Endocytosis Flashcards Preview

Advanced molecular cell biology > Endocytosis > Flashcards

Flashcards in Endocytosis Deck (69)
Loading flashcards...
1
Q

what os the general pH in acidic vesicles?

A

5- a lot lower than in the cytosol

2
Q

what is the genera role of lysosomes?

A

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.

3
Q

what delivers components to lysosomes?

A

endosomes

4
Q

what are the 4 options of processing faced by a membrane receptor? for each, give an example.

A
  • 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)
5
Q

in what type of endosome do recycling events normally happen?

A

early endosome

6
Q

what was the first example of a receptor being recycled and the ligand being processed?

A

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

7
Q

what type of vesicle endocytose receptors at the membrane?

A

clathrin coated vesicles

8
Q

what was the first experiment showing that clathrin was important for receptor endocytosis ?

A
  • 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
9
Q

how was is shown that clathrin is recruited to the receptors rather than the clathrin mediates receptor position?

A
  • 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
10
Q

what are the clathrin adaptor proteins, what is the morphology and how does this relate to their function?

A

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

11
Q

what is the difference in function between AP1 and AP2?

A

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)

12
Q

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?

A
  • 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
13
Q

other than APs and arrestin, what is another large family of clathrin recruiting proteins and how do these function?

A

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

14
Q

how can binding of a receptor recruit clathrin?

A

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

15
Q

during clathrin-mediated endoytosis, what protein is instrumental to driving membrane curvature?

A

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.

16
Q

describe the experiment which demonstrated the role of Bar-domain proteins in membrane sculpting

A

the took liposomes and added amphiphysin, this caused the normally spherical liposomes to form tubular structures

17
Q

as well as Bar-domain proteins like amphiphysin, which also proteins play a role in clathrin membrane bending, when do these act?

A

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

18
Q

once plasma mambranes have a curvature due to clathrin coats, what is the final step and what protein mediates this step?

A

there must be an excision process. This is carried out by Dynamin.

19
Q

how does dynamin function to induce excision?

A

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.

20
Q

what three things are thought to contribute to plasma membrane excision?

A
  • dynamin GTP hydrolysis power stroke and then release
  • synaptogenin PI2P to PI converstion and release of amphyisin
  • actin polymerisation away from the membrane
21
Q

what is a method of endocytosis that is not clathrin mediated?

A

endophilin endocytois

22
Q

why do membrane receptors need to be sorted in the late endosome?

A

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.

23
Q

what complex mediates receptor recycling in the endosome?

A
  • the retromer
24
Q

what are the two main subcomponent of the retromer?

A

the cargo selection domain and the structural role domain

25
Q

what is part of the cargo selection complex in the retromer?

A

VPS26/35/29

26
Q

what is part of the membrane sculpting domain of the retromer? what are these called in mammalian cells? what are their relevant domains?

A

VPS5/17 - SNX in mammalian cells - they have a PI3P (enriched in endosomes) binding PX domain and a bar domain to induce curvature.

27
Q

what is the role of the retromer?

A
  • 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.
28
Q

what is the fate of the receptors which aren’t targeted by the retromer and so are not used for recycling and so need to be degraded?

A

they are tagged by ubiquination and this drives its sorting into clathrin coats then its sorting into the lysosomes. this means they become incorporated into multi vesicular bodies.

29
Q

what is so strange about the budding of vesicles into multivasicular bodies?

A

it is the opposite to endocytosis, it is being budded off away from the cytoplasms rather than in to the cytosplasm, and forms a lumen in the centre. This results in the c terminus being in the lumen of bodies in the endosomes.

30
Q

How was the group of proteins involved in MVB formation first identified?

A

studies in yeast looking at there is a similar mechanism: they tagged a CPS protein with GFP and found that this protein is normally trafficked into the lumen of the vacuole, but in mutants for a group of genes called VPS called E proteins, this was inhibited. This became known as the ESCRT proteins

31
Q

what do ESCRT proteins detect on the receptors?

A

ubiquitin

32
Q

why is MVB an important process?

A

it takes the activated tyrosine kinase domain of receptors away from the cytoplasm and into the lumen so can’t signal anymore.

33
Q

how many ESCRT complexes are there?

A

4 (0-1)- they are recruited in this order

34
Q

what is the role of ESCRT-0?

A

VPS27, Hse etc. These have another lipid binding domain that bind to Pi3P which is enriched on endosomes. This brings the ESCRT machinery on to endosomes. They also have UIM (ubiquitn interacting motif): this is how ESCRT-0 initially selects ubiquitnated cargo molecules to be sorted ino the BVM and eventually lysosomes for degradation

35
Q

what is the role of ESCRT-1?

