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
What did the work of Randy Scheckmann uncover?
Looked at genetics of budding yeast - mutants with active at 25C and inactive at 37C with thermoreversability
- Identified proteins associated with protein trafficking such as SEC1
What did the work of James Rothman discover?
Core complex - SNARE proteins essential for trafficking/fusion at all membranes
- Found that complex NEM could prevent trafficking at the Golgi complex
- Bound to NEM sensitive factor (NSF) and soluble SNF attachment protein (SNAP)
What are the components of the SNARE complex?
- VAMP2 (at the vesicle)
- SNAP25 and syntaxin at plasma membrane
Interact to pull vesicle close and fuse with membrane, form highly conserved coiled-coiled structure
What is the structure of VAMP?
Contains transmembrane domain liking it to the vesicle and SNARE motif
What is the stucture of syntaxin?
Has transmembrane domian, SNARE motif and N-terminal Habc domain
What is the structure of SNAP25
Linked to the membrane via palmitoylated cysteine residues and 2 SNARE motifs
What is the structure of the SNARE complex?
- Four helices bundle together via interactions between amino acid side chains (all except one being hydrophobic the 0-layer)
What is the structure of the 0-layer in the SNARE complex?
Found in the middle and is polar
- 1 x arganine contributed by VAMP (1x R-SNARE)
- 3 x glutamine (3 x Q-SNARE) (2x SNAP25, 1 x Syntaxin)
What is the function of the 0-layer in the SNARE complex?
- Allows strong hydrophobic complex to form
- 0-layer exposure to water causes it to break appart, useful for inactivation
How does the SNARE complex function?
- As vesicle approaches membrane 4 SNARE motifs interact at N-terminal and begin to act as a zip until membranes merge
- Also known as LEUCINE ZIP
What accessory proteins are associated with the SNARE complex?
- Synaptotagmin-1 (Ca2+ sensor)
- Munc-18
These are highly conserved!!
What is the strucutre of Synaptotagmin-1?
- Found on synaptic vesicle
- Has transmembrane domain and C2a and C2b domains which bind to both Ca2+ and phospholipids
- High affinity Ca2+ binding
How does Synaptotagmin-1 function?
- Complexin acts as break just before vesicle fusion
- Ca2+ binds to Synaptotagmin-1 pushing away complexin allowing fusion to take place
- Knockout of this protein abolishes neurotransmitter release
What is the known function of Munc-18?
- Causes N-terminal domain to fold on top of SNARE motif so that syntaxin is closed and cannot interact
- Knock-out however causes no exocytosis therefore cannot be inhibitory
What is the suggested role of Munc-18?
Binds to syntaxin in 2 different modes:
- Inhibitory mode: allowing syntaxin to travel through protein trafficking without interacting with other proteins
- Activating mode: binds only to N-terminal domain of syntaxin, allows interaction of SNARE domains and fusion to occur
