Lecture 9 trafficking 4 Flashcards
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 to GGAs 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?
PIP(4,5)P2 headgroup that interacts with basic domains in the ENTH domain. Helix 0 is an induced helix. When ENTH domain interacts with PI45P2 in the membrane helix 0 forms has a number of basic residues that contributes to the binding.
What are the two families of arrestins?
alpha arrestins
beta arrestins (includes visual arrestin. beta 1 and beta 2 arrestins)
What do beta arrestins recognise?
GPCR- thought to compete with G proteins for GPCR binding. Induces a confirmational shift and ubiquitylation.
Where is the clathrin binding motif in beta arrestin?
In the c terminus.
What happens when receptor gets activated?
Receptor gets phosphorylated on c terminus domain by GRKs (G protein receptor kinases). Now carries negative charge. Important as arrestin wants to recognise receptor. In the closed form of arrestin is a polar core with a number of key conserved basic residues arginines. That polar complex keeps c terminal inside its confirmation. The phosphates from activated receptors insert themselves into polar complex. Negative charge so ionic interactions are disrupted in arrestin causing it to become an open confirmation. The C terminus is flipped out and gets ubiquitin modified. Recruiment via interaction of adaptins or cargo adaptors into vesicles as part of the endocytic process.
What are amphisynins?
Phospholipid adaptors they selectively link clathrin only to phospholipids. Amphiphysins are involved in late stages of CCV budding. They recruit dynamin and synaptojanin. They only respond to curve surfaces. Two genes in humans number 1 is brain specific and number 2 is widely expressed.
How do amphiphysins function and what are their key domains?
Function as dimers. N-terminal BAR domain. C terminal SH3 domain.
What is the BAR domain for?
Interacting with the curved lipid membrane. Helical bundle region forms the dimer interface causing a curvature. the concave side is positively charged to bind to the curved negatively charged phospholipids on the membrane.
What is the SH3 domain for?
Interacts with proline rich motifs. synaptojamin and dymanin also contain them. Dynamin binds for vesicle fission and synaptojamin to facilitate the hydrolysis locally of PIP(4,5)P2. Both for uncoating and loss of adaptins. Leaves cargo present without vesicle.
Where are different adaptins found?
AP1 GGAs. AP2 epsin. AP3 lysosome related organelle.
Why do cargo motifs have low affinity interactions?
For ease to release from the interaction. Many cargo proteins contain multiple low affinity recognition motifs. Multiple low affinity interactions gives an overall high avidity, but facilitates efficient cargo release when required.
