Mitochondria + Chloroplast Flashcards
What is the origin of eukaryotic cells?
Prokaryotic cells arose before and gave rise before eukaryotic cells
What is the endosymbiont theory?
First eukaryotic common ancestor – formed from prokaryotic ‘ancestor’ cell developing internal membrane compartments (e.g., nucleus)
Common ancestor cell engulfed and maintained endosymbiotic organism and other additional changes gave rise to cellular features now common in all eukaryotic cells
Examples:
Mitochondrion – engulfed aerobic prokaryote
Chloroplast – engulfed a photosynthetic cyanobacterium
Nuclear envelope & ER – invagination of the plasma membrane
Other organelles in the endomembrane system – derived from ER
Serve essential & unique roles in eukaryotic cells… generation of metabolic energy (ATP)
What do mitochondria do?
Serve essential & unique roles in eukaryotic cells… generation of metabolic energy (ATP)
Energy production derived from carbohydrate and lipid catabolism (via oxidative phosphorylation)
What do chloroplasts do?
Serve essential & unique roles in eukaryotic cells… generation of metabolic energy (ATP)
Energy production and carbohydrate synthesis via photosynthesis
Also unique from all other organelles… contain their own DNA (genome)
What do the mitochondria and chloroplast serve a purpose for?
Code for some (20-30%) of the factors involved in their biogenesis and functioning
Organelle biogenesis- includes protein targeting, membrane assembly, morphology, motility, replication, degradation, and inheritance during cell division
Mitochondria and chloroplasts are semi-autonomous
Organelle replication is controlled by both the cell (nuclear genome) & the organelle itself
Mitochondria and chloroplast arise only from pre-existing organelles
Do not form de novo (‘a new’) – unlike all other organelles
What is the mitochondria morphology?
Double membrane-bound organelle
Outer membrane – permeable to ions & small molecules
Contains porins – ‘barrel-shaped’ integral membrane proteins with a large internal channel
Intermembrane space – high [H+]
The inner membrane lies adjacent to the outer membrane and forms ‘folds’ (cristae) that extend into the organelle’s interior (increased surface area)
Impermeable – maintains H+ gradient, site of ATP synthesis
Outer and inner mitochondrial membranes differ functionally and in their overall protein and lipid composition
Matrix- aqueous interior
Site of TCA cycle, ATP from oxidative phosphorylation
Contains mitochondrial genome – circular DNA, varies between species in size, copy and gene number
Human: encode 13 proteins, 2 rRNAs and 22 tRNAs
Contains ribosomes – used for translation of mitochondrial genome encoded proteins
All other (most) mitochondrial proteins are encoded by nuclear genes
Traditionally, mitochondria considered to be individual, small, bean-shaped, organelles
Recent live-cell imaging of mitochondria in different tissues/cell types has revealed that the organelle possesses a wide range of shapes and sizes
Mitochondria often form a ‘mitochondrial network’ – highly branched, long, and interconnected series of tubules
Bean shaped images of mitochondria often seen via EM are usually due to sectioning (during specimen preparation) through a single tubule of the mitochondrial network
The mitochondrial network allows for cell-wide co-ordination in terms of what cell needs
More tubule shaped
A mitochondrial network is a highly dynamic structure
Mitochondrial tubules are mobile and can undergo fusion and fission
Occurs in response to the environmental stimuli, developmental status, and/or overall energy requirements of cell
Mitochondrial fission occurs at end of G1 and during cell death
Rates of fission versus fusion control the number, size, and extent of inner-connections of the mitochondrial network (organelle homeostasis)
Defects in the mitochondrial network correlate with the progression of numerous neurodegenerative diseases (Alzheimer’s)
Fission and fusion controlled by distinct protein machinery
What are the steps of mitochondrial fission?
