Vesicle transport Flashcards
What does the endomembrane system do?
Name some of its roles
- divides the cell into different membrane bound compartments
- regulates the translation, modification and trafficking of proteins
- turns on/off signal transduction
What are some components of the endomembrane system?
- nuclear envelope
- endoplasmic reticulum
- Golgi apparatus
- Secretory vesicles
- Endosomes
- Lysosomes
- Autophagosomes
- Plasma membrane
What is a vesicle formed from and what is its function?
- Vesicles bud off of cells and fuse with different compartments
- They carry ‘Cargo’ - cargo is membrane associated soluble molecules
- Each vesicle must be selective for certain cargo and fuse with approproate target membranes
Whats the Secretory pathway?
The flow of membrane bound, and soluble proteins destined for certain organelles or extracellular space flow from ER –> golgi –> plasma membrane via secretory vesicles
Whats the Endocytic pathway?
Plasma membrane capture of extracellular components and internalisation of membrane proteins into vesicles that result in recycling of receptors or degradation of contents in the lysosome
What are the steps of protein transport in the cell?
Why is there a constant bidirectional flow of membrane and proteins?
To ensure integrity of individual organelles i.e. shape/ morphology as well as lipid and protein composition
What pathways are represented by each coloured arrow?
What are the requirements of vesicle transport?
- identification of specific cargo
- Sorting of vesicles and associated cargo
- transport
- Cytoskeletal motor proteins
- transfer of vesticular material
- Fission
- Tethering
- Fusion
The transport vesicles usually have coat proteins that…?
- Provide shape to membranes to “curve” and bud
- Determine the size and shape of the vesicle
- Concentrate the protein in the vesicle
- Provide selectivity for the “cargo”
- Determine the vesicle’s destination
- Coats provide specificity for the destination. Certain coats are only in certain pathways
Name 3 coated vesicles used for vesicle transport?
what locations does each vesicle transport cargo between?
- Clathrin coated vesicles- Trans-Golgi network (TGN) to endosome and plasma membrane (via endocytosis)
- COP I coated vesicles- Golgi complex to the ER (retrieval)
- COP II coated vesicles- ER to golgi
What are the different ways in which proteins associate with the lipid membrane?
What do Clathrin coated vesicles transport cargo between?
Transport material from plasma membrane and between endosomes and Golgi apparatus
What are Clathrin subunits made up of?
3 large (heavy chain) and 3 small polypeptides (light chain) that assemble in ‘triskelions’ at the trans-Golgi network (TGN) or at the plasma membrane
What do Clathrins form?
An outer protein lattice
In the following image of a clathrin, what part is the light and heavy chains?
What’s it called when the triskelion structures overlap?
- Red is the heavy chain
- Grey is the light chain
- End terminal globular domain (edge of arm), region which is the distal domain, proximal domain (inside) on heavy chain
- Trimerization domain is interaction and overlap between triskelion structure
Whats Endocytosis?
The englufment of extracellular molecules occuring at the plasma membrane
What does endocytosis regulate?
It regulates receptor signalling, receptor turnover, nutrient uptake, polarity, cell migration and neurotransmission
What are the types of endocytosis and their subtypes if there are any?
- Receptor- mediated endocytosis
- Clathrin-dependent
- Caveolin-dependent (lipid rafts, sphingolipids, GPI anchored proteins)
- Clathrin and Caveolin independent
- Phagocytosis (uptake of large molecules)
- Pinocytosis (uptake of small molecules)
During endocytosis at plasma membrane what recruitment is required?
Recruitment of AP2 adaptor protein complex is required for clathrin recruitment, coat assembly (formation of clathrin-coated pits), and eventual budding
When AP2 adaptor protein binds to a specific phospholipid, what does it result in?
conformational change that allows binding to cargo receptors on cell surface, triggers membrane curvature
What are cargo receptors involved in?
