Topic 4B - Vesicle Transport Flashcards
1
Q
What 3 pathways are used for vesicular transport
A
- biosynthetic secretory pathway
- endocytic pathway
- retrieval pathway
2
Q
Name and describe the thee characteristics of vesicular traffic?
A
- Organized: there must be a specific pathway (ER - Golgi)
- Balanced: movement in and out of the cell
- Selective: cannot randomly pack the proteins
3
Q
What molecule functions as an organelle and membrane domain marker
A
phosphatidylinositol
4
Q
Into what states can phosphatidylinositol be phosphortylated or dephosphorylated, and which domains on the phosphate specifically?
A
- PI(345)P3
- PI(35)P2
- PI(34)P2
- PI(45)P2
- PI(3)P
- PI(4)P
- PI(5)P
- PI
- sites 3, 4, 5 are always phosphorylated
5
Q
What is the function of the phosphatidylinositol? (3)
A
- lipid signalling
- cell signalling
- membrane transport
6
Q
What i the function of coated vesicles (2)
A
a) these coat proteins help mold the vesicles
b) concentrate specific proteins
7
Q
Give three examples of coated vesicle proteins? (Important slide)
A
- clathrin - trans golgi to secretory vesicles
- COPI - involved in the golgi cisternae
- COPII - involved in ER to golgi
8
Q
Does clathrin coated molecules always pack the same molecules? what is responsible then?
A
NO; there are several different types of adapter proteins specific to the cargo
- the adapter protein is responsible for the cargo that is transported
9
Q
How does the adapter protein help with the formation of the clathrin coat?
A
- AP2 has 4 subunits that must bind a phosphoinositide in the membrane leading to a conformational change on the protein
- now active AP2 can bind to the cargo receptors on the membrane – this will lead to curvature on the membrane
10
Q
Which two monomeric GTPases are involved in the control of coat assembly?
A
- these are two coat-recruitment GTPases
1) Arf1 monomeric GTPase - involved in COPI and clathrin coat assembly
2) Sar1 monomeric GTPase - involved in COPII coat assembly
11
Q
Which two proteins help control the monomeric activation and inactivation?
A
- GEF - causes the GDP to be removed and GTP added for activation
- GAP - causes the GTP to be removed and GDP+Pi to be added for inactivation
12
Q
What is the function of Sar1 GTPase?
A
- a monomeric GTPase involved in COPII vesicles regulation for assembly and disassembly
- Sec23-24 and sec13-31 function as the adapter proteins for the COPII coat formation
13
Q
Arf1 monomeric GTPase one key point.
A
- is activated by the curvature of the membranes
14
Q
What is attached at the N-terminus of Sar1 and Arf1?
A
- an N-terminal amphipathic alpha helix (both hydrophilic and hydrophobic parts)
15
Q
At this N-terminal amphipathic alpha helix, does the affinity to membranes change when there is a polar substitution in the amphipathic helix?
A
- YES; a polar substitution will not want to be added into the membrane
16
Q
what is Rab? and where is it involved? function?
A
- Rab is a monomeric GTPase bound to a cargo vesicle which has just released the coated vesicle and is about to bind to a membrane
- transport to the target membrane
- vesicle docking
- participation in membrane fusion
17
Q
What regulates Rab’s function? how is Rab removed?
A
- GEF will bind a GTP to the monomeric Rab-GTPase which is bound to the vesicle
- the active Rab-GTPase will bind/tether to the Rab-effector protein pulling the vesicle to docking
- GAP will remove the GTP and bind a GDP which will make Rab-GDP bind to a GDI protein (inhibitor)
18
Q
What proteins are involved in vesicle fusion to a membrane?
A
- monomeric Rab-GTPase
- Rab-effector: attached to the membrane (tethering protein)
- v-SNARE on the (vesicle-SNARE)
- t-SNARE on the (target(membrane)-SNARE)
19
Q
What occurs during the docking phase?
A
- v-SNARE will bind to t-SNARE tightly to form a SNARE complex to lead to fusion
20
Q
What occurs during the Fusion step of vesicular SNAREs mediated membrane fusion?
A
- after the SNAREs complex is formed (stalk) all the water will be squeezed out of the way forming a hydrophobic effect to allow the membranes to become continuous
- where first Hemifusion followed by Fusion of the two membranes will occur
21
Q
What protein is involved when two vesicles attempt to bind together or after fusion the SNARE complex must be dissociated
A
- through the NSF proteins which uses ATP hydrolysis to dissociate v-SNARE to t-SNARE to recycle them
22
Q
In what direction to COPI and COPII vesicles travel and why?
A
- due to protein retention signals (retrieval pathway of soluble ER proteins)
- COPI (ER to Golgi - via Arf1)
- COPII (Golgi to ER - via Sar1)
23
Q
What is the role of golgi apparatus?
A
- receiving proteins from the ER @ the cis side, where proteins are sorted (assembly line) via phosphorylation of oligosaccharides and the addition of sugar molecules via glycosylation
- the proteins will be placed in vesicles at the trans side facing the cell membrane where they will be bound to:
1. lysosome
2. plasma membrane
3. secretory vesicle
24
Q
What is the Cisternal Maturation Model?
A
- protein cargo remain in a given compartment and different enzymes arrive in the cisternae to convert a cis to medial to trans cisternae
25
What is the Vesicle Transport Model
- each cisternae remains in place with the same enzymes (unchanged) and the proteins move forward through the stacks in the Golgi via vesicles that move from earlier to later cisternae
26
Explain the glycosylation process?