Mitochondrial division (one mitochondrion into two) Three-step process: 1) ER tubules (change in ER shape) encircle mitochondrial at future fission site and initiate constriction --> MAM subdomain --> involved in fission 2) Drp1 recruited from cytoplasm to constriction site and assemble into helices around the surface of mitochondrial outer membrane Drp = dynamin-related protein – a member of dynamin GTP binding protein family responsible for scission of other cellular membranes (clathrin vesicle formation 2 )Recruitment of Drp1 and assembly into helices is mediated by a lipid microdomain in the mitochondrial outer membrane Enrichment of cardiolipin, a mitochondrial species membrane phospholipid, at fission site in the outer membrane – Drp1 interacts with cardiolipin (cardiolipin normally only found in the inner membrane) 3) Conformational change in Drp1 due to GTP hydrolysis results in membrane constriction and scission – formation of two ‘daughter; mitochondrion
What is mitochondrial fusion?
Often occurs in response to cell stress – an increased need for coordinated functioning of mitochondria network
Requires coordinated fusion of both mitochondrial membranes
First outer membranes fuse and then inner membranes fuse – order essential to maintain mitochondrial sub(compartmentalization) homeostasis
Multistep processes
Involves various mitochondrial membrane proteins, energy (GTP), and remodeling of mitochondrial membrane phospholipids
What are the steps of mitochondrial fusion?
1) Outer membrane ‘tethering’
GTPase mitofusins – Mfn1 and Mfn2- an adjacent mitochondrion link together (dimerize) in a GTP dependent manner to form an ‘organelle tethering complex’
Mfn1 and Mfn2 are integral outer membrane proteins that both possess cytoplasmic facing GTPase domain and long, coiled-coil protein-protein interaction domain
Proper Mfn1/2 binding (e.g., prevention of ‘self-binding’) is regulated by other mitochondrial outer membrane proteins – Bak and Bax (Soluble, ensure proper fusion, and make sure fusion occurs only between correct mitochondria’s)
2) Outer membrane fusion
Formation of outer membrane lipid microdomains at sites of Mfn1/2 ‘tethering’
Phospholipase D converts cardiolipin (moves from inner to the outer membrane) into phosphatidic acid
Phosphatidic acid is a ‘cone-shaped lipid’
Causes outer membrane curvature inward (concave) and promotes Mfn1/2- mediated membrane fusion
3) Inner membrane (cristae) ‘fusion’
Inner membrane fusion mediated by OPA1
OPA1 is an integral inner membrane-bound mitofusin (Mfn1/Mfn2)
Contains an intermembrane space facing GTPase domain
OPA1 proteins on adjacent inner membranes interact in a GTP dependent manner to promote membrane fusion
OPA1 binding is regulated by other mitochondrial inner membrane proteins
E.g., prohibitin – ensures that OPA1 mediated fusion occurs only between ‘different’ inner membranes
Prevents self-fusion of cristae within the same mitochondrion
What is mitochondrial protein targeting?
The majority of the mitochondrial proteins (soluble and membrane-bound) are nuclear-encoded, synthesized on ‘free’ ribosomes in the cytoplasm, and targeted post-translationally to the organelle
Cytoplasm –> mitochondria (after protein is translated, not while it is being translated, like in the endomembrane system
Highly efficient process
How does the cell ensure that a nascent protein is properly targeted to the mitochondrion and then to the correct mitochondrial sub-compartment?
All nuclear-encoded mitochondrial proteins possess unique targeting sequence
Specific sequences of amino acids that serve as a ‘zip code’ to mediate protein targeting from the cytoplasm to the surface of the mitochondrial and to 1 of 4 specific mitochondrial sub compartments
Complex – multiple mitochondrial targeting pathways
Vary depending on the protein’s final location in the mitochondrion
Outer membrane
Intermembrane space
Inner membrane
Matrix
Each pathway relies on different submitochondrial targeting signals and shared and/or unique import machinery
Targeting and import mitochondrial matrix protein is the best-understood process
Most matrix destined proteins possess a 20-50 amino acid long matrix targeting sequences
Located at protein’s N-terminus
Consists of an amphipathic alpha-helix
Enriched in positively charged residues (R/K) on one side of helix and hydroxylated (S/T) residues on other sites
The signal sequence is cleaved following protein import into the matrix
Responsible for targeting nascent matrix protein to the cytoplasmic surface of the mitochondrion and its subsequent translocation across outer and inner membranes
What are the steps of matrix protein targeting & import?
