Lecture 27 Flashcards
What do proteins and lipids move amongst?
Proteins (referred to as “cargo”) and lipids move amongst the endoplasmic reticulum, Golgi Apparatus and the plasma membrane
Where do proteins go from the ER lumen?
Proteins in the ER lumen (or embedded in the ER membrane) move into carrier vesicles where they are sorted and concentrated prior to transport to the next compartment; the Golgi apparatus
What are the 2 types of vesicles used for secretory pathways?
1) Coated vesicles
2) Vesicular tubular carriers (larger) [VTC]
What happens when these vesicles are released from the ER?
These vesicles will fuse with the Golgi membrane
What will happen at the Golgi after fusion of the vesicles?
At the Golgi, proteins are further modified, re-packaged into cargo vesicles and transported to either:
1) the plasma membrane
2) the endosomal/lysosomal system
3) Back to the ER
What is this transport done by?
This transport is done by molecular motors using actin or microtubules as tracks
What are the 3 coat proteins used during protein transport?
1) COPII (Coat protein II)
2) COP1 (Coat protein I)
3) Clathrin
When are COPII and COPI used?
Used during early secretory pathway
What does COPII do?
Coats ER membrane containing cargo
What does COPI do?
Coats Golgi compartments containing cargo
What does clathrin do?
Used late in the secretory pathway at trans-Golgi network at endosomes and at the plasma membrane (endocytosis)
What do the 3 coat proteins form?
The 3 coat proteins ultimately form “cages” around the vesicle
The cage gives the vesicle their shape
How do all 3 coat proteins use adaptor proteins?
All 3 use adaptor proteins to form an inner layer sandwiched between the vesicle and the outer coat proteins
What do COPII vesicles do?
COPII vesicles initially recruit the inner layer adaptor proteins (Sec23p and Sec24p) to the membrane followed by COPII
What does COPI vesicle do?
COPI pre-assembles the adaptors, which are the gamma-beta COP subcomplex (homologous to AP1/AP2) and another adaptor made of alpha-Beta1 subunits (similar to clathrin). Together these subunits make the COATOMER COMPLEX
What does COPII do?
Used for ER vesicle budding
What are the 3 key components of COPII?
Sar1 GTPase, Sec23p/24p, Sec13p/31p
What do these 3 key components of COPII promote?
The proteins form the COPII scaffold and have an intrinsic curve.
This promotes the curvature of the plasma membrane to areas it is bound to
What does hydrolysis of Sar1 do?
Hydrolysis of Sar1 initiation disassembly
Why is Sec24 such a crucial protein?
Sec24 is the crucial protein for concentrating the membrane cargo as it recognizes many different sorting signals
What are the 10 steps for COPII?
1) Sec12 is an integral membrane protein always found in the ER
2) Sec12 specifically recruits Sar1-GDP and enables it to convert to Sar1-GTP. That is because Sec12 is Sar1/s guanine nucleotides exchange factor (GEF)
3) When Sar1 is GTP-loaded it exposes an Alpha helix at its N-terminus that inserts into the ER plasma membrane phospholipids through the hydrophobic residues in the alpha helix. The helix is amphipathic (has both hydrophobic and hydrophilic residues). Insertion of the helix causes the membrane to begin to curve
4) Sar1- GTP then recruits Sec23/24 by the binding of Sec23 to Sar1-GTP
5) Sec23/24 also interacts now with the ER membrane
6) Sec 24 is the major cargo-binding adaptor protein of the COPII coat. It samples/binds cargoes at the ER and concentrates them, then segregates them from other proteins. The Sar1-GTP/Sec23/24 cargo complex is called the Pre-budding complex.
7) Now, Sec 13/31p gets recruited. This makes a cage around the pre-budding complex and curves the membrane to make a vesicle
8) Vesicle formation requires GTP hydrolysis from Sar1-GTP. This is done through Sec23, which is the GTPase activating protein for Sar1
9) The vesicle gets uncoated once budded from the ER membrane. How does this happen? Likely through Sar1-GTP hydrolysis. This hydrolysis can destabilize the COPII components with the membrane.
10) COPII components are recycled
How do vesicles with different shapes and sizes of cargo form?
When Sec 23 binds beneath Sec31 it can do so in a variety of different conformations as there are two binding sites where this interaction can occur at each point where Sec31 and Sec 23 dock around the 3D forming vesicles.
This then influences Sec24’s ability to bind the cargoes (because Sec23 doesn’t bind to cargo directly)
What does COPI do?
Guides protein sorting from the Golgi back to the ER
What are the 3 main components of COPI?
1) The Coatomer complex (multiple subunits)
2) Arf1-GTP
3) Arf-GAP1
What happens when Arf-GAP1 binds to the coatomer?
When Arf-GAP1 binds to the coatomer (following the binding of Arf1-GTP to the coatomer), COPI is formed.
What does the COPI cage do?
The COPI cage sorts proteins ready to be moved back from the Golgi to the ER
What is a dilysine sorting motif?
Most of these proteins have a dilysine sorting motif (so they have 2 lysine’s [or in some protein 2 arginine’s] at their C-termini that bind to COPI)
Where does assembly of the COPI and COPII coats happen?
Assembly of the COPI and COPII coats happens on the periphery of the internal lattice (so things assemble on the membrane first)
How is coat disassembly triggered?
Coat disassembly is triggered from within the internal lattice
What happens if binding of coat components is faster than release?
Coat grows and forms a coated vesicle
What happens if binding of coat components is slowly than release (eg. after a vesicle is formed)?
Coat disassembles
What happens if binding of coat components is equal to release?
Coat will have a curve, but it will not grow or shrink… it is at an equilibrium (AKA. “Metastable”)
What are tethering proteins?
Tethering proteins have long “arms” that project from the organelles to “grab” vesicles
These engage, pull the vesicles into closer proximity to the organelles for SNARE attachment (only 10-15 nm long)
What do SNARE proteins do?
SNARE proteins drive vesicular fusion with the organellar membrane
What are the 2 different types of tethering factors?
1) Coiled-coiled tethering factors
2) Multisubunit tethering factors
What are coiled-coiled tethering factors?
Extend ~150nm (or more) into the cytoplasm from the organelle membrane
Ros-shaped
Globular heads at both ends
What happens when both heads are engaged?
When engaged, they have a hinge region that collapse to bring the vesicle closer to the organellar membrane for SNARES to attack
Does Golgi have coiled-coiled tethering factors?
The Golgi has many of these types of tethering factors called Golgins
Lengths up to 300nm
What are multisubunit tethering factors?
Smaller size (only ~30nm long)
Function like the coiled coiled tethering factors as they grab the vesicles and direct them to the organelle membrane
What is the mechanism of membrane fusion?
1) Vesicle must come in close contact with the accepting membrane
2) The accepting membrane must bend becoming destabilized
3) The outer leaflets of both membranes must initially fuse
Why is membrane fusion important?
Specificity to ensure that the correct vesicle fuses with the correct membrane is mediated through both tethering factors and SNARES
What are SNARES?
Transmembrane proteins
C-termini in the membrane
N-termini in the cytoplasm
Contains a stretch of 60-70 residues containing a heptad repeat called a “SNARE motif”
What are the 4 SNARES needed?
1) 2 v-SNAREs (on the vesicle)
2) 2 t-SNAREs (on the target membrane)
When bound together, they form 4 SNARE complex called a “SNAREpin”
How are SNAREs disassembled?
SNARES are disassembled through SNAP that recruit specific ATP enzymes (ATPases) (eg. NSF)
Causes dissociation of the SNARE helixes
SNAREs get recycled
