Membrane Trafficking Flashcards
1
Q
What are 4 families of Small GTPases and what mechanism are they used for?
A
- Rab
>Endosomal trafficking - Ras
>Cell proliferation (an Oncogene) and migration (lamellipodia formation) - Rho
>Cytoskeleton/ Migration (Actin stress fibre contraction) - Arf
>Membrane budding
2
Q
What is the largest family of Small GTPases?
A
Ras superfamily
3
Q
What is an overview to how Small GTPases a) activate b) signal?
A
a) Activation based off of conformational change
b) Bind and activate downstream effectors (usually a kinase which goes on to phosphorylate)
4
Q
What is the structure of GTP?
A
> Guanine nucleotide bound to ribose to form Guanosine nucleo side
> 3 phosphate groups, alpha, beta, gamma phosphates (tri phosphate groups)
5
Q
Describe why hydrolysis of GTP causes conformational change of a protein.
A
Hydrolysis of a phosphate bond at gamma phosphate, releases energy but also causes change in a molecule making it less charged (more negative) causing a conformational change to bound protein
6
Q
Why is a protein in a GDP bound state unstable?
A
Due to energy source due to cleavage, makes activated GTPase unstable, as is energetically favourable for molecule to want to move back to GTP form.
7
Q
Why are Small GTPases referred to as cycling molecules?
A
GTPase needs to be able to cycle bound nucleotide
8
Q
What is the difference between Signalling active and Hydrolysis active?
A
> Difference between signalling active and hydrolysis active as signalling active is bound to GTP and hydrolysis active is bound to GDP (as has removed phosphates and switching off signalling activity).
> Say GTP bound Rac1 instead of signalling active Rac1 for example, make sure to specify what type of active the GTPase is.
9
Q
Describe the 5 main structures of a Small GTPase and their functions.
A
- Conserved body between most GTPases
- Phosphate coordinated by P-loop
>Phosphate binding loop, phosphate of bound nucleotide are coordinated by P-loop (crucial for nucleotide binding/ controlling shape of GTPase) - Mg2+ essential for nucleotide binding
>Magnesium allows highly negatively charged phosphate groups to bind strongly. - Switch regions
>Switch 1 at bottom, Switch 2 at top; When GTP is bound is that these switch regions tuck in a bit, changing shape so can bind to effector. - Threonine stabilises water facilitating hydrolysis of beta-gamma phosphate bond.
10
Q
What is the effect of GTP bound to a GTPase?
A
When GTP is bound is that these switch regions tuck in a bit, changing shape so it can bind to effector.
11
Q
Why can we not detect an active GTPase via a) phosphorylation b) antibodies
A
a) No phosphorylation event as is not a kinase, it is phosphorylated but does not phosphorylate.
b) Antibody not used due to subtilty in conformational change, as antibodies we design would bind to both the unactive and active form due to minor change.
12
Q
How can we use effectors to distinguish between active and unactive GTPases?
A
Effectors can only bind when GTP is bound to GTPase while effectors cannot bind when GDP is bound to GTPase.
13
Q
How is GTP bound to a GTPase hydrolysed in 3 steps?
A
- Catalytic glutamine residue binds to GTP
- Positioning of attacking water
>Threonine positions water which hydrolyses bond between beta and gamma phosphate - Counteracting of negative charge at phosphates
>P-loop contributes positive charge through lysine residue to neutralize the negative charge a bit and lower the energy barrier
14
Q
What are 2 mutants used to examine GTPase in GTP bound state?
A
- Q61L (Glutamate61) catalytic glutamine mutant
>Used to make GTPase always in GTP bound form, we would substitute the glutamine for a mutant so cannot do catalytic activity - G12V pushes Q61 out of position and disturbs P loop (P-loop mutation)
>Glycine12 in P-loop can be substituted for a mutant to push Glutamate61 out of position so catalysis cannot occur so stuck in GTP form.
15
Q
Describe the cyclic regulation of GTPases by 1) GEF 2) GAP?
A
- GTPase + GDP (inactive signalling)
>GEF (guanine nucleotide exchange factors) stabilise transition state so we can exchange bound nucleotide, accelerates exchange rate of GDP to GTP (switches on signalling of GTPase). - GTPase + GTP (actively signalling)
>GAP (GTPase activating proteins) speeds up hydrolysis of bound GTP to form GDP and stop signalling.
16
Q
What do GEF and GAP stand for?
A
GEF (guanine nucleotide exchange factors)
GAP (GTPase activating proteins)
17
Q
Why are GEF proteins needed to remove GDP from Small GTPases?
A
Small GTPases are unstable so would not remove GDP on their own.
18
Q
Why do GTPases require GAPs to hydrolyse GTP?
A
As small GTPase hydrolysis is slow.
19
Q
What is the function of GDI (Guanine Nucleotide dissociation inhibitor)?
A
Bind to GTPase in GDP bound state and keeps it switched off.
20
Q
In the Rac1 GTPase, what is the role of the Thr35 residue?
A
> Thr35 (Threonine) stabilises water and bound magnesium.
> Restricted freedom -> reduced entropy barrier/ less energy required to change state
(Water has less freedom to move about, which accelerates hydrolysis).
21
Q
How do GAPs contribute to stabilisation during GTP hydrolysis at a GTPase in 2 ways?
A
- GAP Arg85 stabilises position of catalytic glutamine (GIn61) stabilising GTPase more.
- Arg85 is positively charged amino acid which counteracts negative charge phosphate, causes destabilisation of phosphate bond, increasing rate of hydrolysis.
