Lecture 8 - Endocytosis, Cell Sorting and Cargo Recruitment

Question 1

What two types of transport require cargo sorting?

Where does this sorting occur?

Answer 1

1. Receptor-Mediated Endocytosis - receptor recycling or degradation
2. Anterograde Transport

• Sorting typically occurs in the endosomal system

Question 2

What are the two types of cargo recognition mechanism utilised by cells?

Answer 2

1. Primary Sequence Determinants - short, located on cytosolic domains
2. PTMs (e.g., Ub, M6P) - do not necessarily require contiguous sequences

Question 3

State the Four ways in which the Clathrin chain may recruit cargo

Answer 3

• Clathrin itself:
  1. Binds to proteins with NPXY motif (cargo/adaptors)
• Clathrin Terminal Domains - adopt B-propeller, which binds:
  2. Clathrin Box via periphery of propeller
  3. DLL motif
  4. W-box via centre of propeller

Question 4

What are Adaptins?

Describe the Domain organisation of AP2

Answer 4

• Family of Heterotetrameric Cargo Adaptors, each consisting of 2 Large Subunits, 1 Medium Subunit, 1 Small Subunit
• AP2 - consists of a main globular domain, connected to two ear domains (appendages with globular domains)

Question 5

AP2 contains two PIP2 binding domains, however these cannot bind to PIP2 at the same time in the closed conformation. Explain why, and how this is resolved.

(2 Points)

Answer 5

• PIP2 binding sites - not coplanar in closed conformation, requiring the flipping out of the µ subunit to allow interaction with membrane (also exposes cargo binding site)
• This occurs via phosphorylation of Thr-156 in the µ-subunit by AAK, which weakens interaction that holds AP2 in closed conformation

Question 6

Define GGAs in terms of:
(i) What they are
(ii) How they are recruited
(iii) How they recruit Cargo
(iv) How they recruit Coat proteins

Answer 6

(i) GGAs - family of monomeric Golgi-resident cargo adaptors, which mediate transport from trans-Golgi to Late Endosome

(ii) Recruited via their GAT domain, which is a 3 helical bundle containing a hook domain that interacts with ARF1 GTPase

(iii) Recruit cargo with acidic dileucine motif (DLL)

(iv) Recruit Clathrin using a clathrin box in appendage of ear domain

Question 7

Define Epsins in terms of:
(i) Function
(ii) Structure

Answer 7

(i) Family of Ubiquitin Binding Adaptors
(ii) Consist of:
* Terminal ENTH Domain - mediates interaction with membrane (via PIP2, which causes formation of Helix 0)
* Extended Flexible body containing several binding motifs

Question 8

What is the Function of Helix 0 (ENTH)?

Answer 8

Once formed, its hydrophobic side chains insert into the membrane and stabilise the interaction, as well as inducing curvature

Question 9

Describe Hrs/Vps27p in terms of:
(i) Function
(ii) Structure

(5 Points)

Answer 9

(i) Ubiquitin-binding cargo adaptor, which sorts ubiquitylated cargo into the MVB/Late endosome for turnover
(ii) Consists of:
* FYVE domain - localisation to Late endosome (via PI-3P)
* C-terminal UIMs - 2 in Vps27p, 1 in Hrs
* Large unstructured regions

Question 10

What are Arrestins? What is their Structure?

Answer 10

• Cargo specific adaptors responsible for the downregulation of GPCR signalling, competing with G-proteins for binding and inducing internalisation
• Arrestins (e.g., ß-Arrestin) - winged fold structure, with C-terminal tail that carries multiple binding sites (Clathrin-binding, Accessory binding, ubiquitylation site)

Question 11

Describe the mechanism by which the Arrestin C-terminal tail is released into the cytosol

Answer 11

• Arrestin binds to phosphorylated GPCRs, with the -ve charge of phosphate groups interacting with highly conserved positive residues of arrestin
• This Interaction disrupts ionic interactions that hold the tail within the winged fold structure, causing its release into cytosol

Question 12

(i) What are Amphiphysins?

(ii) What is their Structure?

Answer 12

(i) Family of Cargo adaptors that recruit clathrin to phospholipids/membranes
(ii) Each monomer consists of:
* N-terminal BAR domain - phospholipid-binding
* C-terminal SH3 domain - recruits accessory proteins

Question 13

(i) What Cellular processes are Amphiphysin Involved in?
(ii) How is it recruited to its site of action

Answer 13

(i) Involved in Late Stages CCV budding, recruiting Dynamin (severs neck) and Synaptojanin (breaks down PIP2)
(ii) Amphiphysin monomers dimerise together, with the folded BAR domains having an inherent curvature and +vely charged face that facilitates interactions with -vely charge phospholipids
* BAR domain = curvature-sensing, only being recruited when curvature is significant enough

Question 14

(i) What is the Affinity of Cargo-Receptor interactions?

(ii) Why is this Important

Answer 14

(i) Low Affinity (µm), with many cargo proteins containing multiple recognition motifs

(ii) Must be a balance between cargo-binding and cargo release, therefore low affinity = compromise

Question 15

What is the Function of the ESCRT Pathway/Complexes?

Answer 15

Involved in the formation of MVBs, and in the sorting/routing of cargo into the lysosome for turnover/degradation

Question 16

Summarise the steps of the ESCRT Pathway

(4 Points)

Answer 16

1. ESCRT-0 - recruits ubiquitin-modified cargo (via Hrs/Vp27p), and passes it to ESCRT-I
2. ESCRT-I - recognises ubiquitin-modified cargo via UEV domain, and passes it to ESCRT-II
3. ESCRT-II - early endosomal membrane-associated complex, recognises cargo using NZF domain, and passes it to ESCRT-III
4. ESCRT-III - consists of cytosolic proteins, which are recruited following activation (via interaction with ESCRT-II carrying cargo)

Question 17

Compare how the different ESCRT-complexes associate with the membrane

(4 Points)

Answer 17

• ESCRT-0 - via FYVE domain, which recognises PI-3P enriched in the early endosome
• ESCRT-I - via interaction with membrane-inserted cargo
• ESCRT-II - via GLUE domain, which recognises PI-3P enriched in early endosome
• ESCRT-III - via Vps24, which recognises PI-3,5-BP enriched in late endosome

Question 18

Describe the Mechanism of ESCRT-III activation

(3 Points)

Answer 18

• ESCRT-III subunits adopt cytosolic autoinhibited fold, with the a5 helix and MIR domain being folded across structure
• Following Activation, a5 and MIR domains are flipped out, exposing other helices that mediate polymerisation of subunits into a repeating dimer
• Repeating dimers form semi-crystalline array across membrane of MVB/Late endosome

Question 19

How are MVBs produced?

Answer 19

ESCRT-III complex has membrane deformining properties, leading to production of many smaller internal vesicles containing cargo for turnover

Question 20

What is the function of the ESCRT-III MiR domain?

Answer 20

Once Flipped out, it recruits the Vps4 ATPase complex (similar structurally/functionally to NSF/P97), which uses ATP hydrolysis to depolymerise crystalline array

Question 21

What roles do (i) Ubiquitination, and (ii) Deubiquitination play in the ESCRT pathway?

(3 Points)

Answer 21

(i):
* Monoubiquitination - signal for internalisation, as well as recruitment to the ESCRT-0 complex

(ii):
* Deubiquitination - critical step in committal of cargo for trafficking into MVB
* STAM - member of ESCRT-0 complex, that requires deubiquitination of K-48 linked chain by UBPY to prevent its proteosomal degradation (prevents ESCRT-0 recruitment)