Lecture 19 Endocytosis Flashcards
Transport into the cell requires
Cytosolic vesicles
Cytosolic vesicles are
Single membrane bound
Trafficking of vesicles involves
Budding, scisson, fusion
Vesicles are linked to the
ER nd Golgi membranes
Lysosomes
Peroxisomes
3 categories of endocytosis
Pinocytosis
Phagocytosis
Receptor mediated endocytosis
Pinocytosis
‘Cell drinking’
Unspecific, continuous budding of vesicles from the membrane
Micropinocytosis (>0.1um) or macropinocytosis (0.5-5um)
Pinocytosis is the process by which cells
Take up fluids and solutes
Phagocytosis
‘Cell eating’
Occurs in specialized cells (macrophages, neutrophils, dendritic cells)
Receptors involved
Used also for clearance of apoptotic bodies
Phagocytosis is the process by which cells
Engulf solid matter
Receptor mediated endocytosis
Generates small vesicles 60-120nm through invagination
Forms clathrin coated vesicles
Vesicles called endosomes
Can be utilized by viruses
Receptor mediated endocytosis is the process by which cells
Absorb metabolites, hormones and proteins (and in some cases viruses)
Endocytosis can be
Clathrin dependent or clathrin independent
Clathrin mediated endocytosis stages
- Initiation - clathrin coat recruited to the bud
- Assembly - dynamin recruited to the neck deforms membrane
(Dynamin is GTPase forms rings in the neck of the budding vesicle) - Dynamin recruits phosphatase to endocytic site
- PIP2 creates phase segregation
- PIP hydrolysis squeezes the neck, resulting in curvature and finally scisson
Clathrin structure
Triskelion shaped scaffold protein
Three heavy and three light chains
Initiation of the clathrin complex requires
PIP2 and adaptor proteins
Adaptor proteins link
The activated receptors to clathrin
Clathrins act as a
Mould so the vesicle forms without direct association of the membrane
Clathrin is a protein which forms
A coat around the vesicle
Adaptor proteins
AP1-4
Tetrameric adaptor complexes
AP2 is required for
CME
clathrin mediated endocytosis
AP2 sits
Between activated receptor and the clathrin coat
Clathrin cage disassembly
Dissociation rapidly follows scisson of the vesicle from the membrane
Auxilin binds to the triskelion and recruits 3Hsc70:ATP and hydrolyses to 3Hsc:ADP
This energy is used to disassemble the cage
Caveolin mediated endocytosis is
Clathrin independent
Membrane deformation is driven by Caveolin and associated Cavin proteins
50-100nm size
Cav1-3
Caveolin homologues
Caveolin mediated endocytosis requires
GTPase dynamin for vesicle scisson
Each caveolae contains
150 Cav molecules
Caveolin are well described for
Virus internalization
After endocytosis vesicles fuse with the
Early endosome
The early endosome collects cargo and distributes it to a
Recycling endosome or towards the late endosome for degradation
pH of early endosomes
6
RabGTPases can be used to identify
Individual endosomes
Rab4/11 marks
Recycling endosomes
Rab7 marks
Late endosomes
Rab proteins are
GTPases
70 different kinds
Rabs are anchored to the membrane by a
Lipid group ‘tail’
Prenylation
In the inactive state, Rab proteins are
GDP bound
In the active state, Rab proteins are
GTP bound
The conversion from active to inactive Rab is regulated by
GAPs
GTPase activating proteins
The conversion from inactive to active Rab is regulated by
GEFs
Guanine nucleotide exchange factors
Prenylation
The addition of hydrophobic molecules to a protein or chemical compound
Rab proteins occupy distinct
Membrane domains on endosomes called Rab domains
Rab GTPases allow the sequential transport of
Cargo between Rab domains
Rab5 EE -> Rab4 RE-> Rab7 LE
Rather than transport
Maturation of endosomes
The recycling endosome
Rab4/11
Rab4 = fast recycling
Rab11 = slow recycling
Rab11 can be seen as a storage endosome
Late endosomes/MVBs
Contain membrane bound intraluminal vesicles
Vesicles form by budding into the lumen (ESCRT)
The pH of LE/MVB
5-6
MVBs can fuse with
Lysosomes for degradation of content
MVBs can fuse with the plasma
Membrane to release vesicles for exocytosis
Lysosomes
‘Lyse’ cut/digest
pH 4-5 contains hydrolytic enzymes
Break down hormones, signalling proteins
Released into cytoplasm for re use
LDL as an example of RME
Low density lipoproteins
LDL binds to LDL receptors on PM
Receptor and ligand are internalised into an EE by RME
LDL receptor are transported to RE to go back to the membrane
LDLs are routed to the LE then lysosome
Cholesterol is released to build new membranes
Nieman-Pick C disorder (NP)
Mutations in NPC1 gene
NPC1 is a cholesterol sensor/binder protein
Reduced activity of NPC1 leads to lipid build up in LE and lysosomes
Cholesterol gets ‘stuck in traffic’
Less free cholesterol in the cytosol
Affects the trans golgi network (TGN) function and build up in lysosomes eventually kills cells
Causes neurological disease, liver spleen and heart
Cells internalize substances via
Pinocytosis, phagocytosis or RME
Activated receptors interact with adaptor proteins which recruit
A clathrin coat to be internalised in clathrin coated vesicles
Vesicle scisson is achieved by
Dynamin and PIP2
Vesicles need to be dismantled before interacting with
EE
Vesicles fuse with EE to distribute the content to
RE or LE/lysosomes
The endocytic cycle is important as it allows the cell to
Receive nutrition and regulate signalling