A

upon initialy recruiment for ESCRT 0. ESCRT-1 then binds to ESCRT-0. ESCRT1 can also bind to Ub cargo proteins via the same domain that binds to ESCRT-0.

36
Q

what is the role of ESCRT-2?

A

ESCRT-2 is comprised of proteins which have a domain that can also bind to Pi3P on endosomes , localising it to endosomes, and it can also reconise the ubiquinated cargo
ESCRT-2 acts a nucleator. It can take monomers of another set of ESCRT proteins called ESCRT-3, which has VPs proteins which can exist in the cytoosl as monomers, but upon enagement of the ESCRT-2 complex, they are formed into polymers (oliogmers),

37
Q

what is the role of ESCRT-3?

A

ESCRT-3 complex has a unique ability to form these polymers that can act as a cargo correl and capture cargo into a region of the endosome that will form a vescle that will bud into the lumen of the vesicle ESCRT-3, because it can be polymerised into filaments, can form spirals which drive membrane invagination into the lumenof the endosome.

38
Q

how is ESCRT-3 protein thought to work?

A

ESCRT-0: cargo capture, initaly recruitment of ESCRT-1 and 2 which drive the assembly of the escrt-3 polymer into a cargo spiral . Then it is thought that the further polymerisation of the ESCRT-3 molcules can now drive membrane deformation into the lumen of an endosome and possibly drive the excision event (this is debated). There is another protein called VPS4 which comes in at a later step that dissasembles the escrt 3 complex and this maybe be coupled to this fission step. ESCRT 3 assembles on the upper side of the vesicle not the lower side was with previous vesicle formation:

39
Q

the majority of the first ESCRT work was done in yeast, is it conserved?

A

yes- highly conserved processes- all the escrt complexes have been identified in metazoan and mammalian cells
so yeast dont have growth factor receptors- so all the found examples were relevant to g-protein coupled receptors
but in mammals is the same process which drives the degradation of an activated EGFR into intraluminal vesciles which will go on to fuse with the lysosomes

40
Q

have MVBs been seen to fuse with lysosomes?

A

yes- EM picture shows an MVB loaded with gold-particle tagged EGF, fusing with a lysosome (electron dense vacuole)- can see them docking and fusing.

41
Q

how are ESCRT proteins linked to cancer?

A

if these EGFR/ growth factor receptors are not nternalised properly they wil contineut to signal outside of the PM which could lead to out of ocntrolc ell grwoth leading to cancer
a lot these escrt proteins when mutated have been implicated in different types of cancers.
one of the drugs given to patients with cancer- is an antibody to the EGFR which drives its internalisation etc
all of these steps requires sophisticated molecular machines

42
Q

what does dynamic get recruited by?

A

amphiphysin

43
Q

what is the general pathway of endocytosis?

A

plasma membranes bud to form early endosomes, which form later endosomes which then fuse with the lysosomes or the golgi

44
Q

what is the structure of clathrin?

A

three heavy chains and three light chains that together form a legged triskelion

45
Q

what do clahtrin triskelions form?

A

assemble into a basket like framework of hexagons and pentagons to form coated pits

46
Q

what determines which receptors will be endocytosed?

A

the adaptor proteins that bind to them

47
Q

how does AP2 become activated and mediate the formation of the coat?

A

when AP2 binds to PI2P it alters its conformation, exposing the binding sites for cargo receptors in the membrane. The binding of AP2 simultaneously to lipids and to the membrane significantly stabilises the binding. This means that the proteins act as coincidence detectors, only assembly at the right time at the right place.

48
Q

how does AP2 act as coincident detectors?

A

when AP2 binds to PI2P it alters its conformation, exposing the binding sites for cargo receptors in the membrane. The binding of AP2 simultaneously to lipids and to the membrane significantly stabilises the binding. the binding of AP2 induces a curvature in the membrane, making the recruitment of more AP2 more likely.

49
Q

what protein functions to aid the peeling of the of the clathrin coat followingg excision?

A

in addition an hsp70 chaperone proteins functions as an uncoating ATPase, using enegry of ATP hydrolysis to peel of the clathrin coat.
Auxilin, another vesicle protein, is thought to activate the ATPase.

50
Q

when is the ligand released from its receptor?

A

in the early endosome

51
Q

what is special about this type of ubiquitnation?

