Lecture #3 Flashcards
what are MFSN 1&2 and OPA 1?
GTPases
what two GTPases are involved in mitochondrial fusion?
MFSN1 & MFSN 2, and OPA1
what mediates fusion of the OMM?
MFSN 1& 2
what mediates the fusion of the IMM?
OPA1
what is considered to be the core component of the fusion machinery of the OMM?
MFSN 1
what is observed if MFSN 1 is genetically removed?
a highly fragmented network
what is observed if MFSN 2 is genetically removed?
the network is partially impaired but tubular structure are still seen
what particular repeat is important for MFSN1 interaction?
heptad repeat 2 - association induces a conformational change in the structure of the protein that drives GTP hydrolysis and favors the fusion of the membrane
where is MFSN 2 located besides the OMM?
outer membrane of the ER
what alternate role is it hypothesized MFSN 2 might have?
important for the interaction with the plasma membrane
what two finely regulated processes does OPA 1 undergo?
transcriptional regulation and post-translational modification
what are the two proteases involved in OPA 1 post-translational processing?
YMEL1 and OMA1
what role does YME1L play in the IMM?
component of the m-triple A complex
what site does OMA1 act on
cleaves OPA1 at the S1 site
what site does YME1L act on?
cleaves OPA1 at the S2 site
how does the balanced cleavage by OMA1 and YME1L affect OPA1?
results in an equilibrium between long and short forms of OPA1 ensuring morphology of the mitochondrial network
under conditions of stress, what happens to OPA1?
a mitochondrial stress sensor is overactivated and exerts an enhanced processing of L-OPA1 - resulting in an imbalance between long and short forms
what specific form is necessary to mediate fusion?
presence of the long form - the function of the short form is currently unclear
in cases of stress, which protease is overactive, and what occurs?
OMA1 - overprocesses OPA1 so there are no more long forms of the protein attached to the membrane
what is the function of L-OPA1?
mediates the fusion of the IMM through heterotypic cell interactions with cardiolipin of the neighboring mitochondria : lipid-protein interacting
what is the role of S-OPA1?
more debated - help interaction of the long form?
besides fusion, what other function does OPA1 have in mitochondria?
important for the maintenance of mitochondrial cristae shape
what is the specific function of L-OPA1 in regards to mitochondrial cristae?
can oligomerize and generate tight junctions at the base of cristae as well as important in the confinement of Cytochrome C
List the three potential models for the organization of the respiratory chain in cristae?
fluid model (complexes randomly distrinuted and floating around), solid model (complexes are closely packed), and plasticity model (combination of both - most accepted)
describe the assumed formation based on the plasticity model of the respiratory chain in cristae
optimal arrangement in order to maximize the electron flux - all complexes are arranged to converge at the tip of the cristae where there are dimers of ATP-synthesis
what protein mediates mitochondrial fission?
dynamin related protein 1 (Drp1)
describe Drp1:
cytosolic protein that is recurited to the OMM upon fission stimulus as well as physiological stimulus
describe how Drp1 acts on mitochondria
after binding it starts to oligomerize forming a contractile ring around the OMM, upon GTP hydrolysis Drp1 mediates separation of the OMM
what occurs at the OMM before Drp1 is recruited?
contact between the ER and mitochondria forming a pre-constriciton site that marks the region where Drp1 will be recruited
after the ER meets the mitochondria and forms the preconstriction site, what happens?
nucleation of the actin cytoskeleton, then myosin II is recruited to the fission site and mitochondria is constricted generating two daughter mitochondria
when mitochondrial fusion is not functioning properly and hyper-fragmented mitochondrial networks are formed, what might be the cause?
due to OPA1 or mitofusion mutations
when mitochondrial fission does not function properly and giant mitochondria are formed, what might be the cause?
due to Drp1 mutations which are able to prevent the fission process, resulting in the formation of huge mitochondria that accumulate lots of ROS - very dangerous for cells especially neurons
in relation to cellular mechanisms, what are mitochondrial dynamics important for?
maintenence of cellular metabolism - if working properly can help maximize ATP production
what must mitochondria be labeled with to tag them for degredation?
ubiquitin
how do conditions such as hypoxia and ROS lead to mitophagy?
they can alter and dissipate the mitochondrial membrane potential
where is PINK1 located?
protein normally integrated into the IMM - delivered through a presequence recognized by TOM / TIM
what occurs when damage depolarizes the IMM causing PINK1 to accumulate in the outer membrane?
leads to the dimerization and auto-phosphorylation of PINK1 molecules
once dimerization of PINK1 has occurred, what is recruited and what happens?
