Clathrin-mediated endocytosis Block C L3

What is endocytosis?

• morphological opposite of exocytosis

• describes the de novo production of internal membranes from the plasma membrane

• internalises plasma membrane proteins and lipids and extracellular fluid

What is pinocytosis?

• a non-specific process where the cell takes up extracellular fluid

What is receptor-mediated endocytosis?

• a process whereby a receptor and its ligand are taken into the cell

• is a form of clathrin-mediated endocytosis

why is endocytosis important?

• allows precise control of plasma membrane composition and regulates interaction of the cell with the external environment

• uptake of nutrients e.g. iron

• destruction of pathogens and cellular debris

• retrieval of vesicle/granule proteins following exocytosis

How does endocytosis regulate interaction of the cell with the external environment?

• endocytosis of receptors at the plasma membrane regulates the sensitivity of cell to their ligands -

• insulin binding to insulin receptor triggers endocytosis of the receptor, reducing subsequent sensitivity of the cells to insulin and any over-response

What is clathrin-mediated endocytosis?

• major endocytic pathway

• requires a large number of proteins

• involves the formation of clathrin-coated vesicles at the plasma membrane

Requirements for clathrin-mediated endocytosis?

• curvature of the plasma membrane to form membrane-attached vesicle

• recruitment of clathrin, cargo proteins and accessory factors

• scission of the clathrin-coated vesicle from the plasma membrane

• removal of the clathrin coat

role of PIP2 in clathrin-dependent endocytosis?

• phosphorylated lipids PIP2 (phosphatidylinositol 4,5-biphosphate) acts as a nucleation point for clathrin-dependent endocytosis as many components of the machinery bind to this lipid

Role of epsin?

• epsin recruits clathrin to the membrane and plays a role in membrane invagination

• binds PIP2 on the membrane

• inserts an amphipathic helix into the membrane to promote membrane curvature

• recruits clathrin to the membrane

• structural changes occur in epsin upon binding to PIPs

membrane curvature by epsin? (and related proteins)

• the insertion of amphipathic helix as shown into one leaflet of the membrane bilayer causes the phospholipids to splay and increases curvature

How is membrane curvature stabilised?

• stabilised by proteins containing BAR domains (Bin-Amphiphysin-Rvs167)

• domains dimerize to form a crescent-shaped homodimer

• bind to membranes via concave face

• stretch of positively-charged residues bind to negatively-charged phospholipids

example of BAR domains

• amphiphysin binds to membranes of high positive curvature (required at a later step of clathrin-mediated endocytosis)

• FCHo2 binds to membranes with shallow curvature (required at an early stage of endocytosis)

what are adaptor proteins?

• recruit cargo and clathrin to the membrane

• AP1-AP4

• AP2 functions in endocytosis

• AP1, 3 and 4 are generally involved in budding events from intracellular membranes

• all are hetero-tetramers (composed of 4 different subunits)

How does AP2 function in endocytosis?

• AP2 binds to membrane phospholipid

• recruits cargo proteins and clathrin

• cargo typically contain dileucine and tyrosine-based sorting signals that are recognised by AP2

structure of clathrin?

• 3 legged structure - triskelion

• 3 heavy chains (192kDa), each bound to a light chain

• able to spontaneously assemble into clathrin cages

• contain a trimerisation domain

How is scission of clathrin-coated vesicles from the plasma membrane mediated?

• GTPase dynamin

• dynamin self-assembles into a helical collar around the neck of budding vesicle

• GTP hydrolysis by dynamin is thought to result in a lengthwise extension of the helical collar, which results in the vesicle being ‘pinched’ or popped off the membrane

What happens if a mutation occurs in dynamin

• blocks GTPase activity

• vesicles won’t be cut off and will remain attached to the membrane

what is uncoating of clathrin-coated vesicles mediated by?

• auxilin

• HSC70

• ATP → ADP during this process

cellular uptake of Fe3+

• transferrin receptor is endocytosed and recycles constitutively - with or without bound transferrin