Dr Gokhale Flashcards
What are the structures within the mitochondria?
- Double membrane
- Outer membrane
- Inner membrane (folded to form cristae)
- Matrix
- Inter membrane space
What is the structure of the outer membrane?
- Porins for large proteins to enter
- Enzymes embedded for mitochondrial lipid synthesis
What happens in the inter membrane space?
- Contains enzymes which use ATP to phosphorylate other nucleotides
- Where H+ is pumped into to create electrochemical gradient for oxidative phosphorylation
What is the structure of the inner membrane?
- Folded into cristae (increases SA)
- Contains redox proteins of the electron transport chain
- ATPases
- Transport proteins to move things in and out of matrix
What does the matrix contain?
- Enzymes for the Krebs cycle
- mDNA
- Ribosomes
- tRNAs
- Metabolites (ions)
What are the features of mDNA?
- Circular
- Double stranded
- Inherited from mother
What is mitophagy?
Destruction of damaged mitochondria by an autophagosome
How are new mitochondria made?
- Must be made from fission of existing mitochondria, grow (biogenesis)
- Damaged portions are segregated and destroyed
- Damage can be rescued under stress
How do proteins get into the endoplasmic reticulum?
- Proteins translocate as they are synthesised (so unfolded)
- Signal peptide binds to signal recognition particle (SRP)
- Docks with SRP receptor protein in translocation complex
- Protein fed through channel as it is made
- Signal peptide cleaved
Which transport complexes are present in the outer membrane?
- TOM complex
- SAM complex
What does the TOM complex stand for?
Translocator of the Outer Membrane
What does the SAM complex stand for?
Sorting and Assembly Machinery
Which transport complexes are present on the inner membrane?
- TIM 22 and 23 complexes
- OXA complex
What does the TIM complex stand for?
Translocator of the Inner Membrane
What does the OXA complex stand for?
Cytochrome Oxidase Activity
What is different about TIM 23?
Embedded in the inner membrane but bridges across to the outer membrane
How do proteins get into the mitochondrial matrix?
- N-terminal signal sequence binds to TOM complex receptors, polypeptide kept unfolded by chaperones
- Fully synthesised protein translocates through TOM and the TIM23 (inner) to enter the matrix, chaperones again keeping unfolded through TIM23
- Signal peptide cleaved off by signal peptidases in the matrix
In what form are proteins transported into the mitochondria?
Fully synthesised (kept unfolded by chaperone proteins)
What are examples of chaperone proteins
Cytosolic and mitochondrial Hsp70
How are chaperones removed?
Using energy from ATP
What effect does the electrochemical gradient in the inter membrane space have on the signal sequence?
Drives the peptide through the inter membrane space into the matrix since the signal sequence and the inter membrane space are both positively charged
How do proteins get embedded in the outer membrane (bilayer)?
- Enter inter membrane space via TOM
- Kept unfolded by chaperones in IMM
- Folded and inserted into the bilayer by the SAM complex
- SAM removes chaperones
What proteins are embedded in the outer membrane?
Porins (beta-barrel proteins)
How do proteins get embedded in the inner membrane? (method 1)
- Through TOM and into TIM 23
- Signal peptide cleaved off at TIM 23 to reveal the stop transfer sequence (can’t be put through into matrix)
- TIM 23 inserts the protein into inner membrane
How do proteins get embedded in the inner membrane? (method 2)
- Protein enters matrix completely via TOM and TIM 23
- Signal peptide cleaved to reveal a second signal which causes insertion into OXA complex
- OXA inserts protein into inner membrane
Which complex inserts mitochondrially translated proteins into the inner membrane?
OXA complex
How do you get soluble proteins to stay in the inter membrane space?
Cleave from the stop transfer sequence after insertion into inner membrane
How are multipass proteins inserted into the inner membrane?
- Loops through TOM
- Chaperones bind to guide the protein towards TIM 22 which inserts the protein in the correct orientation
What are the features of peroxisomes? (4)
- Single membrane
- No DNA
- No ribosomes
- Found in all eukaryotic cells
What is the function of peroxisomes?
- Contain oxidative enzymes catalase and uric oxidase
- Removes hydrogen atoms to form hydrogen peroxide
- Peroxidases use peroxide to form water
What is the role of peroxisomes in the liver?
Detoxification
How are new peroxisomes made?
Fission of existing peroxisomes with peroxisomal precursor vesicles from the endoplasmic reticulum
How do proteins get into peroxisomes?