Ligand interactions
Clathrin coat formation diagram
What shape is the AP2 adaptor protein complex?
How many subunits make up the complex?
The AP2 protein complex is a heterotetrameric, multi subunit structure
Its made up of 4 main subunits
What are the four main subunits making up the AP2 complex?
- alpha adaptin
- beta2 adaptin
- omega 2 chain
- µ2 chain
AP2 on clathrin coated vesicles originate from where?
The plasma membrane (endocytosis)
Whats another adaptor complex, where is it specifically found and what does it help provide?
AP-1 adaptor complex (golgi)- helps provide specificity for certain cargos
What does the AP adaptor protein complex recognise?
It recognises specific peptide motifs on the cargo receptor (endocytosis signals)
What does the AP adaptor protein complex interact with?
plasma membrane lipids, cargo and clathrin
Whats the dominant mechanisms of endocytic clathrin coated vesicle formation
AP-2 complex interaction with phospholipid (Pidins (4,5)P2)
Where is the Clathrin binding site buried in the AP2 complex in?
Its ‘locked’, soluble state (not bound to membrane)
Binding to the PIP2 on the membrane exposes what?
The Clathrin binding motif in beta2-adaptin leading to a transition to the AP-2 ‘open’ conformation
When the PIP2 is bound to on the membrane, what happens with the µ2-subunit?
the µ2-subunit at the same time interacts with cargo which further stabilises AP-2 complex ‘open’ conformation and the dwell time of AP-2 at plasma membrane- thus facilitating clathrin coat assembly
How is a clathrin coat assembled?
- Protein “cargo” binds to a membrane bound receptor protein (e.g. Mannose-6-P receptor on the golgi)
- The receptors only selectivity recruit the correct cargo for the vesicle
- The receptor also binds adaptor proteins (e.g. AP-1 complex), which in turns binds to the triskelion cathrin
- Many “cargo-receptor-adaptin-clathrin” complexes form in a clathrin-coated vesicle (clathrin helps to shape and bend the membrane)
What does the AP complex link?
It is the link between the cargo proteins and lipids to clathrin, at vesicle binding sites, as well as binding accessory proteins that regulate coat assembly and disassembly
Vesicle formation and budding is assited by what?
dynamic (requires GTP)
When the clathrin coat dissociates and the components are recycled, what is left behind?
An uncoated vesicle that is transported to its destination
Membrane fission by dynamin
- Dynamic wraps around neck of vesicle
- Requires hydrolysis of GTP –> GDP + Pi
How does dynamin facilitate membrane fission?
- dynamin oligomerises and forms a helical ring around the neck of the bud (vesicle), it recruits other proteins and tethers itself to the membrane through lipid binding domains
- Dynamic constricts the presence of GTP
- GTP hydrolysis of dynamin results in the lengthwise extension of helix, and fission of membrane
Whats used to help understand dynamin function in vesicle?
Give an example of a mutation in dynamic that halts fission?
- Use of temperature sensitive mutants to understand dynamin function in vesicle scission
- Ts mutations in dynamin (e.g. Drosophila “Shibire”) halt vesicle fission and allow visualisation of arrested buds (function normally at the permissive temperature
- Results in immediate paralysis of the flies – but is reversible upon return to permissive temperature
How are clathrin-coated vesicles transported to lysosomes?
- Acid hydrolase enzymes are N-glycosylated, then phosphorylated on mannose-6 in the Golgi
- This allows binding to M6P-receptor and trafficking to lysosome
Questions to consider for revision…
- What are the requirements for vesicle transport?
- What is the role of the clathrin coat?
- How is the clathrin-coat assembled?
- Explain the mechanisms of AP-2 adaptor regulation and function during clathrin mediated vesicle formation
- How does dynamin regulate vesicle fission?
How mant subunits does COP II have?
What else is it associated with?
The COP II has 5 protein subunits and also has an associated GTPase
COP II interactions carry bulk protein but also specifically recruit what?
