN2) for the fusion of the OMM
OPA1 for the fusion of the IMM
What can be observed in mitochondria if MFN1 is genetically removed?
the mitochondrial network becomes highly fragmented
What is the main player between MFN1 & MFN2?
MFN1
What are heptad repeats (HR)?
found in MFN1 and MFN2, and they are helical structures important in trans interactions between MFN1 proteins localized in nighboring mitochondria
What kind of proteins are MFN1 & MFN2?
transmembrane proteins
Describe OPA 1:
!!!
GTPase protein that undergoes a finely regulated process of transcriptional regulation and post-translational regulation
How many splicing variants of OPA1 are there for humans? for mice?
8
4
What do the OPA1 splicing variants depend on?
inclusion or exclusion of specific exons that determine the cleavage site doe post0translational processing
Where are OPA1 proteins imported?
into the mitochondria and inserted into the IMM
What do the upper bands of this western blot on HeLa cells lysate represent? lower bands?
long forms of OPA1 (L-OPA1)
isoforms generated upon cleavage
**this is a healthy cell
What mediates the fusion of mitochondria?
L-OPA1
What are OMA1 and YME1L? What do they do?
transmembrane proteins that protrude the intermembrane space
cleave OPA1 and give soluble short forms that accumulate in the IM space
In cases of stress, describe what happens with OMA1:
OMA1 is over-activated and it over-processes OPA1 so there are no more L-OPA1
*eventually OMA1 is degraded upon cleavage of OPA1 since its activity would result in a danger for the cell
How can OPA1 mediated the fusion of the IMM?
L-OPA1 mediates the fusion of the IMM through heterotypic cell interactions with cardiolipin (only found in IMM) of the neighboring mitochondrion
*through protein lipid interactions
MFN mediates the fusion of…
OMM
L-OPA1 mediates the fusion of…
IMM
S-OPA1 mediates the fusion of…
not defined
What can L-OPA1 do at the base of cristae?
oligomerize and generate tight junctions at the base of the cristae when combined with a lot of proteins as MICOS complex and cardiolipin
Why is cristae formation important?
necessary for oxidative phosphorylation
confinement of the cytochrome c
What are the 3 models that describe the organization of the respiratory chain?
fluid model
solid model
plasticity model
What is the fluid model of the respiratory chain?
complexes are randomly distributed and they float around the membrane
What is the solid model of the respiratory chain?
all complexes are closely packed
What is the plasticity model of the respiratory chain?
combination of the fluid model and the solid model
it is the most accepted and confirmed by the isolation of complexes with a protocol of Blue native PAGE›
Are respiratory chain complexes organized?
yes, they are organized in an optimal arrangement in order to maximize electron flux and ATP production
What do mutations on the proteins of that maintain cristae shape result in?
widening of the cristae structure, with a consequential loss of ATP synthesis and release of cytochrome c
What mediates mitochondrial fission?
Drp1
What do patients with a mutation in Drp1 have?
encephalopathies and motor behaviors
*most die before 10 years of age
What is Drp1?
cytosolic protein that is recruited to OMM upon fission or physiological stimulus
What receptors does Drp1 bind to?
Fis1
Mff
MiD49
MiD51
What happens after Drp1 binds to a receptor?
forms a contractile ring around the OMM after the oligomerization
Upon GTP hydrolysis, Drp1 mediates _______.
the separation of the OMM
Where are the Drp1 receptors located?
all over the OMM
What is mitokinesis?
division of the mitochondria
mitochondria uses the cytoskeleton and actin myosin fibers to contract to divide into 2 mitochondria
once the mitochondria meets with the ER, a preconstriction site is generated and there is nucleation of the actin cytoskeleton
myosin 2 is recruited to fission site
myosin 2 activity results in the constriction of the mitochondria and the generation of 2 daughter mitochondria
dyamin (Dyn2) may be impacted
What could happen if the mitochondria cannot control fusion and fission events?
hyper-fragmentation of the mitochondrial network
formation of giant mitochondria
What causes hyper-fragmentation of the mitochondrial network?
OPA1 or mitofusion mutations
What causes the formation of the giant mitochondria?
Drp1, which prevents the fission process
*results in a huge mitochondria that accumulates ROS
**very dangerous for all cells but especially neurons
What have the mitochondria shown in terms to adaptation?
they are able to adapt in response to the cell’s nutrients
What happens if the cell is in a condition of ss deprivation?
mitochondrial network has the tendency to hyperfuse
What happens if the cell is in a condition with high levels of nutrients?
mitochondrial network has the tendency to be fragmented
What ahppens if the mitochondria are completely depolarized?
they are targeted to mitophagy
How are cells targeted for autophagy/mitophagy?
the cargo (in this case: mitochondria), is ubiquitinated which allows for the interaction with specific receptors
What triggers mitophagy?
hypoxia and ROS, which can alter and dissipate the mitochondrial membrane potential
Where is PINK1 integrated?
inner membrane
Hw is PINK1 delivered to the inner membrane?
through a pre-sequence, which is recognized by TOM/TIM pathway
pre-sequence is then cleaved and the PINK1 is imported and localized in the inner membrane where it can phosphorylate its substrate
What is PINK1 involved in?
mitophagy
What are the 2 proteins involved in mitophagy?
