Lec 14 Flashcards
What protein preassembles and forms three-armed “triskelions”?
clathrin
Separation of proteins found in vesicles isolated from “Torpedo” and mammalian brain membranes led to the discovery of what 2 vesicle proteins?
synaptobrevin and synaptotagmin
The molecule NEM (N-ethylmaleimide) is an inhibitor of what process?
vesicle trafficking through the Golgi complex
What protein does Tetanus cleave?
synaptobrevin
What is the protein “dynamin” used for?
the pinching off, or fission, process of a vesicle in endocytosis (in the golgi or surface of the cell membrane)
what “coat” protein mediates the formation of secretory vesicles that bud off of the trans Golgi as well as the internalization of membrane from the cell surface during endocytosis?
Clathrin
The discovery of what two vesicle proteins led to the immunoprecipitation experiments?
synaptobrevin and synaptotagmin
what synaptic vesicle membrane protein senses Ca2+ levels and triggers exocytosis of docked vesicles?
Synaptotagmin
What is the function of the membrane associated protein of synaptic vesicles of SV2 ?
the function of SV2 is unknown
what synaptic vesicle membrane protein is a GTPase and involved with cellular membrane trafficking?
Rab3
What type of enzymes are Botulinum toxins?
endopeptidases (cleave the middle)
What do endopeptidases do?
cleave the middle of a protein.
What part of a clathrin complex provides flexibility and strength?
overlapping arrangement of triskelion
What Synaptic vesicle protein senses Ca2+ concentration and initiates the fusion process?
synaptotagmin
what synaptic vesicle membrane proteins have only 1 transmembrane region?
Synaptobrevin (VAMP) and Synaptotagmin
Synaptobrevin is cleaved by what proteases?
Botulinum B,D,F,G & Tetanus
Formation of Clatherin coated vesicles: What happens following the binding of triskelions to the surface of the membrane?
triskelions assemble spontaneously to form a complete cage that attaches to the underlying membrane and pulls it up into a spherical configuration. completion of the cage occurs simultaneously with the pinching off of the evaginated membrane from the planar surface.. closing the sphere.
The SNARE complex consists of how many coils?
4
Dissolution of a clathrin cage requires:
hydrolysis of ATP from cytoplasmic enzymes.
After the crude purification synaptic vesicles, it is further purified by what?
permeation chromotography over a controlled-pore glass (CPG) bead column.
vesicle fusion to the presynaptic membrane: What happens following SNARE Complex Disassembly?
Recycling of SNAREs: with the endocytosis of the vesicle, the synaptobrevin is effectively recycled. The syntaxin and SNAP-25 are now free for an additional cycle of vesicle fusion.
Where does the coat proteins (clathrin) preassemble to form three-armed “triskelions”?
cytoplasm
Immunoprecipitation experiments discovered the proteins on the synaptic terminal membrane such as _________
syntaxin
Where are tSNARES located?
located on the target membrane of the presynaptic membrane
What are the two types of tSNARES?
syntaxin and SNAP-25
List the presynaptic membrane proteins involved with exocytosis:
SNAP-25 Syntaxin (n-Sec-1 attached)
What is the order of operations to make a CPG column from a Brain or electric organ homogenate?
spin 30,000xg > SUP > spin 100,000xg > Pellet > sucrose gradient is created containing the CPG Column
Formation of Clatherin coated vesicles: The first step:
triskelions of clathrin attach indirectly to the surface of the membrane that is to be excised by binding to the cytosolic tail of membrane proteins.
vesicle fusion to the presynaptic membrane: what is the first step (or Initial State) of vesicle fusion to the presynaptic membrane?
Vesicles with synaptotagmin and synaptobrevin(v-SNARE or VAMP) move to the nerve terminal membrane which contains syntaxin and SNAP25 ( both tSNARES)
Botulinum A and E cleaves what protein?
SNAP25
In Exocytotic release of NT, how many coils does synaptobrevin contribute?
one
What two proteins are required to disassociate the SNARE complex to allow the cycle to begin again?
NSF and a-SNAP (and ATP)
Following synaptic membrane and vesicle fusion, what proteins bind to the SNARE complex to disassemble it?
NSF and a-SNAP (along with ATP)
what synaptic vesicle membrane protein appears to be the molecular switch for exocytotic fusion?
Rab3
A “triskelion” resembles the adjoining edges of what shape?
tetrahedron
What mediates the binding between Triskelions and cell membrane receptors?
adaptin
The specificity for triskelions to bind to particular membrane proteins is conferred by what structure?
adaptin
In order to purify a synaptic vesicle what general steps need to be performed?
- differential centrifugation 2. flotation equilibrium in sucrose density gradient
What 2 processes is the protein, Clathrin, used for?
- mediates formation of secretory vesicles that bud off of the trans Golgi 2. used in the internalization of membrane from the cell surface during endocytosis
List the membrane associated proteins of synaptic vesicles:
- voltage-type H+ pumps 2. NT-H+ exchangers 3. SV 2 4. Synaptobrevin (VAMP Vessicle associated membrane protein) 5. Rab 3 6. Synaptophysin 7. synapsin
vesicle fusion to the presynaptic membrane: What happens as the Ternary complex (SNAREs) begin to form? (following Formation of Ternary Complex of SNAREs)
Tightening of Ternary Snare Complex: The three SNAREs continue to form a tight bundle of a-helicies drawing the vesicle and presynaptic membranes into a close apposition to promote fusion.