22
Q
How do GEFs accelerate exchange of GDP for GTP at GTPases?
A
By Stabilising nucleotide-free, Mg2+-free GTPase form, so GDP can fall off so more GTP can come in.
23
Q
Describe the T17N dominant negative mutant of Rac and the effect on GEFs
A
> Mutant that disrupts ability of nucleotide to bind at all, pushes to nucleotide free GTPase (which is inactive)
> This Rac will also effect all GEFs so all GTPases in cell will be switched off (GEFs bind in attempt to swap GDP to GTP but as can’t bind nucleotide the GEF never dissociates)
24
Q
What are 3 types of GEFs?
A
- Dbl-homology domain
- DOCK-family
- Sec7 domain
25
What is an example of a GEF which binds to many different GTPases?
Vav 1,2,3 are not specific to a GTPase, but assist to GDP exchange in many different GTPases
26
What is the effect of having so many GEFs?
Gives a lot of capacity for regulation
27
Give an example of a GEF which is specific to a GTPase and what would a mutation in this GTPase cause?
>Tiam1: GEF that regulates Rac
>Mutant in Rac can no longer bind to Tiam1 but will bind to another GEF; shows the subtilty between different GTPase structure.
28
Describe the role of 1)Cdc42 b)Rac1 c)RhoA in actin-based motility of a cell
1. Allows cell to adhered to matrix coated surface, stimulation with a growth factor for example, which induce migration towards this by Filopodia
>Through activation of Cdc42 (Rho family GTPase)
2. Formation of lamellipodia protruding forward and forming adhesions.
>Regulated by Rac1 (Rho family GTPase).
3. Formation of actin stress fibres, contraction and dissembley of adhesions at the rear to pull forward body of the cell
>RhoA (Rho family GTPase) responsibility for contractility
29
Describe the pathway of GTPases in activation of actomyosin contraction
GTP-RhoA -> Rho Kinase (effector molecule) -> Myosin light chain phosphorylation -> Actomyosin contraction
30
Why are Cdc42/Rac signals and RhoA signals antagonised against each other
1.Cdc42/Rac are protrusive signals
>Pushes front of cell forwards
2. RhoA is a contractile signal
>Pulls back of cell forward
As they have opposite functions, the signals must be active at opposite times.
31
When RhoA is GTP bound state, what is happening to the cell and what state are Cdc42/Rac in?
>Actin stress fibres active, so back of the cell pulled towards the front.
>Cdc42 and Rac are in GDP bound states, so will not signal for protrusions.
32
What is the difference in movements of cells in a a) 2D environment (on a plate) b) 3D matrix (like in our body)
a) Cells move randomly in a 2D setting, but are fast
b) Cells move in a specific direction, but more slowly (do translocate more efficiently despite a low speed as have direction).
33
Describe how the position of Rac1 in a cell is important for cell motility.
>When Rac1 is found only at the front of the cell, when active, protrusions will only be from the front. So the cell moves in a linear fashion across matrix fibres (has direction).
>When Rac1 is found all over the cell, when active, protrusions will be from all directions. So the cell will move without direction so will not translocate across the matrix fibres (has no direction).
34
Why would a mouse with fibroblasts where Rac1 is found all over the cell have a healing defect?
As a cell with Rac1 all over lacks direction in movement, fibroblasts will not be able to migrate to wounds quickly (slow translocation), so the mice's skin will not heal quickly.
35
What are sequential modifications?
Modifications that are made in a particular order or sequence, where each modification is dependent on the previous one.
36
Why is membrane trafficking needed for sequential modifications of proteins?
Membrane trafficking allows for compartmentalisation: As specific enzymes can be contained in separate compartments so proteins can undergo different sequential modifications in different cellular compartments.
37
What is membrane trafficking?
Membrane trafficking is a process by which membranes and the proteins and molecules they contain are transported within and between cells.
38
What are the 2 major pathways of membrane trafficking and their function?
1. Secretory/Exocytic (biosynthetic) pathway
>Peptides enter ER lumen then trafficked to Golgi to Plasma Membrane/endosome/lysosome
2. Endocytic pathway
>recycling or degradative/ downregulate signals by downregulate receptors
39
What are the 3 types of secretion from the trans Golgi network in the Secretory/Exocytic membrane trafficking pathway?
1. Constitutive secretion: At trans Golgi network proteins can be constitutively secreted: Don't need to have signals on the proteins to end up at membrane
2. Regulated secretion: sorted into secretary vesicle and held until a signal from the cell
3. Enzymes pass through and are sorted within a lysosome
40
What are the 2 outcomes of the endocytic pathway?
1. Internalize protein and degrade it by lysosome
2. Can move receptor to different area of cell by recycling.
41
Where do the Exocytic and Endocytic pathways intersect?
Vesicles from both pathways intersect on the way to the lysosome.
42
What are 2 post transcription modifications that can be done to proteins as they transit the ER and Golgi?
Proteins can be modified (glycosylated by addition of oligosaccharides (many sugars added at once) and proteolytically cleaved as they transit the ER and Golgi
43
What organelle is not apart of the secretory pathway?
Mitochondria
44
What processing occurs for proteins at 1) ER lumen 2) Early Golgi 3) Later Golgi?
1) ER lumen
>Addition of pre-formed oligosaccharide to an asparagine amino acid in a consensus sequence (glocalization)
>Sugar group is similar size to amino acid, so multiple additions can weigh more than the initial polypeptide, so impact what the protein folds like and becomes in the ER and Golgi
2) Early Golgi
>The oligosaccharide group is trimmed
3) Late Golgi
>Further sugars are added and the structure can be further branched
45
What are 2 purposes of glycolisation?