- enzymes for Golgi processing such as glycosyltransferases
- docking and fusion proteins
- integral proteins that bind to specific “cargo”
Tell me about the assembly of COP II vesicles?
- Sar1-GEF (Sec12) is embedded in donor membrane
- Recruits and activates Sar1 – loading with GTP
- Sar1-GTP recruits Sec23/24 which interacts with cargo, forming inner coat
- Sec13/31 forms the outer coat
What type of transport does COP I undertake and between what locations?
Retrograde transport from Golgi to ER
What do ER proteins have at the C-terminus what does this mean for its transport?
ER proteins have KDEL (Lys-Asp-Glu-Leu) at C-terminus which is recognised by a KDEL receptor in cis-Golgi, and retrieved by interaction with COP I
What does COP I comprised of?
It consists of 7 core subunits;
- alpha COP
- beta’ COP
- epsilon COP
- beta COP
- delta COP
- gamma COP
- Zeta COP
Where does COP I form?
In the cytoplasm prior to interaction with the membrane
How is COP I organised?
Organised as a cytoplasmic heptamer called coatomer and is recruited en blac to the membrane
i.e. coat organised first in cytosol and then recruited at membrane sites to form coated vesicle
What required for the coatomer recruitment?
Whats it activated by?
ARF1 GTPase is required for coatomer recruitment
Which is recruited and activated by Golgi-localised GEF proteins
These GEFs replace GDP to activate ARF1
COP I vesicles and retrograde transport
Key protein complexes and GTPases associated with each type of coated vesicles
Each type of coated vesicles has an associated what?
GTPase
e.g. Sar1 in COP II vesicles, ARF in COP I and clathrin-coated vesicles
GTP loaded ARF or Sar1 binds to what and therefore facilitates what?
Binds to effector proteins which facilitates coat assembly
In the presence of non-hydrolysable GTP analogous what do all types of coated vesicles do?
Why?
accumulate
This is because GTP hydolysis is required for coat assembly
Also, dynamin is a GTPase so budding cannot be completed
Numerous GTPases are involved in vesicle transport (draw a diagram which represents the GTPase proteins involved during the reactions between protein-GTP and protein-GDP)
(Rab family of GTPases)
What is the Ras family of small GTPases?
Small guanosine triphosphates (GTPases)- Over 150 human members
The Ras family of small GTPases is divided into 5 major branches based on sequence. What are these branches?
- Ras
- Rho
- Rab
- Ran
- Arf
In the Ras family of small GTPases, binary molecular switches occur between those which share common biochemical mechanisms. Name two in which these occur between and what it depends on
- Rab and Ras can switch between active and inactive form depending on whether is bound to GDP or GTP
- Function as monomeric G proteins- GDP/GTP regulated molecular switches
What do post-translational modifications control?
Subcellular localisation and interaction with the proteins that act as regulators and effectors
Whats the largest branch of Ras family?
The Rab GTPase family
it has roughly 61 members
What does the Rab GTPase family regulate?
How does it do this?
intracellular transport of vesicles and transport of proteins between organelles of the endocytic and biosynthetic pathway
It does this through interactions with effector proteins- facilitate vesicle formation, budding, transport, and vesicle fusion at acceptor site
Subcellular localisation and specificity for different intracellular compartments of each Rab is dependent on what?
The post-translational modifications (prenylation) and effector interactions
The Rab GTPase family
Rab GTPase and vesicle transport
Vesicles which originate from the plasma membrane aquire what Rab?
Rab5
The transition to early and recycling endosome requires what Rab?
Rab4/11
When do you aquire Rab7?
During vesicle transport and maturation towards late endosome/lysosome
Variation in Rab GTPase localisation during endocytosis
What keeps Rab inactive in the cytosol?
GDI keeps Rab inactive in the cytosol, sequestered away from membrane
What does GDF do?