- Signal sequences
- Translocators in the membrane called peroxins which use ATP
- Proteins don’t need to be unfolded
- Pex5 binds to signal sequence and accompanies proteins through the membrane
What syndrome is caused by a mutation in Pex5?
Zellweger syndrome
What are the stages of the cell cycle?
- G1
- S
- G2
- M
What happens during G1?
- Cell increases in size
- Ribosomes and RNA produced
- Preparation for DNA synthesis
What happens during S phase?
DNA replication
What happens during G2?
- DNA checks
- Prepare for division
What is the restriction point?
Point of no return at the end of G1 which is a check for favourable conditions
When in the cell cycle does the cell respond to growth factor signals?
G1
What is the purpose of the checkpoint in S phase?
Check for DNA damage during synthesis
What is the purpose of the checkpoint in G2?
Check for unreplicated DNA
What is the purpose of the checkpoint during mitosis?
Check for chromosome attachment to the spindle fibres
What is quiescence?
G0 phase
Cells can exit into G0 before the restriction point if growth factors are withdrawn (reversible)
What are the stages of mitosis?
- Prophase
- Metaphase
- Anaphase
- Telophase
What does CDK stand for?
Cyclin dependent kinase
Which proteins control transitions between cell cycle phases?
Cyclin and CDK
How do kinases activate proteins?
Phosphorylate to turn on using ATP
How do cyclin and CDK work together in the cell cycle?
- CDK is inactive until cyclin binds
- Then active so can phosphorylate target proteins
- Cyclin then destroyed leaving CDK inactive again
- Multiple cyclins and CDKs associated with different phases of the cycle
Which cyclin controls transition into mitosis?
Cyclin B
Which cyclin controls transition into S phase?
Cyclin A
Which cyclin increases after the restriction point?
Cyclin E
What drives the cell cycle?
Collapse of cyclin levels
How are cyclins and CDKs regulated?
CDK inhibitors
What is the complex which drives the transition into mitosis?
M-CDK (cyclin B with a CDK)
What processes does M-CDK trigger?
- Assembly of mitotic spindle
- Sister chromatids attached to go to opposite poles
- Chromosome condensation
- Breakdown of nuclear envelope
- Rearrangement of the actin cytoskeleton and golgi apparatus
How is M-CDK activated?
- CAK attaches an activating phosphate
- Wee1 attaches an inhibitory phosphate (still inactive)
- Cdc25 removes the inhibitory phosphate =active
- Active M-CDK inhibits Wee1 = more active M-CDK
- Active M-CDK activates Cdc25= more active M-CDK
What is CAK?
CDK activating kinase
Phosphorylates to activate M-CDK
What is Wee1?
CDK inhibitory kinase
(Phosphorylates to inhibit M-CDK
What is Cdc25?
A phosphatase
Removes a phosphate
What is believed to activate Cdc25?
S-CDK complexes
What does APC stand for?
Anaphase promoting complex
What controls the transition from metaphase to anaphase?
APC
Driven by protein destruction
What does APC do?
- Ubiquitin ligase
- Adds ubiquitin to the cyclin in M-CDK which targets it for destruction (S and M cyclins)
- Leaves the CDKs inactive so can’t phosphorylate their target proteins
- Also destroys securin which causes the separation of sister chromatids
What is securin?
Holds sister chromatids together
What are the 2 targets of APC?
- S/M cyclins
- Securin
What is loss of heterozygosity?
Loss of one copy of the wildtype allele (LOH)
What is hemizygosity?
- Physical loss of one copy of a chromosome
- Dangerous if you are left with a mutant allele with no wildtype active copy to balance it out
What is chromosome non-disjunction?
When chromosomes end up in the wrong daughter cell
What are astral microtubules?
Connects the cell cortex to the mitotic spindle
What is a kinetochore?
Where spindle fibres attach to separate the sister chromatids
(Centromere holds the sister chromatids together)
What are kinetochore microtubules?
Attach at the kinetochore of chromosomes
What are interpolar microtubules?
Overlap in the middle to aid separation
How do kinetochore microtubules attach to chromosomes?
- Trial and error
- When they are correctly attached the correct level of tension is detected
- If incorrectly attached the tension is lower which triggers inhibitory signals, try again
What activates APC?
Cdc20
What does APC do to securin?
- Ubiquitinates securin which releases and activates the separase which is bound to securin
- Separase cleaves cohesin proteins which hold sister chromatids together during anaphase
How can LOH occur in normal cells?
- Non-disjunction
- Mitotic recombination
- Gene conversion
What is LOH by non-disjunction?