1. To assist the folding of the protein
2. For a ligand:
>Intracellular for trafficking/sorting
>Outside the cell for interactions with extracellular matrix and with proteins/sugars on other cells.
46
How are proteins from the same gene be slightly differet?
All proteins from a gene will be the same, but the modifications will be slightly different due to the range of interactions with enzymes; they're similar but not exactly the same
47
What are Saccharomyces cerevisiae?
Budding yeast
48
What are two factors which make a model suitable for membrane trafficking?
1. Simplicity - trafficking occurs on a cellular scale so a single celled organism is likely to provide information.
2. Analysis of specific types of secretion e.g regulated secretion, would need a model system that is able to perform this function.
49
Why can Saccharomyces cerevisiae (budding yeast) not be used to study regulated secretion?
As budding yeast do not do regulated secretion.
50
What are 4 advantages to Yeast as a model organism for membrane trafficking?
1. Amenable for genetic studies (can grow as haploid and diploid cells)
2. Entire genome sequence known since 1996 (and is fully annotated), cheap and easy to grow in large quantities (good for biochemical studies),
3. limited gene diversity (both ±)
> Not much redundancy (take away one protein not another doing the same job)
4. fundamental pathways conserved
51
What are 3 disadvantages to Yeast as a model organism for membrane trafficking?
1. Limited cell-cell contact so unlikely to be informative about multicellularity
2. Small (5µm), so high resolution imaging studies of intracellular compartments is difficult.
3. Has a cell wall which can preclude some types of studies
>E.g. effects micro injection work.
52
What were the 3 membrane trafficking pathways studied in yeast and which screens were used for them>
1. Secretory pathway by the SEC screen
>Screening Identifying things which could not be secreted
2. Vacuolar protein screen (VPS) for secretory pathway leading to lysosomes (in yeast lysosome is the vacuole)
3. Endocytic pathway by END screen
53
What did Novick and Schekman study in yeast about membrane trafficking?
Investigated the secretory pathway using SEC screening to identify the SEC genes involved.
54
What was observed in the genetic screen "SEC"?
>Disrupted membrane trafficking getting into ER, getting out of ER, entering Golgi, leaving trans Golgi network, docking with membrane.
>So if secretion was disrupted (without SEC genes), the cell would increase its density as vesicles carrying proteins would accumulate.
55
How did Novick and Schekman define their SEC mutants?
They defined secretory mutants as those strains which fail to export active Invertase and Acid Phosphatase (enzymes), but continued to synthesize protein under restrictive growth conditions.
56
How did Novick and Schekman measure the accumulation of vesicles (density)?
By electron microscopy
57
What were the results of Novick's and Schekman's SEC screens on yeast?
1. Wild-Type
>Yeast had no build up secretory vesicles.
2. Sec- mutants
>Many vesicles accumulated (shows these SEC genes mediate the secretory pathway).
58
How many SEC genes did Novick and Schekman discover and what were they used for?
>23 genes were identified by grouping mutants with similar phenotypes (genetic crossing).
>At least 23 distinct gene products are required to ensure the transport of proteins from the ER to the plasma membrane.
59
How did Novick and Schekman put different SEC- mutants in sequential order?
Mutant groups were placed in sequential order by combining mutants from different classes and by use of more detailed analysis of protein modifications.
60
Describe the a) fate of secreted proteins b) the defective function for the 5 classes of SEC genes
1. Class A
a) Accumulation of vesicles in cytosol
b) Defective transport into the ER
2. Class B
a) Accumulation of vesicles in rough ER
b) Defective budding of vesicles from the rough ER
3. Class C
a) Accumulation in ER-to-Golgi transport vesicles
b) Defective fusion of transport vesicles with Golgi
4. Class D
a) Accumulation in Golgi
b) Defective transport from Golgi to secretory vesicles
5. Class E
a) Accumulation in secretory vesicles
b) Defective transport from secretory vesicles to cell surface.
61
How did Novick and Schekman use the pheromone "Alpha factor" to measure were each where SEC- mutant failed post-transcriptional modifications?
>Alpha factor is modified at each stage of the secretory pathway in yeast:
1. In ER it gets modified by oligosaccharides
2. Moved to Golgi, more sugars added
3. Moved to later Golgi, lots of sugars added
4. Late Golgi, proteolytically cleaved into small peptides
5. Alpha then secreted
Could detect if Alpha factor is secreted and if not, could look for each SEC mutant at which stage of modification it stopped based off of its size (how many sugars added) using a western blot.
62
What were three reason to why Novick and Schekman couldn't identify all the SEC genes/proteins involved in the secretory/exocytic pathway?
1. They only identified temperature sensitive mutants. Not all genes when mutated will cause this phenotype.
2. They only considered secretion to the plasma membrane so defects in transport to endosome or vacuole/ lysosome would not be identified.
3. Any ‘redundantly’ functioning genes would not be identified though yeast has relatively low gene redundancy which underpinned the success of these approaches.
63
What decision is made at the Trans Golgi Network (TGN) for membrane trafficking?
A decision is made in TGN (trans Golgi network) whether to traffic to surface of cell or towards lysosome.
64
What do mutations in trafficking from the endosome to lysosome effect?
Both the biosynthetic (secretory) and degradative (endocytic) pathways.
65
What is endocytosis?