GDF:
GDI displacement factors –> displaces GDI from GDP bound form of Rab, this allowing membrane anchor with its hydrophobic prenyl group
What does GEF do?
GEF mediated GDP to GTP exchange triggers a conformational change in the switch 1 and 2 regions of Rab allowing interactions with effector proteins
Tell me about Rab GTPase activation and effector recruitment
- Rab5-GDP (kept inactive in the cytoplasm)
- A Rab5-GEF binds Rab5 and activates it by exchanging GDP for GTP
- GDI is lost and GTP binding induces conformational change that exposes prenyl lipid group anchoring it to the membrane
- Active Rab5 can now bind effector proteins (e.g. PI3kinase)
- This changes the lipid composition which works collectively with Rab5 to recruit effector proteins (e.g. vesicle tethering proteins, signalling proteins, scaffold proteins)
Whats the Rab cascade and vesicle identity (vesicle maturation)
- As vesicles traffic along the pathway, they change composition and mature – acquiring different components required for function (e.g. SNAREs)
- Activation of a RabA-GEF to membrane, locally activates RabA
- RabA then activates effector proteins, one of which is a RabB-GEF
- Activation of RabB by the RabB-GEF, activates RabB effector proteins, one of which is a RabA-GAP
- This RabA-GAP inactivates RabA
- Thus, this cycle replaces a RabA domain on a membrane with RabB
- This sequence will continue via recruitment of the next GEF by RabB
Vesicles have to fuse with the correct target membrane
what happens once vesicles are made and budded off?
- Vesicles have to be uncoated to expose the v-SNARE protein
- Each v-SNARE protein on the surface of the vesicle has a corresponding t-SNARE protein on the target membrane
What facilitates SNARE assembly and vesicle fusion?
GTP-bound Rab protein binds an effector protein –> tethering protein (e.g. multi-subunit HOPS complex)
This facilitates SNARE assembly and vesicle fusion
What do V- and t- SNAREs have?
What do these interact with and what can these sorts of interactions also be used in?
Helices that interact with one another and dock the vesicle to the target membrane
The interaction is initiated by a vesicle specific Rab GTPase
These sorts of interactions can also be used in bringing vesicles destined for extracellular space into contact with the plasma membrane e.g. synaptic vesicles containing neurotransmitters during exocytosis
Tell me about vesicle docking
- Rab-GTP protein on vesicle surface binds to specific Rab-effector in target membrane
- This brings v-SNAREs and t-SNAREs into close proximity -allowing docking
- a-helices of v-SNARE and t- SNARE form coiled-coils (trans-SNARE complex)
- exerts inward force that brings the two membranes close together
The mechanism of membrane fusion is unknown, but may be opposite of dynamic model. What is this model?
- Lipid bilayers fuse by flowing into each other after being forced into close proximity
- A complex of two proteins (NSF and alpha-SNAP) binds to the “empty” SNARE complexes (cis-SNARE complex)
- ATP hydrolysis (catalysed by NSF) causes disassembly of the SNARE complexes and recycling
Mechanism of coordinated synaptic membrane fusion and neurotransmitter release
What does calcium induce in synaptic membrane fusion?
Calcium induces a conformational change in the complex allowing coordinated vesicle fusion with plasma membrane leading to neurotransmitter release
Endocytosis
Tell me about the vesicles transport to the lysosome…
- Golgi derived vesicle
- plasma membrane derived vesicles
- What they both require
- Eventual fusion of transport vesicles with the lysosome for degradation and recycling of contents
- Golgi derived lysosomal enzymes traffic through sub compartments before making way to lysosome
- Plasma membrna derived endocytic vesicles traffic through intermediate compartments before fusing with lysosome
- both require cargo transfer and machinery to become competent for fusion with lysosome
When vesicles are produced from the plasma membrane, what is their route of transport in order to get to the lysosome?
