Endocytosis Flashcards
what os the general pH in acidic vesicles?
5- a lot lower than in the cytosol
what is the genera role of lysosomes?
they are considered to be the garbage bins of the cell, it is where misfolded proteins go to be degraded and where other compounds of the cell of in order to be degraded, allowing the components to be recycled.
what delivers components to lysosomes?
endosomes
what are the 4 options of processing faced by a membrane receptor? for each, give an example.
- the receptor is recycled and the ligand is degraded (LDL receptor and cholesterol)
- the receptor is recycled and the ligand is recycled too (transferring receptor and iron)
- the receptor is degraded and the ligand is degraded (EGFR and EGF)
- the receptor is transported and the ligand is transported (other side of the cell) (maternal IgG)
in what type of endosome do recycling events normally happen?
early endosome
what was the first example of a receptor being recycled and the ligand being processed?
cholesterol and the LDL receptor- the receptor-ligand is internalised and then there is a sorting decision, the receptor is sent back to the membrane and the cholesterol goes into the lysosome where it is recycled and processed
what type of vesicle endocytose receptors at the membrane?
clathrin coated vesicles
what was the first experiment showing that clathrin was important for receptor endocytosis ?
- they depleted the express of clathrin heavy chain using antisense RNA in BHK cells, they then analysed the internalisation of transferring during internalisation and found that its uptake was significantly reduced
how was is shown that clathrin is recruited to the receptors rather than the clathrin mediates receptor position?
- they used a virus that dependent on clathrin mediated endocytosis to infect cells. They found that once the virus had bound to the membrane, clathrin accumulates at the plasma membrane at the site where the virus is
what are the clathrin adaptor proteins, what is the morphology and how does this relate to their function?
AP1 and AP2 are enriched at clathrin coats. AP2 is the one involved in endocytosis at the plasma membrane. AP2 have two larger alpha and beta (adoption) subunits, and two smaller mu and gamma subunits. The role of the adaptor proteins is to connect lipids and cargo to the clatherin coat. The adoption subunits bind to the clathrin via their ‘clathrin box’. Mu recognises tyrosine-based sorting signals within the cytoplasmic domains of transmembrane cargo proteins. AP2 also binds PIP2 which is enriched at the plasma membrane.
AP1 binds PIP4
what is the difference in function between AP1 and AP2?
AP1 is generally involved in the binding of proteins, which have been translated by ribosomes and processed in the golgi, at the golgi membrane (such as mannose 6-phosphate receptors) to the trans-golgi network to endosomes and lysosomes where they are then sorted. (PIP4)
AP2 functions to transport receptors and cargo from the plasma membrane to endosome and lysosomes. (PIP2)
if the internalised receptor does not have a tyrosine motif for AP2 to bind, what can replace AP2 to recruit clathrin coat and to what kind of receptors is this relevant?
- arrestin can bind to the C terminals of G protein coupled receptors, and it also has a clathrin box which will recruit a clathrin coat
other than APs and arrestin, what is another large family of clathrin recruiting proteins and how do these function?
the ENTH and ANTH domain proteins, these domains binds PIP2 at their N terminal and and at the c terminus there is a clahtrin box and other domains that will bind directly to receptor so to ubiquitin tags on receptors
how can binding of a receptor recruit clathrin?
the binding induced a conformational change that exposes a C terminal domain sorting motif which can act to drive the binding of adaptor proteins which recruit clathrin
during clathrin-mediated endoytosis, what protein is instrumental to driving membrane curvature?
Bar-domain protein, specificlaly Amphiphysin . These bar domain proteins form dimers and then oligomers that polymerise. They have an inherent curvature and also they have an amphiphatic helix which has a hydrophobic nature on one side, inserting into the plasma membrane and acting as a wedge, which can further drive deformation of the membrane.
describe the experiment which demonstrated the role of Bar-domain proteins in membrane sculpting
the took liposomes and added amphiphysin, this caused the normally spherical liposomes to form tubular structures
as well as Bar-domain proteins like amphiphysin, which also proteins play a role in clathrin membrane bending, when do these act?
F-Bar proteins, they induce a different type of curvature. As soon as activated receptors begin to cluster, it is thought that adaptor protein bring clathrin and also F-bar proteins, these induce a slight curvature. Then amphiphysin is recruited to this membrane with a higher curvature and increases the curvature e
once plasma mambranes have a curvature due to clathrin coats, what is the final step and what protein mediates this step?
there must be an excision process. This is carried out by Dynamin.
how does dynamin function to induce excision?
i it is a modular protein and have an n-terminal GTPase domain. When bound to GTP or GDP it undergoes a conformational change. when GTP is bound, it will drive the dimerisation of dynamin protein into a protein scaffold which will bind around the neck and constrict it further. it forms like a necklace around the neck. There is also a Stalk domain and a PH domain The PH domain binds PI2P at the membrane. the Ph and stalk domain are bound on the inside to the membrane and the GTPase domain is on the outside. Its assembly is thought to cause super constriction and when two plasma membranes become 4nm apart they spontaneously fuse- so maybe this happens. It is thought that the hydrolysis of GTP causes a power stroke, inducing fusion nd then the release of dynamin. Furthermore, synaptogenin converts PIP2 to PI, releasing amphiphysin.
what three things are thought to contribute to plasma membrane excision?
- dynamin GTP hydrolysis power stroke and then release
- synaptogenin PI2P to PI converstion and release of amphyisin
- actin polymerisation away from the membrane
what is a method of endocytosis that is not clathrin mediated?
endophilin endocytois
why do membrane receptors need to be sorted in the late endosome?
because their signalling kinase domain is still facing the cytosol and so can still mediate signalling within the cell. This means they need to be sorted and degraded or recycled.
what complex mediates receptor recycling in the endosome?
- the retromer
what are the two main subcomponent of the retromer?
the cargo selection domain and the structural role domain
what is part of the cargo selection complex in the retromer?
VPS26/35/29
what is part of the membrane sculpting domain of the retromer? what are these called in mammalian cells? what are their relevant domains?
VPS5/17 - SNX in mammalian cells - they have a PI3P (enriched in endosomes) binding PX domain and a bar domain to induce curvature.
what is the role of the retromer?
- for recycling of receptors. it can polymerise and bind multiple receptors while causing elongation of tubule structures from the endosomes that can present their bound receptors to be recognised to be recycled back to the golgi or back to the PM. These tubules will undergo excision.