Endocytosis is the process through which the plasma membrane invaginates into the cell resulting in the production of a vesicle that is then able to fuse with endosomes and enter the endo-lysosomal membrane system.
66
What are 3 reasons why endocytosis is important?
1. Retrieval of molecules that formed part of the secretory vesicle for recycling
2. Downregulation of signals
>E.g. change receptors
3. Remodelling cell surface lipid and protein composition
67
What are the 4 stages of the endocytic pathway?
1. Plasma membrane to endocytic vesicle (invagination)
2. Endocytic vesicle to early endosome
3. Early endosome to late endosome (MVB) or recycling to the plasma membrane
4. Late endosome to Golgi or vacuole/ Lysosome (recycled)
68
Why is the actin cytoskeleton important for invagination during endocytosis and when the cell is under which conditions is this important?
>The cytoskeleton helps to drive the formation of the endocytic vesicle by generating force and pulling the plasma membrane inward.
>Actin cytoskeleton role for invagination is important for cells under pressure (Yeast cells are high in pressure as contain a lot of sugar, so is very useful for them).
69
What are the 2 major functions of lysosomes (which are the vacuole in yeast)?
1. Degradation of extracellular material taken up by endocytosis.
2. Degradation of intracellular components by autophagy (When organelles fuse with lysosome and contents get degraded).
70
Why must lysozymes be kept compartmentalised inside lysosomes by the Trans Golgi network?
As they would degrade organelles and proteins if in the cytoplasm.
71
Describe the Vacuolar Sorting screens (VPS) in yeast
>Carboxypeptidase Y (CPY is an enzyme found in vacuole/ lysosome) is normally transported to the lysosome/vacuole having been trafficked through the ER and Golgi.
>Using colour based assays, it was measured which mutated cells secreted CPY to the vacuole for degradation or if the vesicles built up.
72
How many Vacuolar Protein Sorting (VPS) genes were identified by VPS screens in yeast?
60 vacuolar protein sorting (vps) genes
73
Like with Alpha factor in the SEC screen how was the phase of Carboxypeptidase Y (CPY) processed measured in different VPS- mutant yeast cells?
>Like Alpha factor, CPY had sugars added at different stages so the size could be measured of the enzyme to know at which stage each VPS- mutant disrupted post-transcriptional modification:
1. In ER
>Sugars added (called P1 form)
2. In Golgi
>More sugars added (P2 form)
3. Vacuole
>Sequence gets cut off forming active enzyme in the vacuole.
74
What is a multivesicular body (MVB) and its function?
When the whole endosome (containing what we want to be degraded) begins to invaginate and pinch off, a MVB forms: an organelle packed full of vesicles and fuse with lysosome to be degraded.
75
What are the 4 possible destinations of a vesicle from the Trans Golgi network and which one does CPY enzyme take?
1. To plasma membrane
2. To early endosome
3. To late endosome/MVB
>CPY does this pathway
4. To vacuole
76
Describe the CPY pathway to the late endosome in 4 steps
1. When in late Golgi, P2 form of CPY is recognised by receptor protein Vps10.
2. Vps10 tail is recognised by cytoplasmic factors and is then sorted into a vesicle allowing it to traffic to late endosome
3. In late endosome, the CPY is dissociated and traffics onto the vacuole/ lysosome
4. When Vps10 is released, its tail is recognised by an adaptor protein which sorts it into vesicles to be returned to late Golgi
77
What would be the effect of having not retrieval pathway for Vps10 at the late endosome?
If without retrieval pathway, Vps10 would accumulate at the late endosome
78
What are the 3 types of protein transport?
1. Gated transport
>Between nucleus and cytoplasm
2. Between chloroplasts, mitochondria, peroxisomes
>Proteins are important in organelles/ cross membranes
3. Packaging proteins into vesicles or tubules (vesicular transport)
79
What is the role of a nuclear pore?
Allows for complex regulation of what comes in and out of nucleus.
80
Where do nuclear pores form?
Formed at the junction of the inner and outer membranes of nuclear envelope
81
What is the structure of a nuclear pore?
>Made of 8 subunits (50 proteins overall)
>Centre pore can gate open or be closed.
82
How are nuclear pores important for a) DNA synthsis b) Protein production?
a) In the synthesis of DNA histone molecules are needed to package the new DNA. These are transported from the cytoplasm into the nucleus
b) For protein production to occur ribosomes are needed. The ribosomal subunits formed in the nucleolus have to enter the cytoplasm (out of nucleus)
83
What are the 2 processes that transport can occur by at the nuclear pore and what mediates the use them?
1. Diffusion
>Molecules below 60,000Kda
2. Active transport
>Molecules above 60,000Kda require signal and energy to expand size of the pore so protein can translocate into nucleus.
84
What is the effect of molecule size on translocation into a nucleus?
As molecules get larger it takes longer to equilibrate into the nucleus.
85
What is the most commonly used signal for active transport through nuclear pores and their structure?
>Nuclear localization signal (NLS)
>Short sequence of amino acids, rich in positively charged amino acids (lysine (Lys) and arginine (Arg)) which are recognised by nuclear transport receptors causing the pore to increase in size.
86
What is the effect of a mutation changing an amino acid in a signal for active transport through a nuclear pore?
Disrupts the amino acid sequence, changing the charge and therefore not being recognised by a transport receptor so the nuclear pore size stays the same.
