Lecture 9 trafficking 4 Flashcards
(34 cards)
Where does sorting of cargo mainly occur?
Endosome system
What are the two mechanisms for specific cargo recognition?
Primary (mainly short) sequence determinants or PTMs of cargo.
Where are primary cargo determinants?
Cytosolic domains. E.g., C terminus.
What does cargo recruitment require?
Multiple adaptors.
What domain do adaptins interact with of clathrin?
Terminal domains as they point inwards towards the membrane.
What motifs do clathrins recognise?
NPXY
What structure does clathrin adopt?
A beta propellor structure which binds to the clathrin box this is a peptide motif. Any structures with this motif will interact with clathrin. Also recognise proteins with the DLL motif or SDLL motif (the variant clathrin box). Also recognise the W-box (PWxxW). The W box sits on top where the axel is. The clathrin and variant box bind on the blade of one of the propellers Not a mutually exclusive binding site so can bind to multiple at once.
What are the main adaptins?
1-5. They are heterotetramers.
Where do APs show similarities?
To COPI both have beta propellor domains. There is sequence similarity between 4 of the chains in COPI and adaptins.
What are adaptins made up of?
Heterotetrametric. Two large subunits (around 125kDa: Beta and one of alpha gamma etc), one medium (around 50kDa), one small (around 18kDa).
What are some of the AP2 binding sites?
Clathrin binding site, cargo motif present in beta subunits, the alpha and beta subunits share structural homology they have a solenoid structure made up of continuous alpha helices with a loop at the end of the helices.
What was realised when AP2 structure was crystallised?
The PIP2 inositde binding sites are not coplanar. The cargo binding site is blocked.
How does AP2 then function?
The alpha subunit rotates out. Brings the two PIP binding sites into a co planar and reveal the cargo binding site. A clathrin activating kinase phosphorylates the U2 subunit the contains the PIP2 binding site to open up the structure. The PI head groups have electrostatic interactions attraction of them to the U2 subunit. Believe other APs have a similar mechanism.
What cell component do GGAs (Golgi localised, gamma ear containing ARF binding proteins) transport proteins across?
The golgi, lysosomes, retrieval pathways back to golgi.
Where GGAs discovered?
Two in yeast, three in mammals
What are GGAs structure?
Monomeric, clathrin and cargo binding adaptors. Have an N terminal VHS domain. Proline rich linker. GAT domain. C-terminal GAE domain. Bind to clathrin via clathrin box. Binds cargo molecules carrying DxxLL motifs.
What does the GAT domain do?
1 end called a hook region and helicle bundle. The hook region interacts with ARF GTP bound. The helicle bundle interacts with Rab actin 5 effector protien. This interacts with rab X 5.
Why is ubiquitylation essential in yeast?
Essential for receptor internalisation.
What are some examples of ubiquitin binding domains that cargo proteins contain?
UIM, UBA, CUE, NZF, GAT
How many members of epsins are in higher eukaryotes?
Epsin 1-3 and epsin R
What are epsin 1 and 2 for?
Clathrin-mediated endocytosis.
What is epsin R for?
TGN-endosome transport
What architecture do epsin motifs share?
terminal ENTH domain. extended flexible body containing many binding motifs.
What is in the ENTH domain?
The ENTH domain of epsin binds to the membrane lipid PIP(4,5)P₂ through electrostatic interactions between the lipid’s negatively charged headgroup and basic residues in the ENTH domain. Upon binding, an amphipathic α-helix called Helix 0 is induced to form, which then inserts into the membrane. This not only stabilizes epsin at the membrane but also helps to bend the membrane, promoting clathrin-coated vesicle formation during endocytosis.