1) In the cytoplasm, precursor (matrix destined) protein synthesized on free ribosomes and recognized by cytoplasmic molecular chaperones
E.g., cytosolic Hsp70 (heat-shock protein of 70 kDa)
Maintain a conformation of the nascent protein in a partially unfolded, important-competent state
Nascent mitochondrial proteins are enriched in the vicinity of mitochondria surface due to diffusion and mRNA localization
2)At the surface of the mitochondrion, protein’s matrix-targeting sequence recognized (bound) by the import receptor complex
Consist primarily of two integral outer membrane proteins – Tom20/22
Also, consist of several accessory proteins
Serve as ‘scaffold’ to mediate subsequent precursor protein transfer from import receptor complex to general import pore
3) Precursor protein passed from import receptor to general import pore in the outer membrane
Consist primarily of integral outer membrane protein Tom40
Referred to as ‘general’ import pore
All mitochondrial proteins (both matrix and membrane-bound) access mitochondria initially through Tom40
Tom40 forms a transmembrane channel with a pore that allows for protein translocation across (or into, for membrane proteins) the outer membrane
4 and 5) Precursor protein transferred through general import pore and then through an adjacent inner membrane channel
Consists of integral membrane proteins Tim23 and Tim17
General import pore and inner membrane channel are adjacent to each other at contact sites
Places where outer and inner membranes are closely appressed – intermembrane space reduce or absent at contact sites
Contact sites maintained by interactions of Tom40 and Tim23/17 intermembrane space facing domains
Precursor protein translocation occurs across both membranes sequentially
N-terminal matrix-targeting sequence of precursor proteins exits inner membrane channel into the matrix
Matrix targeting sequence cleaved by matrix processing protease
Emerging precursor protein also recognized and bound by matrix Hsp70
Located at matrix face of inner membrane channel via binding to Tim44 – inner membrane channel accessory protein
Matrix Hsp70 acts as a molecular motor (‘rachet’)
Tim44 bound Hsp70 undergoes ATP dependent conformational changes that ‘pulls’ protein into the matrix and prevents ‘back sliding, of protein back into the cytoplasm
•Requires energy, expends energy, and prevents the protein from going back
4 and 5) Two other steps in mitochondrial matrix protein import require energy input
In cytoplasm (step 1), ATP hydrolysis required for cytosolic Hsp70 to maintain bound precursor protein in a partially unfolded, import- competent state
During protein translocation (step 4 and 5), import driven partially by H+ electrochemical gradient across the inner membrane – established during electron transport
[H] in intermembrane space > [H] in the matrix
Positively charged residues in amphipathic matrix targeting sequence are attracted (‘pulled’) to less positively chard matrix
Energy from gradient allows proteins to enter/be pulled into the matrix
6 and 7) imported, cleaved (mature) protein into matrix folds without further assistance into final, active conformation or if this does not occur then final folding of the imported, cleaved protein in a matrix requires additional matrix localized molecular chaperones and ATP hydrolysis
Represents an additional step in mitochondrial matrix protein import that requires energy input
Requires additional energy
What is Mitochondrial mRNA localization?
mRNAs encoding mitochondrial proteins are often enriched in the cytoplasm surrounding mitochondria – ‘mitochondrial RNA cloud’
Mediated by RNA binding proteins located on a mitochondrial outer surface
e.g., unique sequences in mitochondrial mRNA UTRs bound by cytoplasmic facing domains of certain mitochondrial outer membrane proteins that serve as mRNA ‘anchors’
Results in mitochondrial protein synthesis (translation) usually taking place adjacent to the mitochondrial surface
mRNA localization allows for site specific (spatial) control of mitochondrial protein gene expression
Facilitates efficient (post-translational) protein targeting to mitochondria