- precursor oligosaccharides are added to proteins in the ER
- modifications of the of these oligosaccharides occur in the golgi
- these oligosaccharides CORE REGION consist of 3 branched mannose and 2 N-acetylglcuosamine
27
What is the purpose of glycosylation?
1. promotes protein folding
2. proteins more resistant to digestion
3. regulatory roles (signalling)
4. protection against pathogens
28
In vesicles that contain lysosomes (Acid Hydrolases) why is the pH lower than compared to the the cytosol, and how is this done?
- through V-type ATPases (a H+ pump that pumps protons into the cell lower the pH)
- this is done to form a failsafe if these proteins leak out they will not be active and degrade the cellular machinery
- low pH's are required for the lysosomes to become active an function
29
What is unique about lysosomal membrane proteins?
- they will be highly glycosylated so they do not degrade
30
What is the equivalence of lysosomes in plant cells?
- plant cells do not have lysosomes, they have the central vacuole
31
What does the transport look like between Golgi to Lysosomes?
- via vesicular transport
| - Golgi to Late Endosome to Lysosome
32
What are the 4 pathways that deliver vesicles to lysosomes?
- Endocytosis (late endosome)
- Exocytosis
- MacroPinocytosis (late endosome)
- Autophagy
33
What is endocytosis?
- a form of active transport where a cell transports molecules into the cell by engulfing them in an energy-rich process
34
What is pinocytosis (as a form of endocytosis), and what forms are there?
- cell drinking
1. a constituitive process
2. macropinocytosis
3. receptor-mediated pinocytosis
35
Explain the 3 types of pinocytosis
- Constituitive process: where this process maintains the cells size by having a endocytic-exocytic cycle & maints fluidity
- Macropinocytosis: occurs non-specifically OR may be induced
- Receptor-mediated endocytosis: via LDL protein particles and their receptors
36
What occurs during receptor mediated endocytosis?
- example LDL particle and receptors
- the LDL particle binds the receptor and an endocytotic vesicle will form with clathrin coated vesicle forming
- the vesicle will then uncoat and fuse to the early endosome where the LDL particle and receptor dissociate -- the receptor is recycled by budding off in a transport vesicle bound for the plasma membrane
- the LDL is transferred to the lysosome a degraded
37
What are 3 possible fates after pinocytosis (endocytosis)
- TRANSCYTOSIS (as vesicle transport of a receptor)
- RECYCLING (of the receptor that bound its ligand that caused the endocytosis process)
- DEGRADATION (in a lysosome the receptor and its ligand are sent to an endolysosome for degradation)
38
What are two pathways for sorting Plasma Membrane Proteins (via exocytosis and endocytosis)?
1 - Direct sorting in the trans golgi network; endocytosed transmembrane proteins will be sorted in the trans golgi, packed up a sent to its target site on the plasma membrane
2. Indirect Sorting at Early Lysosomes; as the vesicle enters the cell it will bind to a lysosome where the proteins will be separated and packed into new vesicles bound to the plasma membrane
39
What are 2 functions of Phagocytosis?
1. unicellular consume a molecule and digest it for food
| 2. multicellular cells will take up dead cells and recycle its components
40
What initiates phagocytosis?
Rho-GTPases (monomeric)
- functions by accumulating PI(4,5)P2 & localized actin polymerization to all the engulfing process
- to seal the phagasome a PI3Kinase will depolymerize PI(4,5)P2 --> PI(3,4,5)P3
41
What are the 3 forms of Exocytosis?
1. Constituitive Secretory Pathway (proteins just accumulate in the golgi LACKING a signal)
2. Signal-Mediated Diversion to Lysosome (regulated by a extracellular signal)
3. Signal-Mediated Diversion to Secretory Pathway
- signal-mediated: will only occur when a signal is received before releasing it contents
42
What is unique about the Constituitive Secretory Pathway?
- found in all cells
| - ex. the golgi released vesicles for proteins required at the membrane
43
What is special about Regulatory secretory pathway?
- specific only to cells that ex. secrete hormones
- this will only occur when a signal is received
- so some vesicles may wait at the membrane waiting for a signal before exocytosis occurs
44
Explain proteolytic proccessing?
1) Cargo concentration - as the vesicle moves to its target site it becomes smaller as components of the vesicle are sent back to the golgi and the contents of the vesicle increase in concentration
2) Proteolytic processing examines how the vesicle becomes more acidic as the protein/enzyme undergoes maturation (activation) - since proteins are made as polyproteins
45
Why is proteolytic processing a common process?
- This method helps a cell avoid damage to the inside of the cell
46
An example of Exocytosis is the synaptic vesicle - explain the 6 steps.
1. from the Golgi a vesicle arrives at the presynaptic plasma membrane with containing its components
2. the endocytosis of synaptic vesicle membrane components to form a new synaptic vesicle
3. Endocytosis of synaptic vesicle membrane components and delivery to endosome
4. budding of the synaptic vesicle from an endosome
5. neurotranmitters are loaded into the synaptic vesicle
6. the secretion of the neurotransmitters due to an action potential
47
What is special about synaptic vesicles during endocytosis?
- as the vesicle approaches the membrane DOCKING myst occur between v-SNARE and t-SNARE
- a partial SNARE complex forms called PRIMING I since a complexin protein inhibits the SNARE bundle at PRIMING II
- released Ca2+ will come in an bind to synaptobrevin and a complete FUSION PORE OPENING
- as complexin is removed with Ca2+ FUSION COMPLETEs as the neurotransmitters are released
48
4 types of exoctysosis to enlarge the plasma membrane?
- cytokinesis
- phagocytosis
- plasma membrane repair
- cellularization
