Membrane Trafficking Flashcards
Give a brief description of the secretory pathway
- All proteins produced by mRNA, majority then translated on RER before entering the endoplasmic reticulum and Golgi complex where they undergo post-translational modifications, can then enter membrane or are secreted
- Some proteins produced on free ribosomes destined for nucleus or cytosol or peroxisomes where they are folded. If not folded enter mitochondria
What are the 3 principles of membrane trafficking?
- Occurs between membrane bound organelles
- Donor compartment to target compartment
- Must form bud or secretory vesicle on donor compartment which contains everything that will be leaving. This vesicle must break off the donor membrane before fusing with the target compartment allowing the merging of contents.
What are the requirements for clathirin-dependent endocytosis?
- Nucleation - Envagenation of membrane (curving)
- Cargo selection - cargo must then be brought in and associated with cargo receptors
- Coating
4 Uncoating -can then fuse with target
Which proteins mediate nucleation?
FCHO1,2 create the bud by inducing membrane curvature, Intersectin and EPS15 form scaffolding
Which proteins mediate cargo selection?
AP2 or adaptor protein 2 which links the cargo e.g receptors to the clathirin coat protein
Which protein forms the endocytosis coat?
clathirin coat forms lattice structure with protein dynamin recruited, responsible for cutting vesicle away from the plasma membrane by tightening and constricting
Which proteins mediate uncoating?
Auxlin and GAK
Describe the structure of AP-2
- made from 4 polypeptide adaptins
- 2 ‘ear’ domains (mikey mouse protein) - alpha and beta
- beta binds clathrin coat, alpha subunit binds various proteins such as AP180 and amphiphysin
- PIP2 is a key protein in endocytosis, interacts with sigma and mu subunits. Cargo also interact with these subunits
Describe clathrin coat assembly
- Made of 2 chains with a heavy chain (structural) and a light chain (regulatory)
- Triskelion interact to form hexagons (8) and pentagons (12)
- AP2 links cargo to coat by binding to both heavy and light chain
How is the cell membrane deformed to make a bud?
- ENTH (Epsin) and ANTH (AP180) both interact with PI(94,5)P2
- Insert helical domain into membrane driving a wedge into the membrane forcing phospholipids apart and forming a curvature
What is the role of BAR domain containing proteins aphiphysin and syndapin?
- Aphiphysin is a homodimer with BAR domain gives it an arch structure allowing it to fit outside of the budding vesicle
- Syndapin has a flatter surface and so binds to flatter membranes
- These proteins therefore bind to different parts of the protein and are responsible for different parts of endocytosis
- Aphiphysin binds to clathrin coat bringing dynamin into correct position where it will remove vesicle from the plasma membrane
What is the structure of dynamin?
- Uses GTPase to hydrolyse GTP and scisssion the membrane, also has PH domain which binds to PIP2 and has N-domain (PRD) which binds to aphiphysin, syndapin and endophilin
- Wraps around neck of budding vesicle
What are the 3 models of dynamin action?
- Constriction model – wraps around neck and as it hydrolyses GTP becomes tighter and tighter
- Spring model
- Molecular switch
How do proteins mediate uncoupling of the endocytosis coat?
- Mediated by synaptojanin (de-phosphorylates PIP2 causes coat proteins to fall off)
- Auxilin binds ATPas Hsc70 which uncoats the vesicles, breaking down coat proteins and releasing them
What did Geoge Emil Palade discover through the use of radiolabelled proteins in pulse chase experiments?
6 steps in secretion: 1) synthesis (proteins synthesised on ribosomes) 2) segregated (movement through ER) 3) Intracellular transport (movement in Golgi) 4) Condensation (in condensation vacuoles, or the end of the golgi network) 5) Intracellular storage (intracellular granules) and 6) discharge