87
Describe experimental evidence that transport is active into the nucleus a) in vivo (in cells) b) in vitro?
a) Chill a cell to 4 °C, ATP hydrolysis is inhibited, which inhibits mRNA transport out of the nucleus.
b) Take isolated nucleus, in absence of ATP, a large protein will bind to the pore complex but remain outside the nucleus; then add ATP and the proteins start to appear inside the nucleus.
88
What happens to proteins like growth factors after being secreted from the cell they were synthesised in?
Many proteins which needed to be synthesised in a cell (e.g. growth factors) are secreted from cell and translocated into the ER for secretion.
89
What % of the human genome encodes for membrane proteins?
30%
90
Describe the association of ribosomes with ER and why is this needed?
>Ribosomes tightly associated in close proximity of ribosome to ER membrane
>Ribosomes are needed close to ER or co-translational translocation of protein.
91
Describe the two ways newly synthesised proteins can be translocated into organelles
1. Co-translationally
>Coupling of translation to translocation into ER lumen from ribosome.
2. Post-translationally
>Proteins synthesised in cytoplasm then moved to ER after (more rare).
92
How does the signal hypothesis suggest a soluble protein destined for secretion (e.g. GF) is synthesised co-translationally in the ER in 3 steps?
1. Ribosome sits on top of translocator, signal is recognised, newly translated protein is fed through the translocator pore into the lumen of ER while being translated.
2. The peptide signal is then removed by signal peptidase and is degraded
3. The fully translated soluble protein (linear at entry) is in the lumen of ER, is folded due to many chaperones and protein disulphide isomerases (allowing disulphide linkages forming)
93
Why are a lot of proteins involved in protein translocation called SEC?
As their protein homologues were discovered in yeast.
94
What is a chaperone protein's function?
Help other proteins to fold and prevent from aggregation
95
What is an isolated ER membrane called
Microsomes
96
What is SEC61 an example of and its function?
SEC61 is a translocator on the ER membrane, it is closed tightly if no ribosome is associated with it open when a ribosome is associated allowing protein entry into ER lumen.
97
How do we know translocator proteins on ER membranes close very tightly?
We know translocators are closed tightly as the lumen of ER has very different ionic composition to cytoplasm of cell, without mixing.
98
What is the difference between signals for protein entry into ER and Nucleus?
Receptors on ER are very plastic and response to many signals, while nuclear import receptors respond to very few signals (mainly nuclear localization signals).
99
How does a type I transmembrane protein (not soluble) get inserted into the ER in 4 steps?
1. Signal sequence starts transfer recognised by SEC61 which opens
2. Newly translated protein fed through translocator, but encounters a stop transfer sequence (very hydrophobic part of protein)
3. Allows the N-terminus to remain anchored into ER membrane
4. The C-terminus is left exposed to the cytoplasm.
100
What is a Type 1 transmembrane protein?
Where the N terminus is in lumen of ER, and C terminus is exposed to the cytoplasm.
101
What is a Type II transmembrane protein?
Where the C terminus is in lumen of ER, and N terminus is exposed to the cytoplasm.
102
How does a type II transmembrane protein (not soluble) get inserted into the ER?
C terminus fed to ER lumen, N terminus stays outside in cytoplasm with peptide signal acting as a transmembrane domain
103
How does a multi-spanning transmembrane protein (e.g. GPCR) get inserted into the ER?
For multi-spanning proteins like a GPCR, many stop start transfers so the protein is weaved in and out the ER.
104
What is an example Chaperone in the ER lumen and what is its function?
BiP associates with newly synthesised proteins to ensure they fold correctly, it won't unbind until folded correctly.
105
What is the function of glycosylation in the ER?
In ER ensures the quality control, recognition of these sugars show if the protein is correctly folded or not
106
What happens to incorrectly folded proteins which have been secreted from the ER lumen?
Proteins will be reverse translocated into cytoplasm and broken down.
107
How can defects n protein folding give rise to cystic fibrosis type CFTRΔ508 and how can we treat it?
>Misfolded CFTRΔ508 proteins means dysfunctional chloride channels leading to excess mucus.
>Enhance delivery and opening time at the cell surface of the chloride channels fixes this.
108
What is the effect of a ER containing many misfolded proteins?
1. Upregulation of chaperones to increase rate of correct folding.
2. If there are too many, the apoptotic pathway is activated.
109
When is post-translational translocation used more often?
For organelles like mitochondria and chloroplasts, post-translational translocation must be done to transport proteins to them that are already folded
110
How do you get protein into the mitochondrial matrix via post-translational translocation in 3 steps?
1. Protein completely translated and associated with chaperon proteins
2. Recognised by TOM/TIM complex on mitochondrial membrane
3. Translocated through mitochondrial matrix and signal is cleaved off.
111
Describe how the nature of the cytochrome oxidase signal sequence found on the alcohol dehydrogenase enzyme, allows translocation into the mitochondria
For cytochrome-oxidase (cytochrome c) signal sequence, one side of the helix is hydrophobic while the other is hydrophilic. The hydrophobic residues are found in a hydrophobic groove and are recognised by a receptor associated with TOM complex allowing for translocation of Alcohol dehydrogenase.
112
How proteins are translocated into the outer membrane of Mitochondria to form porin in 3 steps?
1. TOM complex translocates in the polypeptide chains
2. Chaperons within intramembrane space help polypeptide to assemble
3. SAM complex helps assemble the porin in the outer membrane
113
How proteins are translocated into the outer membrane of Gram negative bacteria to form porins in 3 steps?
>Similar to mitochondria process:
1. Polypeptide translocated into periplasmic space
2. Chaperons associate with polypeptide
3. Porin inserted into outer membrane
114
What are 2 similarities and 2 differences between translocation of proteins into mitochondria and chloroplasts?