A

Unlike polyubiquitylation, which adds a chain of ubiquitins that typically targets a protein for degradation in proteasomes, ubiquitin tagging for sorting into the clathrin-dependent endocytic pathway adds just one or a few single ubiquitin molecules to the protein—a process called monoubiquitylation or multiubiquitylation, respectively.

52
Q

how does ubiquitination have an effect?

A

it marks the receptor for lysosomal delivery rather than recycling- they have multiple functions- first to trigger endocytosis then for delivery to the lumen

53
Q

what is the yeast retromer and what is the mammalian homologue?

A
  • in yeast, the retromer is has the membrane shaping (VPS5p and VPS17p) and the cargo sorting subunits (VPS260/29p and 35p).
  • in mammals the, homologue of VPS5 are SNX1 and SNX2, and the homologue of VPS17p is SNX3, 5 or 6. These bind to form heterodimers
  • the trimeric subunit for shaping is highly conserved in mammals, but contains more paralogues.
54
Q

what is the difference between the mammalian retromer and the yeast retromer in terms of functioning of each domain

A

given the apparently weak assocaition of the SNX-BAR subcomplex with the mammalian heterotrimeric subcomplex and the large number of additional proteins that interact with mammalian retromer compared with yeast retromer, the SNX-BAR complex cannot be regarded as an inetrgal component mammalian retromer complex.

55
Q

what receptor has been shown to be sorted via the retromer in mammals?

A

sortilin and sortilin-related receptor, also thought WNTless form endosomes to the trans gold network
and has been found to sort proteins from endosome to the plasma membrane in conjunction with SNX27

56
Q

which SNX is thought to allow the retromer to shuttle receptors from the endosome to the plasma membrane rather than the TGN? and what receptor has this been shown for?

A

beta-2 adrenergic receptor- SNX27- binds to the SNX 1,2,5,6 proteins

57
Q

what are the specific functions of each of the cargo selection retromers?

A

VPS35 and VPS26 have been shown to have binding sites for c-terminals of cargo

58
Q

how has the retromer been linked to disease?

A
  • Ko of components of the retromer in mice is generally lethal
  • alzheimers disease model mice that bear only a single alle of VPS35 display earlier onset of alz phenotypes.
  • generally linked to neuropathologies
59
Q

what is the structure of the cell membrane shaping subunits of the retromer?

A

they have bar domains and PX domains which bind to PI3P

60
Q

how do the SNX proteins form membrane tubules?

A

BAR domains can oligomerise to form a stable helical array that induces and stabilises curved membrane stuctyres such as tubules and vesicles.

61
Q

how have the SNX-bar domain been shown to form tubules?

A

Many, but not all, of the 12 different mammalian SNX-BAR proteins will induce extensive endosome tubulation when over- expressed in cells, and can reshape liposomes into tubular structures in vitro

62
Q

what receptor is recycled by the retromer in mammals?

A

mannose 6-phopshate

63
Q

what ensures that the ESCRT proteins are only assembled transiently?

A

AAA+ ATPase VPS4

64
Q

what viruses recruit the ESCRTs and how?

A

HIV-1 uses a protein called Gag which they use to recruit ESCRT-1 and assemble the ESCRT complex, thereby allowing the virus to bus out of the cell

65
Q

in what cellular rpcoess does ESCRT play a role in? what showed this? which specific sub units are involved?

A
  • cytokinesis, the division of two daughters- it is a similar process- the budding away of the cell from the original cell
  • subunits of ESCRT-1 (TSG101 and ALIX) were shown to be recruited to the middy of the dividing cell
  • ESCRT-1 and ESCRT-3 and VPS4 are required
66
Q

what is the role of ESCRT-0:

A
  • binds to ubiquinated cargo and to PIP3
  • heterodimer
  • has 5 cargo recognition sites
    recruits ESCRT-1 via TSG101
67
Q

what is the role of ESCRT-1:

A
  • binds to 0 and 2

- contains the essential component TSG101

68
Q

what is the role of ESCRT-2:

A

can bind PI3P, ubiquitn cargo and ESCRT-1

69
Q

what is the role of ESCRT-3:

A
  • activated CHMP4 can recruit downstream ESCRT-III subunits such as CHMP2, CHMP3, CHMP1, and IST1 that have been shown to individually polymerize into filaments in vitro.
  • differs in that its components are soluble monomeric that polymerise when recruited and form filaments that form a loose and flat circular or spiral carry on the membrane.
  • It is also thought that VPS4 remodelling of the filaments causes tension on the membrane which leads to scission
  • VPS4 rapidly depolymerizes ESCRT-III filaments by unfolding and threading individual subunits through its central pore