Block C Lecture 3 - Clathrin-mediated endocytosis Flashcards
What is endocytosis?
The production of internal membranes from the plasma membrane
(Slide 3)
What does endocytosis internalise?
Plasma membrane proteins, lipids and extracellular fluid
(Slide 3)
What happens after phagocytosis removes pathogens via exocytosis?
The phagosome fuses with a lysosome, where enzymes then digest the pathogen
(Slide 4)
What is a phagosome?
A membrane-bound vesicle formed inside a cell as a result of phagocytosis
(Slide 4)
What is pinocytosis?
A non-specific process where a cell takes up extracellular fluid
(Slide 4)
What is receptor mediated endocytosis?
A process where a receptor and its ligand are taken into the cell
(Slide 4)
What is receptor mediated endocytosis a form of?
Clathrin-mediated endocytosis
(Slide 4)
What are 4 functions of endocytosis?
It allows precise control of the plasma membrane composition and regulates interaction of the cell with the external
It allows for the uptake of nutrients
It allows for destruction of pathogens an cellular debris
It allows retrieval of vesicle / granule proteins following exocytosis
(Slide 5)
What is clathrin-mediated endocytosis?
It’s a major endocytic pathway which involves the formation of clathrin-coated vesicles at the plasma membrane
(Slide 6)
What are the 4 steps of clathrin-mediated endocytosis?
- Curvature of the plasma membrane to form membrane-attached vesicle
- Recruitment of clathrin, cargo proteins and accessory factors
- Separation of the clathrin-coated vesicle from the plasma membrane
- Removal of the clathrin coat
(Slide 7)
How is phosphatidylinositol 4,5-bisphosphate (PIP2) important for the initiation of clathrin dependent endocytosis?
As it acts as a nucleation point as many components of the machinery bind to this ligand
(Slide 8)
What is a nucleation point?
A specific location where a new phase or structure begins to form within a substance
(Slide 8)
What is the role of Epsin in clathrin-mediated endocytosis?
It binds to PIP2 on the membrane and inserts an amphipathic helix into the membrane to promote membrane curvature, therefore recruiting clathrin
(Slide 9)
How does the amphipathic helix inserted into the membrane by Epsin promote membrane curvature?
It causes phospholipids to spread and increases curvature
(Slide 11)
What stabilises membrane curvature?
BAR (Bin-Amphiphysin-Rvs167) domains
(Slide 12)
How do BAR domains stabilise membrane curvature?
They dimerise to form a crescent-shaped homodimer and bind to membranes via its concave face, with a stretch of positively-charged residues residues binding to negatively-charged phospholipids, with their crescent shape being able to stabilise or promote membrane curvature
(Slides 12 and 13)
What are 2 examples of BAR domain-containing proteins which are implicated in membrane sculpting events during endocytosis?
FCHo2
Amphiphysin
Endophilin
(Slide 13)
What is the difference between Amphiphysin and FCHo2?
Amphiphysin binds to membranes of high positive curvature and is required at a later step of clathrin mediated endocytosis
FCHo2 binds to membranes with shallow curvature and is required at an early stage of clathrin-mediated endocytosis
(Slide 14)
What do adaptor proteins do?
They recruit cargo and clathrin to the membrane
(Slide 15)
What are the 4 main types of adaptor protein complexes and what are they involved in?
4 types are labelled AP1-4
AP2 functions in endocytosis whereas AP1,3 and 4 are generally involved in budding events from intracellular membranes
(Slide 15)
What is the structure of AP1, 2, 3 and 4 adaptor protein complexes?
They are all hetero-tetramers (composed of 4 different subunits)
(Slide 15)
What does AP2 bind to?
PIP2
(Slide 16)
What 2 sorting signals do cargo typically contain?
Dileucine (D/ExxxLL)
Tyrosine (YxxΦ)
x is any amino acid D/E is Aspartate/Glutamate and Φ refers to any amino acid (e.g Leucine, Isoleucine, Methionine… etc)
(Slide 16)
What is the structure of clathrin?
It’s a 3-legged structure called a “triskelion”, which is composed of 3 heavy chains each bound to a light chain which is able to spontaneously assemble into clathrin cages
(Slide 17)
What is dynamin?
A GTPase which mediates separation of clathrin-coated vesicles from the plasma membrane
(Slide 18)
What is a GTPase?
An enzyme that hydrolyses GTP (guanosine triphosphate) to GDP (guanosine diphosphate) and inorganic phosphate (Pi)
(Slide 18)
How does dynamin mediate scission of clathrin-coated vesicle from the plasma membrane?
It self-assembles into a helical collar around the neck of the budding vesicle, and then GTP hydrolysis results in a lengthwise extension of the helical collar, which results in the vesicle being “pinched” or “popped” off the membrane
(Slide 18)
What is a shibire mutant and what causes it?
It is a temperature-sensitive phenotype which arsies due to point mutations in dynamin which block GTPase activity
(Slide 19)
What 2 proteins mediate uncoating of clathrin-coated vesicles?
Auxilin and HSC70
(Slide 21)
How does clatherin mediated endocytosis allow cellular uptake of Fe3+?
As the transferrin (iron transporter) receptor is endocytosed and is constantly recycled; with or without bound transferrin
(Slide 22)
What can a transferrin receptor assay be used to diagnose and why does this work?
It can be used to diagnose anaemia as the level of the receptor is increased on cells when iron is low
(Slide 22)