1. Similarities
>Both occurs post translation
>Both require ATP
2. Differences
>Plant cells have chloroplasts and mitochondria and have correct sorting of the proteins for each
>The signals are similar but are different between mitochondria and chloroplasts.
115
What is a) Anterograde b) Retrograde movement between organelles?
a) Anterograde movement (e.g. ER to Golgi)
b) Retrograde movement (e.g. Golgi back to ER)
116
What mediate movement of newly synthesised molecules through the secretary pathway?
Being packaged into transport vesicles and tubules
117
What mediates movement of proteins from ER to the Golgi?
Process of going from ER to Golgi is mediated by cytoskeleton (microtubules carry cargo between).
118
Why is cargo not lost during vesicle fusion to target membranes?
The fusion is non-leaky
119
What proteins act as a guide for vesicles?
SNARE proteins on transport vesicle surface ensure the right vesicle fuses with the right target membrane.
120
What are the 3 types of coated vesicles?
1. Clathrin
>Hexagonal structure
2. COPI
3. COPII
121
What are the 3 essential components of all transport vesicle formation?
1. GTPase
2. Adaptor proteins (to recognise cargo)
3. Coat (Clathrin, COPI, COPII)
122
Why is there a lot of GTP bound Ran (active) in the nucleus?
As the GEF for Ran is localised in the nucleus
123
How is the small GTPase Ran used for nuclear transport?
1. Protein with nuclear localisation signal binds to nuclear import receptor
2. When cargo enters the nucleus it brings nuclear import receptor with it, Ran (in GTP active form) binds to the cargo
3. Cargo dissociates from nuclear import receptor and stays in the nucleus.
3. Ran leaves the nucleus and is inactivates (to GDP form) by GAPs in the cytoplasm and dissociates from the nuclear localisation signal.
124
Why is the segregation for GEF for Ran inside the nucleus and GAP in the cytoplasm important for nuclear transport?
>So Ran is in active GTP form inside nucleus, to dissociate nuclear import receptor from cargo.
>So ran is found in GDP inactive state in cytoplasm so it dissociates with nuclear import receptor so it can be used again.
125
What are the components of the COPII coats?
1. GTPase: Sar1 (member of ARf family)
2. Adaptor protein: SEC23/24
3. Coat: SEC13/31
126
How are COPII vesicles formed?
1. Activation of Sar1(GTPase) by GEF allowing recruitment of adaptor complex (sec23/24) to an already budding membrane (can bind to a curved membrane due to bow tie shape)
2. Sec23 binds to Sar1 and Sec24 recognises and binds to cargo receptor on ER membrane
>(Adaptor complex recognises and binds to cargo receptor)
3. Adaptor protein recruits coated proteins
4. Formation is coupled with quality control mechanisms in ER
5. Vesicle buds off
127
What is the role of an outer coat for a vesicle?
Coats stabilises the disforming membrane so the bud forms correctly.
128
How do transport vesicles not take up too many ER resident proteins and mainly take the wanted proteins?
Due to having a high surface area to volume ratio of the bud.
129
What is a Er reconstitution assay?
Break open a cell, run in centrifuge on a sucrose gradient, allows us to isolate an ER membrane (called microsomes).
130
How were reconstitution studies used to show what the minimal components required for COPII formation?
1. Took an ER membrane preparation and identified a known ER membrane localised protein (defines starting material)
2. Added different variables, like cytosol or ATP
3. Centrifuged solution on sucrose gradient to measure if COPII vesicles were produced.
131
What did the results of reconstitution assays show were the minimum components required for COPII fomration?
Cytosol, ATP and GTP added to ER membrane
132
What allows Sar-1 GTPase to recognise a budding membrane?
SAR24/23 complex is curved and bow tie shape so can recognise the curved budding membrane
133
Why do vesicles have to be uncoated?
After the vesicle buds the coat has to be uncoated to reveal SNAREs, so fusion can occur
134
How does the adaptor protein SEC23 acting as a GAP for the GTPase SAR1 effect the COPII coat disassembly?
GAP activity enhances following recruitment of the coat, so the coat only remains on the vesicle for a short time as the GTPase (SAR1) enters a GDP inactive form, allowing for SNAREs to be viable.
135
What are the 2 forms of mutated GTPases and their effects?
1) Constitutively active (always in active GTP form)
>Without GTPase cycle activity will not carry out functions correctly.
2) GDP mutant can't exchange GDP for GTP
>Will sequester all GEFs (will bind to the GDP form but as will never be exchanged are stuck) so prevents other GTPases exchanging GDP for GTP.
136
What is the effect of mutating Ras to constantly be in GTP active form?
Nuclear transport will be inhibited as Ras will never dissociate from nuclear import receptor in the cytoplasm.
137
What are vesicles coated in a) COPII b) COPI c) Clathrin used for?
a) COPII important for ER budding to Golgi (retrograde)
b) COPI delivers material in anterograde and retrograde direction from Golgi
c) Clathrin take from TGN (trans Golgi network) to lysosomes and also bud from the cell surface
138
What cargo is contained by a) COPII b) COPI c) Clathrin TGN d) Clathrin PM coated vesicles?
a) COPII: Newly synthesised proteins into transport vesicles that bud from ER and move through secretory pathway
b) COPI: Retrieved (retrograde: resident ER proteins that are returned to ER) and newly synthesised protein (anterograde: moving forwards through Golgi), move proteins forward and backwards (antro and retrograde)
c) Clathrin TGN: Lysosomal proteins (to endosomal pathway) or Regulatory secreted proteins
d) Clathrin PM: Endocytosed material (Material taken up by endocytosis is taken up).