Plasma membrane –> early endosome –> late endosome/ multivesicular body –> lysosome
If the cargo i.e. receptor is recycled, how do it get to the lysosome? whats its endocytic pathway?
cargo –> early endosome –> recycling endosome –> plasma membrane
Golgi derived vesicles transport to lysosome
What do multivesicular body’s provide a mechanism for?
What do they require?
Provide a mechanism to shield receptors from cytosol, thereby turning off potential signal transduction
Require a specialised set of machinery for intraluminal vesicle formation
How do receptors at the plasma membrane get activated?
What does this lead to and how is this process controlled?
Receptors at the plasma membrane get activated by ligand-binding (e.g. Growth factors)
This leads to activation of a signalling cascade that results in changes in cell function (e.g. proliferation, changes in cell shape or gene expression)
These signals need to be controlled
One method of control is the internalisation and inactivation of the ligand activated receptor
What happens to ligand-receptor when its internalised?
What does this result in?
It gets sequestered (isolated) and transported to lysosome
This results in degradation of receptor and inactivation of signalling cascade
Activated signalling receptors at plasma membrane get internalised and trafficked via what?
What happens to these activated receptors?
What does this act as a signal for?
Activated signalling receptors at plasma membrane get internalised and trafficked via an endosome
These activated receptors get ubiquitylated
Ubiquitylation acts as signal for receptor sequestration within an intraluminal vesicle
Once MVB forms, it fuses with the degradative lysosome to breakdown and recycle components into building blocks (lipids, amino acids)
Tell me the binding domains associated with ESCRT-0 and what they interact with?
ESCRT-0 contains ubiquitin binding domain which interacts with ubiquitylated receptor cargo
ESCRT-0 also contains binding domain for interaction with PI3P rich phospholipid on endosomal membrane
Cargo (e.g. membrane receptor) ubiquitylated cargo and endocytosis initiates the process. What do the different ESCRT comples do?
ESCRT-0, -I, -II complexes bind to ubiquitylated cargo and the membrane phospholipid PI3P
ESCRT-III complex and Vps4 are necessary for membrane budding and scission
Whats VPS4?
Vascular protein sorting-associated protein 4
This is an ATPase that hydrolyses ATP to disassemble ESCRT complex allowing intraluminal vesicle to form
What do Virions bud off from?
What do they require?
Whats their shape similar to?
- Virions bud off from plasma membrane surface using the ESCRT machinery
- Requires similar membrane bending to intraluminal vesicle formation
- Shape of budding membranes are similar when comparing viral shedding from plasma membrane and intraluminal vesicle formation in multivesicular bodies
The lipid membranes that form the autophagosome are derived from what?
Once they’re sealed what happens?
Other intracellular membranes (e.g. ER, plasma membrane, Golgi)
Once sealed, its transported to the lysosome and fuses with the lysosome.
Onced fused, it contents gets broken down into building blockes which are lipids and amino acids
Whats Atg8 (aka LC3)?
A membrane protein that decorates inner and outer leaflets of an autophagosome
Following closure of the autophagosome, there are fusion events with the endosomes and MVBs to form what?
What does this result in and eventually lead to?
Following closure of the autophagosome, there are fusion events with endosomes and MVBs to form an amphisome
This also results in a gradual reduction in internal pH and acquisition of machinery to facilitate fusion with the lysosome (e.g. SNARE components)
Leading to eventual fusion with the lysosome to form an autolysosome, resulting in proteolytic degradation of components
Non-selective vs. selective autophagy
Autophagy receptors can interact with ubiquitin on cargo and also with Atg8 on autophagosome membrane to connect the two together
cytoskeleton
Actin cytoskeleton (review)
- How are they polarised?
- What are they composed of?
- What do they undergo?
