Vesicular Transport Flashcards
Describe the structure of clathrin coated vesicle.
Clathrin-coated vesicles consist of 36 overlapping triskelions arranged into pentagonal and hexagonal mesh-like structures, resembling a basket. A triskelion consist of clathrin protein subunits, each having 3 large (heavy) and 3 small (light) polypeptide chains. Each chain of the triskelion faces inward (into the vesicle, away from the cytosol) whereas the N-terminus of each chain forms an intermediate shell which harbours adaptor proteins, adaptins. The overlapping segments of triskelions provide strength and flexibility.
Describe clathrin-mediated endocytosis.
Upon binding of cargo with a respective receptor, clathrin triskelion is assembled by the adaptin protein (AP2) which connects it with the cargo-bound receptor. The plasma membrane region is curved inwards and with the increasing density of cargo-bound receptor, an invaginating bud forms. This bud is pinched off by a ring-like protein dynamin. Dynamin is a GTP-binding protein that recruits other proteins to the vesicle neck, whit together with dynamin destabilize the membrane so that the non-cytoplasmic leaflets of the lipid bilayers flow together. A newly formed vesicle is formed and clathrin triskelions is rapidly removed.
The formation of a clathrin coated pit is driven by forces
generated by the successive assembly of adaptins and the clathrin coat on the cytosolic surface of the membrane . The lateral interactions between adaptins and between clathrin molecules then aid in bud formation.
How many adaptin types are there?
Adaptin protein 1-4, each adaptin is recruted in different type of cargo, receptors and membrane types.
What transporting vesicles are used to transport cargo from:
- ER->Golgi
- pre Golgi-> Golgi cisterane
- COP II
- COP I
Describe the function of small GTPase and molecules that control its activity. What processes are the small GTPases involved in?
Hydrolyse proteins capable of cleaving GTP to GDP. Similar to alpha subunits of heterotrimeric G-proteins but they can function independently.
Active = bound to GTP Inactive = bound to GDP (hydrolysed GTP to GDP) GEF = guanine nucleotide exchange factor exchanges GDP to GTP and activates it GAP = GTPase activating protein hydrolyses the GTP to GDP GDI = guanine nucleotide dissociation inhibitors keeps the GTPase in the inactive state GDF = GDI displacement factor displaces the GDI
Growth, cellular differentiation, cell movement and lipid vesicle transport.
How do COP II coated vesicles function?
The Sar1 protein is a coat recruitment GTPase.
Inactive, soluble Sar1 GDP binds to a GEF (called Sec12) in the ER membrane, causing the Sar1 to release its GDP and bind GTP. A GTP triggered conformational change in Sar1 exposes its hydrophobic tail , which inserts into the ER membrane. Membrane bound, active Sar1 GTP recruits COPII subunits to the membrane. This causes the membrane to form a bud, pinch off and releases the coated vesicle. Other coated vesicles are thought to form in a similar way.
Sar1 protein is responsible for COP II assembly, which protein works similarly in COP I and clathrin coats at the Golgi and PM?
ARF proteins are responsible for both COPI coat assembly and clathrin coat assembly at Golgi membranes. Clathrin coat assembly at the plasma membrane GTPase is unknown.
What are SNARE proteins and how do they work?
V-SNAREs (vesicular) and t-SNAREs (target membrane, complementary). V-SNAREs are packed together in the vesicle and co-localise with t-SNAREs and remain associated until prayed open. SNAREs facilitate the membrane fusion which requires water exclusion and great closure of both membranes.
The v-SNARE synaptobrevin + t-SNARE syntaxin are transmembrane proteins and each contributes one a-helix to the strong trans-SNARE complex.
The t-SNARE Snap25 is a peripheral membrane protein that contributes two a-helices.
How are the v-SNAREs and t-SNAREs dissolved?
The complexes have to be disassembled before the SNAREs can mediate new rounds of transport. NSF (ATPase) binds to the SNARE complex via adaptor proteins and hydrolyzes ATP to pry the SNAREs apart.
SNAREs facilitate the fusion of the membranes, what proteins facilitate the docking?
Rab proteins work together with other proteins to regulate the initial docking and tethering of the vesicle to the target
membrane.
Explain the function of Rab proteins in docking and transport.
A GEF in the donor membrane recognizes a specific Rab protein and induces it to exchange GDP for GTP. GTP binding alters the conformation of the Rab protein, exposing a covalently attached lipid group, which helps anchor the protein in the membrane. The Rab GTP remains bound to the surface of the transport vesicle after it pinches off from the donor membrane, and it then binds to varying Rab effector proteins on the target membrane. The Rab protein and its effectors help the vesicle dock and thereby facilitate the pairing of the appropriate v-SNAREs and t-SNAREs. After the vesicle has fused with the target membrane, the Rab protein hydrolyzes its bound GTP, releasing Rab GDP into the cytosol, from where it can be reused in a new round of transport.
Rab GDP in the cytosol is bound to a GDI, which prevents the Rab from releasing its bound GDP until it has interacted with appropriate proteins in the donor membrane.
Name exaples of Rab proteins and their targeted membrane.
Rab1 ER and Golgi complex
Rab2 cis Golgi network
Rab3A synaptic vesicles, secretory granules
Rab4 early endosomes
Rab5A plasma membrane, clathrin coated vesicles
Rab5C early endosomes
Rab6 medial and trans Golgi cisternae
Rab7 late endosomes
Rab8 secretory vesicles (basolateral)
Rab9 late endosomes, trans Golgi network
How does a HIV virus enter the cell?
HIV binds first to the CD4 protein on the surface of the lymphocytes. This interaction is mediated by the viral gp120
protein bound to the HIV fusion protein. A second cell surface protein on the host cell, which normally serves as a
receptor for chemokines, now interacts with gp120. This interaction releases the HIV fusion protein from gp120
allowing the previously buried hydrophobic fusion peptide, to insert into the plasma membrane. The fusion protein,
which is a trimer, thus becomes transiently anchored as an integral membrane protein in two opposing membranes.
The fusion protein then spontaneously rearranges, collapsing into a tightly packed six helix bundle. The energy
released by this rearrangement in multiple copies of the fusion protein is used to pull the two membranes together,
overcoming the high activation energy barrier that normally prevents membrane fusion. Thus, like a mouse trap, the
HIV fusion protein contains a reservoir of potential energy, which is released and harnessed to do mechanical.
What are vesicular tubular clusters and where are they fromed?
Condensates of vesicles forming tubullar clusters by homotypic fusion. Vesicular tubular clusters move along microtubules with the help of motor protein to carry proteins from the ER to the Golgi apparatus.
COP II = ER -> vesicular tubular clusters -> cis Golgi
COP I = cis Golgi/vesicular tubular clusters -> small vesicles -> ER
How are soluble resident ER protein returned back to the ER after shutling cargo to the Golgi?
The KDEL receptor (in vesicular tubular clusters and the Golgi) captures the soluble ER resident proteins and carries them in COP I coated transport vesicles back to the ER. Upon binding its ligands in this low pH environment, the KDEL receptor may change conformation, so as to facilitate its recruitment into budding COP I coated vesicles. The retrieval of ER proteins begins in vesicular tubular clusters and continues from all parts of the Golgi apparatus. In the neutral pH environment of the ER, the ER proteins dissociate from the KDEL receptor , which is then returned to the Golgi for reuse.