139
What decision is made at the trans Golgi network?
At trans Golgi network decision is made: either send material to plasma membrane directly via constitutive secretion or targeted to endosomal pathway (e.g. lysosomal proteins), or targeting proteins that undergo regulated secretion (e.g. released by Ca2+ stimulus)
140
What are the 3 functions of adaptor proteins during vesicle formation?
1. Recognise and select cargo ensuring specificity
2. Link the coat to the ER membrane
3. Adaptors recognise motifs (signals) in the cytoplasmic domains of membrane proteins on target membrane (membrane where budding is occurring)
141
Where are the adaptor protein families a) AP1 b) AP2 c) AP3 found?
a) AP1 on endosomes, trans golgi network (TGN)
b) AP2 (Clathrin coated surface) at the plasma membrane allowing for selective endocytosis (just need to know this one).
c) AP3 on TGN (trans Golgi network) and lysosomal related organelles
142
Despite having a similar structure, what is different about adaptor proteins in vesicular coat formation?
Each adaptor protein family is very specific for their cargo and location/ target membrane (where membrane is budding).
143
What allows adaptor proteins to go to the correct budding membrane?
The cytoplasmic domain contains different motifs which allow interactions with different adaptor proteins.
144
What is the structure of the adaptor protein AP2 and what does each section do?
>2 large subunits (alpha and beta2), beta2 binds Clathrin.
>2 smaller subunits (μ2 and sigma2) recognise signals in transmembrane proteins/ cargo receptor (where the membrane is budding).
145
What is a major Clathrin adaptor protein for endocytosis?
AP2
146
Why are there many different adaptor proteins in Clathrin coated pits in the plasma membrane?
Having different adaptors allows us to select what we internalise via endocytosis.
147
Why does interfering with COPII machinery, why do problems with large cargo appear despite COPII being smaller than large cargo like Procollagen?
Modifications in COPII coat is what helps sort things like procollagen into tubular structures (vesicle is modified for large cargo sorting), so COPII does transport large filaments via tubular transport
148
What is Rab required for during membrane trafficking?
Required for fusion between vesicle (SNAREs present) and membrane and many other trafficking functions (e.g. signalling, how vesicles associate with cytoskeleton)
149
What tells us that Rab is important for regulation in trafficking in most cells?
Most Rab GTPases are ubiquitously expressed (in most cells), showing they are important for trafficking in almost all cells in our body.
150
What are Rabs associated with and give an example?
>All Rabs are associated with particular intracellular membranes
>E.g. Rab1 associated with ER membrane
151
How are Rabs involved in Vesicle fusion to target membrane in 6 steps?
1. Vesicle approaches target membrane
2. Rab in GTP form is recruited, helps recruits tethering protein
3. Tethering protein is associated with organelles and have long coiled sequences
4. Rab on vesicle interacts with one part of tether while other Rab on target membrane interacts with the other part of the same tether
5. Tether facilitates SNARE complexes to form as brings vesicle close to the membrane. Promotes SNARE complex assembly, so are major regulators of all membrane trafficking pathways
152
What is the role of Rab5 and Rab7 cascade in the endocytic pathway?
>Rab5 is associated with early endosomes, and Rab7 is associated with late endosomes
1. GEFs activate Rab5 on the early endosome.
2. When cargo has bene selected, GAPs inactivate Rab5 and activate Rab7 on the late endosome
(So the GAP of one Rab can activate the GEF of the next Rab in the pathway, so these cascades allow movement of cargo between organelles. )
153
What would be the effect of excess Rab7 activation?
Would reduce autophagy by lysosomes as less late endosomes would deliver cargo for degradation.
154
How does the bacterium Legionella pneumophila use Rabs to promote their replication?
Legionella pneumophila can recruit rab1 to the cell surface to create an ER like compartment where it can replicate (how Legionella survives in macrophages),
155
What is the most common cargo for transport vesicles?
Macromolecules
156
What are the roles of a) Rough ER b) Smooth ER?
a) Protein synthesis site
b) Lipid synthesis site, Roles in Ca2+ storage
157
What are the roles of a) Rough ER b) Smooth ER?
a) Protein synthesis site
b) Lipid synthesis site, Roles in Ca2+ storage
158
What does an electron dense area usually represent?
A lot of proteins present
159
What did electron microscopy show at sarcoplasmic reticulum in muscle?
Close connection between SR and plasma membrane
160
What is the relationship between the ER and all of the organelles in a cell?
ER is very reticular and connects to different organelles throughout a cell and plasma membrane via contact sites where Ca2+ and lipids are transferred.
161
At contact sites between ER, organelles and plasma membrane is there fusion?
No, the membranes of the ER and its target do not fuse.
162
What type of ER interacts with organelles and the plasma membrane and what is the evidence for this?
Everywhere where ER is in contact with another organelle there is an exclusion of ribosomes, it is only smooth ER that contacts
163
What is the role of smooth ER during mitochondrial fission?
During mitochondria fission, the ER winds itself around mitochondria forming a loop around it, the ER then contributes to the breaking apart of mitochondria during fission.
164
What techniques showed us how dynamic membrane contact sites between ER and its target are?
Combination of Electron microscopy and live cell imaging
165
What technique allows for the localisation of fluorescently labelled proteins at MCS (inter-membrane contact sites)?