Parkin (E3 ubiquitin ligase) - amplifies the process by ubiquinating other outer membrane proteins
after Parkin acts on the omm and there is a high level of ubiquination and phosphorylation, what is recruited?
p62 - receptor for the autophagosome
it has been discovered that what type of fission is needed for mitochondrial biogenesis?
mid-zone fission
what are mid-zone fission events mediated by?
interactions of the mitochondria with the ER
in regards to mitophagy, how is its interaction in mid-zone fission events different
fission sites are located at the periphery of the mitochondria are full of damaged proteins - allows for fission with lysosomes as well as the ER
what is fission with lysosomes mediated by?
mitochondrial fission 1 protein (FIS1) which binds to Drp1
what is the organelle most involved in calcium regulation?
ER
what is calcium defined as in many pathways?
the second messenger
when a massive calcium influx occurs in the cytosol, what four players are involved in regulating its distribution?
antiporters at the plasma membrane, SERCA pumps on the ER membrane, calcium binding proteins, and the mitochondria (Mitochondiral Calcium Uniporter - MCU)
What happens to bring calcium to the mitochondria when there is a large flux in the cytosol?
1) ligand binds to a G-coupled receptor at the pm which leads to the release of IP3
2) IP3 acts as a ligand for the IP3 receptor on the ER membrane
3) when interaction occurs calcium is released from the ER lumen
4) a flux is generated which moves from the ER to the mitochondria
where is the MCU located?
imm
where does calcium enter through on the omm?
VDAC
what happens to calcium when it is present in the inner memebrane space?
it is internalized unidirectionally inside the matrix through the MCU
why is calcium entrance inside of the mitochondria unique?
the MCU has a very low affinity for calcium, meaning very high concentrations of calcium are needed to open the MCU
What is needed to measure calcium levels inside of mitochondria?
a sensor protein with a presequence which can penetrate the mitochondria and reach the matrix
what two types of sensor proteins have been generated in the lab to measure calcium levels?
photoproteins and GFP-based calcium sensors
what are photoproteins?
molecules that emit light upon calcium binding
what type of photoprotein is used?
aequorins - isolated from the jellyfish Aequorea Victoria
describe the mechanism of photoproteins:
upon calcium bindind the prosthetic group is oxidized and a conformational change occurs leading to the release of CO2 and the emission of photons
where does the light from photoproteins come from?
the release of photons (not fluorescence)
describe the structure of GFP-based calcium sensors:
two fluorescent proteins CFP and YFP that are connected by a bridge composed of calmodulin and the M13 peptide
describe the mechanism of GFP-based calcium sensors:
calcium binding in the calmodulin motifs leads to a conformational change bringing CFP and YFP closer together, leading to a transfer of energy between the two molecules that can actively be measured as fluorescence
which type of Ca sensor protein is typically used when working with primary cultures?
GFP-based sensors
what is unique about the MCU?
it is able to oligomerize
what two types of subunits compose the MCU?
structural subunits or gatekeepers
how do the structural components of the MCU act?
as dominant-negative subunits of the complex
name the structural components of the MCU:
MCU, MCUb, EMRE
name the gatekeeper components of the MCU:
MICU1, MICU2, MICU3 - act as regulators
if MCUb exerts a dominant-negative regulator effect on the complex, what occurs?
it abolishes the overall activity of the entire complex - if the complex is composed of only MCU subunits calcium would be able to enter, but if there are one or more MCUb subunits present, they would inhibit the entrance of Ca
describe the distribution and stoichiometry of MCU and MCUb subunits:
tissue-specific and tightly regulated
what does the stoichiometry of MCU+MCUb establish?
the magnitude of the uniporter permeability
If the concentration of Ca is low, which two gatekeepers keep the channel closed?
MICU1 and MICU2
what occurs if the concentration of calcium in the ims is too high?
MICU1 (positive regualtor) allows the removal of MICU2 and leads to the opening of the channel, allowing the passage of calcium
describe MICU3:
a very specific positive activator of the channel
what occurs when calcium levels exceed a certain threshold?
mitochondrial calcium overload - triggers a permeability transition leading to apoptosis
what occurs in a permeability transition?
dramatic alteration in the permeability of the IMM allowing water, ions, and solutes to enter
in a low conductance mode, how do permeability transition pores work?
pores open and close very fast releasing small calcium waves outside the organelle - flickering
in a high conductance mode, what would you observe in permeability transition pores?
an alteration of the inner membrane’s permeability leading to apoptosis
describe an example of a low conductance mode:
normal physiological condition
describe an example of high conductance mode:
condition of cell damage
what is the most recent proposition regarding the components of a mitochondrial permeability transition pore?
composed of the aggregation of ATP synthase subunits