- Are polarised, with (+) and (-) end
- Composed of G-actin subunits that assemble into F-actin filament
- ATP-G-actin gets loaded on filament and undergoes hydrolysis before release from filament
- Undergo actin treadmilling
- At physiological concentrations – G-actin gets preferentially added to (+) end, while being preferentially disassembled from (-) end
- Going onto +ve end faster than coming off from -ve end, which is why it looks like it moving towards a certain end, where it’s not just the rate of assembly and disassembly is different
What does the Arp2/3 complex promote?
Promotes actin polymerisation which drives internalised vesicles away from the plasma membrane
How does WASP activate Arp2/3 ?
Following interaction with active GTPase (Cdc42-GTP) through RBD motif, intramolecular interaction is relieved and W domain is exposed to bind actin and the A domain activates Arp2/3
When WASP binds to the Arp2/3 complex, what does this result in?
- The conformational change in complex and allows Arp2/3 binding to pre-existing actin filaments
- Actin subunit gets brought in by W domain of WASP and together binds to Arp2/3 to initiate actin nucleation
- Nucleation at (+) end occurs and filament extends
- Angle between old and new filament is 70˚
Relationship to Arp2/3 function during cell motility
What do the Actin motor proteins known as myosins tether?
Myosins either tether vesicles to actin cytoskeleton or transport vesicles along the actin cytoseleton and facilitate cargo delivery and fusion events
What are the common classes of myosin’s?
Whats their function?
Whats each of their step sizes?
Where do class V myosins function?
What do they move towards?
Whats its step size?
What is it essential for in budding yeast?
Myosin V function in organelle transport
Moves towards the +ve end of the actin filament
Has a step size of 30-40nm, which is ATP dependent
In budding yeast, MyoV is essential to transport mitochondria to newly formed bud
Whats the function of the microtubule network?
What is it involved in?
Its function is to transport vesicles or organelles
It’s involved in long range and fast transport (compared to short range and slower transport of myosin’s)
What does the vestibular tubular cluster serve as?
This organelle serves as a sorting station for cargo between the ER and golgi
What are dynein motors essential for?
Golgi positioning- aligns in perinuclear region and along the axis of cell polarity
What are Golgins?
What are they involved in?
What do they act as?
What do they interact with?
- Large proteins (over 30 genes), with coiled-coil domains adopting a rod like shape
- Golgins involved in transport and vesicle tethering around regions of the Golgi
- Act as Rab effector proteins
- Golgins interact directly with microtubules, with microtubule associated proteins or microtubule motors, such as dynein
- Contribute to Golgi positioning and morphology
Where are lysosomes positioned?
What can a loss of dynein lead to?
Lysosomes are positioned in perinuclear regions
Loss of dynein leads to a dispersal of lysosomes throughout the cytoplasm
What does the dynein heavy chain have that allows it to bind cargo or adaptors?
Dynein heavy chain has an ATP-dependent motor (head), Microtubule binding stalk region, and N-terminal stem that binds cargo or adaptors
What does the conformational change of the dynein motor head result in?
The conformational change of head relative to stem, leads to the movement of stalk domain which triggers the power stroke and therefore results in movement
Tell me how dynein-mediated movement results in a ‘power stroke’ ?
Stalk region interacts with microtubules and the ATP dependent motor domain performs the work
ATP hydrolysis by AAA domains in motor head leads to a conformational change, resulting in 8nm step size of dynein
Release of Pi results in ‘power stroke’
How does 1 dynein gene regulate the transport of many distinct cargo?
Dynein cannot function by itself; transport requires dynactin – a large complex linking dynein to cargo and regulating dynein activity
This complex can interact with a range of adaptor proteins, thus providing specificity for different cargo
Different motors can associate with the same organelle or vesicle. Why do bi-directional transport mechanisms exist?
To turn off one motor while turning on another e.g. dynein vs. kinesin, to allow movement towards different regions of the cell
This is especially important for long range movement in neurons
What does cAMP activate?
Downstream signalling via pKa, may influence motor protein activity/ interactions