What protein is inhibited by NEM (N-ethylmaleimide) ?
NEM-sensitive protein or NSF
After permeation chromotography, purified synaptic vesicles are monitored by absorbance at 310nm what does this do?
measures the total light scattering material.
vesicle fusion to the presynaptic membrane: What happens following the complete formation of the Ternary SNARE Complex and the membranes are in close proximity to one another? (After the tightening of Ternary Snare complex)
Fusion and Exocytosis: the entry of Ca2+ and its binding to synaptotagmin triggers the fusion event.
Where are the vSNARES located?
located on the vesicles in the presynaptic membrane
In Exocytotic release of NT, how many coils does syntaxin contribute?
one
What type of complex is created with the winding of synaptobrevin around the syntaxin/SNAP25 complex?
Ternary complex
vesicle fusion to the presynaptic membrane: What happens following Ca2+ entry into the presynaptic terminal and fusion has been triggered (after Fusion and Exocytosis)?
Disassembly of Ternary SNARE Complex: Neurotransmitters are released from vesicle and Disassembly of Ternary SNARE Complex begins by a-SNAP and the ATPase NSF binding to the SNARE complex and by the use of ATP hydrolysis disassembly is accomplished.
What protein was discovered by immunoprecipitation experiments and is located on the synaptic terminal membrane that interacts with previously discovered synaptobrevin and synaptotagmin?
syntaxin
Isolation of synaptic vesicles was used for what purpose?
isolation is used to identify vesicle proteins involved in exocytotic fusion
what synaptic vesicle membrane protein is may mediate the attachment of the vesicle to the actin cytoskeleton? (anchor)
Synapsin
Tetanus toxin cleaves what protein?
synaptobrevin
What provides the “fuel” for synaptic exocytotic fusion?
ATP
Are “triskelions” planar?
no
What protein is required in endocytosis, for the internalization of membrane from the cell surface?
Clathrin
syntaxin is cleaved by what proteases?
Botulinum C1
Formation of Clatherin coated vesicles: After the bud is formed, the pinching off or fission process appears to involve the action of what protein?
a GTP-binding protein called dynamin
what synaptic vesicle membrane protein is a v-SNARE and is essential for transmitter release?
Synaptobrevin (VAMP)
“SNAP” In SNAP-25 stands for?.
syNaptic associated proteins!
What protein was discovered by the addition of an inhibitor called NEM (N-ethylmaleimide) ?
NEM-sensitive protein or NSF
When you monitor a pool of synaptic vesicle proteins by absorbance at 310nm, how are you able to find the “vesicle peak”?
the vesicle peak is identified by its high ATP content. (i.e. where there is the most concentrated absorbance)
What are the 2 types of SNAREs?
vSNAREs and tSNAREs
vesicle fusion to the presynaptic membrane, when is the ternary complex created?
with the winding of synaptobrevin around the syntaxin/SNAP25 complex.
Botulinum C1 cleaves what protein?
syntaxin
At the presynaptic membrane, what 3 protein structures are involved in a coiled-coil structure?
Synaptobrevin VAMP syntaxin SNAP-25
The end of each triskelion leg turns inward so that..
the amino-terminal domain forms an intermediate shell.
Botulinum B, D, F, and G cleaves what protein?
synaptobrevin
After you identify the vesicle peak by its high ATP content, how is the particular protein identified?
you must compare the absorbance concentration to a Western blot , when a match is found the protein is identified.
vesicle fusion to the presynaptic membrane: What happens following the “Initial State” or after a vesicle is brought near to the nerve terminal membrane that contains tSNAREs?
Formation of Ternary Complex of SNAREs: 1. n-Sec-1 dissociates from syntaxin, allowing the syntaxin and SNAP25 to from a complex. 2. The distal end of the synaptobrevin begins to wind around the syntaxin/SNAP25 complex, forming a Ternary complex.
In Exocytotic release of NT, how many coils does SNAP-25 contribute?
two
what synaptic vesicle membrane protein is part of a complex that drives exocytotic fusion?
Synaptobrevin (VAMP - Vesicle associated membrane protein)
what synaptic vesicle membrane protein plays a role in the formation of a fusion pore?
Synaptophysin
SNAP25 is cleaved by what proteases?
Botulinum A,E
What are adapter proteins that link the membrane and protein tails to the triskelion scaffold?
adaptin
what synaptic vesicle membrane protein has two external repetitive domains that appear to mediate binding of Ca2+ ?
Synaptotagmin
clathrin coats surrounding vesicles have how many pentagons?
12
Before fusion of vesicles can begin in the presynaptic terminal, what peripheral protein must release from syntaxin?
n-Sec-1
Formation of Clathrin coated vesicles: What happens after the fission or pinching off of the coated vesicle?
The coated vesicle remains coated until removed by the hydrolysis of ATP creating an uncoated vesicle. The triskelions and adaptins are recycled.
Each triskelion is composed of what?
3 clathrin light chains and 3 clathrin heavy chains
What protein(s) does Botulinum cleave?
synaptobrevin, SNAP25, and syntaxin
Proteins that interact with SNAPs are called..
SNAREs (SNAP receptors)
dynamin was discovered how?
dynamin was identified through temperature dependent drosophila mutant
what synaptic vesicle membrane protein is the synaptic vesicle Ca2+ receptor?
Synaptotagmin
proteins that interact with NSF are called..
SNAPs (for Soluble NSF Attachment Proteins)