PINK1 and Parkin
What 2 proteins are mutated in the genetic form of Parkinson’s disease?
PINK1 and Parkin
What happens in a mitochondrial damage condition that depolarizes the membrane?
PINK1 is no longer imported → accumulation of OM → dimerization and auto-phosphorylation of PINK1 → OM proteins are ubiquitinated → proteins are retrotranslocated to the cytosol for degradation
*PINK1 and the proteins of the OM phosphorylate themselves in this process
**this combined post-transcriptional modification recruits Parkin, an E3ubiquitin-ligase, to the OMM
What does the combined post-transcriptional modification process result in?
further ubiquitination of the OM proteins
the high ubiquitination and phosphorylation of the OM act as a recruitment signal for p62, which is the receptor for the autophagosome
What is the receptor of the autophagosome?
p62
Why is fission important?
leads to the biogenesis of mitochondria
pre-requisite for the degradation of mitochondria
What was discovered last year about a fission event needed for mitochondrial biogenesis?
a mid-zone fission is needed, which is mediated by the interaction of the mitochondria and ER
How does a cell know when fission leads to mitophagy?
fission is located at the periphery where there is lots of rubbish like damaged and oxidized proteins
What is the mechanism where fission occurs at the periphery to signal for mitophagy through the interaction of the mitochondria and lysosomes mediated by?
Mitochondrial fission 1 protein (FIS1)
What does FIS1 bind to?
Drp1
Where is calcium abundant?
outside the cells
Why is the fine control of calcium important?
calcium is a second messenger and involved in lots of controlled pathways
*loss of its control can destabilize homeostasis
What can anti-porters do?
extrude calcium outside the cell
What can ATPases (aka SERCA) do?
internalize large amounts of calcium inside the ER
How can mitochondria internalize calcum?
they can internalize calcium through a uniporter called Mitochondrial Calcium Uniporter (MCU) located in the IMMM
How does MCU work?
ligand binds to G-coupled protein receptor at the plasma membrane that leads to the release of inositol-3-phosphate (IP3), which acts as a ligand for IP3 receptor located on the ER membrane
*this interaction leads to the release of calcium from the ER lumen, where it will generate a flux that moves from the ER to the mitochondria
How can calcium enter the mitochondria?
using VDAC proteins, which are large β-barrel proteins that are porins that are poorly selective for ions
How can calcium be exported outside of the mitochondria?
using antiporters like Na+/Ca2+ or proton/Ca2+
Why is the Ca2+ entrance inside the mitochondria peculiar?
MCU has a low affinity for calcium
*this means high conc. of calcium is needed to open the MCU
How can we measure calcium inside the mitochondria?
we need a sensor protein and a pre-sequence
What are 2 sensor proteins developed to measure the calcium inside the mitochondria?
photoproteins: emits light upon calcium binding and isolated from jellyfish
GFP-based calcium sensors: composed of 2 fluorescent proteins (CFP and YFP) that are connected thanks to a bridge composed of calmodulin and M13 peptide
What is the MCU complex?
small protein of the IMM, which is aable to oligomerize
What is the MCU composed of?
many subunits (structural or gatekeepers)
What are the structural components of the MCU?
MCUb and EMRE
What are the gatekeepers of MCU?
MICU1
MicU2
MICU3
*act as regulators
What does the structural component of the MCU complex, MCUb act as?
dominant-negative subunit
*this means it abolishes the overall activity of the complex
What is the distribution pattern of MCI and MCUb?
tightly regulated and tissue specific
The stoichiometry of MCU + MCUb establishes ____.
the magnitude of the uniporter permeability, which is tissue-specific.
Describe how the gatekeepers would work if the concentration of calcium is low:
MICU2 will interact with MICU1 to keep the channel closed
Describe how the gatekeepers would work if the concentration of calcium is high:
MICU1 will remove MICU2, which leads to the opening of the channell
What is MICU3 in regards to the type of activator?
MICU 3 is a positive regulator like MICU1
Why is calcium needed in the mitochondria?
calcium flux is needed to maximize ATP production
What is mitochondrial calcium overload?
it occurs when calcium conc. exceeds a certain threshold, which triggers the permeability transition
What does mitochondrial calcium overload lead to?
apoptosis because the excess of calcium opens the permeability transition pores located in the IMM
What to modes can the IMM pores act in?
both high and low conductance mode
What is a low conductance mode?
in physiological condition
at is a high conductance mode?
condition of cell damage
What happens in low conductance mode (physiological condition)?
pores open and close very fast and release small calcium waves outside the organelle
*called flickering beacuse it gives the idea of periodic release from inside the cell to the outside
What happens in high conductance mode (cell damage)?
there is an alteration of the IM permeability that leads to apoptosis
What has been recently proposed i terms of mitochondrial permeability transition pores?
they could be composed of the aggregation of ATP synthase subunits