- One daughter cell ends up with 3 chromosome copies, other ends up with 1
- Extra chromosome is deleted
- Ends up with 2 copies of the mutant allele after losing the normal allele
- Or the cell with 1 could end up hemizygous for the mutant which can cause problems
What is LOH by mitotic recombination?
Bits of chromosomes swapped during G2 or M phase so could end up homozygous for mutant alleles
What is LOH by gene conversion?
- DNA polymerase jumps to the template strand of the wrong homologous chromosome during translation so the product ends up as a combination of both
- Causes a problem if it happened to copy a mutation so now both chromosomes carry the mutant allele
What is a bivalent?
A pair of homologous chromosomes
What is a chiasma?
Point of contact between 2 chromosomes during crossing over in meiosis
How are homologous chromosomes paired up during meiosis 1?
- Synaptonemal complex:
- Cohesin connects chromatin to the axial core
- Transverse filaments hold the axial cores together
What is the difference between meiosis 1 and 2?
In meiosis 1, homologous chromosomes are separated. In meiosis 2, sister chromatids are separated =haploid
What is aneuploidy?
Wrong number of chromosomes
What is the p arm of a chromosome?
Shorter arm
What is the q arm of a chromosome?
Longer arm
What is monosomy?
Only 1 copy of a chromosome present
What is trisomy?
3 copies of a chromosome
What is polyploidy?
A whole extra set of chromosomes (i.e. 3n, 4n etc)
When during meiosis is non-disjunction most severe?
Meiosis 1
What is haploinsuffiency?
When you lose one copy of an allele and having only one copy of the allele is not sufficient to produce the amount of the protein product required (dose matters)
What is monoallelic expression?
When a phenotype is the result of the expression of one allele and the other is silent. Can lead to problems if an allele is lost
What are the 2 main routes of cell death?
- Apoptosis
- Necrosis
What is necrosis?
Uncontrolled cell death from physical damage e.g. infection, hypoxia, ischaemia, bacterial toxins etc.
What is apoptosis?
Controlled cell death
What is hypoxia?
Low oxygen concentration
What is ischaemia?
Loss of blood supply
When does apoptosis occur? (7)
- Tissue size maintenance
- Removal of developmental cells (growth factors)
- Removal of immune cells
- Hormonal (menstrual cycle)
- Anoikis (inappropriate interactions)
- DNA damage
- Virally infected cells
What are the characteristics of necrosis?
- Membrane integrity compromised
- Organelle and cell swelling
- Increased intracellular calcium
- Autolysis
- Cell lysis (bursting)
- Causes an inflammatory response
What is autolysis?
Enzymatic digestion of cells by the action of its own enzymes
What are the characteristics of apoptosis?
- Shrinkage
- Nuclear breakdown
- DNA fragmentation
- Apoptotic bodies
- Phagocytosis
- No inflammatory response
- Energy required
What are apoptotic bodies?
Vesicles containing dying bits of the cell
What is autophagy?
Controlled degradation/recycling of damaged cell components
Which genes control apoptosis?
Ced genes
What mediates apoptosis?
Caspases
Which amino acid is in the active site of caspases?
Cysteine
Which amino acid is in the cleavage site of caspase target proteins?
Aspartic acid
How do caspases work?
- Apoptotic signal activates adaptor proteins
- Adaptor proteins activate inactive monomers of initiator caspases
- Active initiator caspases activate executioner caspases which cleave target proteins leading to apoptosis
Which number caspases are initiator caspases?
8 and 9
Which number caspases are executioner caspases?
3, 6, 7
3 is the main one
What do caspases target?
- Nuclear lamins (breakdown nuclear lamina)
- PARP (DNA repair enzyme)
- Actin cytoskeleton
What does PARP stand for?
Poly ADP-ribose poylmerase
DNA repair enzyme
What are the two pathways which initiate apoptosis?
- Extrinsic
- Intrinsic
What is the extrinsic pathway of initiating apoptosis?
- Killer lymphocyte with a Fas ligand binds to Fas death receptor on target cell
- Triggers assembly of the initiator caspase complex via the DISC
- Activates initiator caspase 8
What is the DISC in apoptosis?
Death-inducing signalling complex
What is the intrinsic pathway of initiating apoptosis?
- Triggered by developmental/ stress signals
- Causes release of cytochrome C from mitochondrial inter membrane space
- Triggers assembly of apoptosome which recruits and activates initiator caspase 9
What are Bcl2 proteins?
Apoptosis regulators kept in balance in the mitochondria (pro and anti)