CLEM (corrective light and electron microscopy) allowed comparison of light and electron microscopy.
166
Describe the structure of the ER contact sites?
>Ribosomes excluded from contact sites.
>Membranes are connected by long multi domain proteins called tethers that hold the 2 membranes together, proteins are made of many domains that have different functions such as for lipid recognition.
167
How long can ER contacts last?
Can be long or short lived.
168
Give an example of a long-lived ER contact?
Muscle SR plasma membrane contact is long lasting, whole life time of cell as muscle needs to continuously make use of the connection
169
What are the 4 roles of tethers in MCS (inter-membrane contact sites)?
1. Protein to protein interaction
>Could be proteins on both membranes interacting
2. Protein to lipid interaction
>Could be protein on one membrane which has lipid binding domain which can bind with lipid on other membrane
3. Distance usually around 30nm but no less than 10nm
>Important as membrane contact sites never fuse.
4. Inhibit fusion of membranes
>Membrane fusion occurs when membranes are 1-2nm together and needs disruption of lipid bilayer, so having a minimum of 10nm difference it means the membranes are not close enough together to fuse and water is not excluded
170
What role does the Osbp tether have as well as acting as a tether?
Transfers cholesterol into ER, holds ER to mitochondria as well.
171
What are membrane microdomains?
Sections of membrane with specific compositions e.g. contact sites
172
Describe a raft microdomain of a membrane
Enriched in sterols; lipids that give rise to local rigidity.
173
What are 4 functions of contact sites?
1. calcium mobilisation
2. lipid transfer
3. signalling
4. organelle division
174
What is Ca2+ mobilisation needed for?
Muscle contraction
175
What is the Ca2+ conc in a) ER b) Cytosol c) Mitochondria?
a) Very high
b) Low
c) Low
176
Describe the long lived contact site of the sarcoplasmic reticulum and muscle cells for muscle contraction
Aps moving a long axon, causes mobilisation of Ca at neuromuscular junction, only works due to close connection of T tubules of muscle cells and Ca stores in SR. This Ca2+ mobilisation allows muscle contraction to occur.
177
In both skeletal and cardiac muscles what are found in close connection?
T tubules and SR have close connection
178
What is Stim1?
An ER transmembrane protein
179
Describe the process of calcium sensing by Stim1 in the ER?
1. Under resting conditions (lots of Ca2+ in ER) Stim1 is monomeric.
2. When Ca2+ levels drop in ER, Stim1 forms an oligomer which goes to top of ER tubule and interacts with ORAI-1 channel on plasma membrane.
3. ORAI-1 channel is enriched with PIP2 lipids, which open so Ca2+ can be transported into ER to replenish stores.
All possible due to the close connection of SR and the plasma membrane (as allows extracellular Ca2+ to move in through PIP2 which can then enter into ER stores).
180
What are the function of Phosphoinositide and an example?
1. Initiating cell signalling cascades (Ca2+)
2. Confirming organelle identity for example at a contact site
>E.g. PIP2
181
What is the role of PIP2 in Er Ca2+ replenishment?
PIP2 defines contact site, recruits ORAI-1 channel which forms contact site with STIM1, CA2+ can be transported into ER allowing for ER replenishment. (An example of how a membrane contact site is used for Ca movement and replenishment as extracellular Ca2+ moves through PIP2 into cytoplasm and then into ER via STIM1 at contact site with plasma membrane).
182
What type of transport is done for lipids at contact sites?
Allow unidirectional non vesicular transfer of lipid
183
What are LTPs and what protects them from soluble environment of the cytosol?
Lipid transfer proteins have hydrophobic grooves which Protects them from soluble environment of the cytosol
184
What are LTPs and what protects them form the soluble environment of the cytosol?
Lipid transfer proteins, have hydrophobic grooves which protects them from soluble environment of the cytosol.
185
How do LTPs use concentration gradients of lipids to promote lipid transfer?
Use counter transport: Couple the movement of ions and lipids so lipids can move against their conc-gradient as are carried by ions moving to a high conc.
186
What allows for lipid transfer on ER?
Lipid transfer proteins (LTPs)
187
How does Niemann Pick Disease C effect lipid transfer from lysosomes and what causes it?
>Lipids accumulate in lysosomes, not removed after they are taken up.
>Due to mutation in a transmembrane protein which is important for membrane contact sites allowing movement of cholesterol from lysosome into the ER.
188
How is the tether connection important for signalling by EGFR (epidermal growth factor receptor)?
>Membrane contact sites help regulate movement of EGFR into MVB (Multivesicular bodies)
189
What is EGFR (epidermal growth factor receptor) and how are they inhibited?
> EGFR is a receptor tyrosine kinase, when RTKs are activated by ligands they become phosphorylated and cause signalling events
> Stop signals are caused by dephosphorylation and downregulating them in lysosome.
190
Describe the generic way coated vesicles form in 6 steps?
1. GTPase in active GTP bound form at budding membrane.
2. Recruits adaptor proteins which binds to the GTPase and the cargo receptors on the budding membrane due to specific (and variable) motifs.
3. Adaptor protein recruits coat proteins to the ER budding membrane (helps stabilise the membrane)
4. Once vesicle has formed, GAP function of Adaptor protein increases
5. Causes GTP to be hydrolysed to GDP so GTPase inactivates.
6. Vesicle is uncoated, presenting SNAREs for binding.
191
What is tubular transport?
COPII vesicles extending to allow collagen pre-cursers to be transported by vesicles.
