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PHPY 301 > Lec 14 > Flashcards

Lec 14 Flashcards

1
Q

What protein preassembles and forms three-armed “triskelions”?

A

clathrin

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2
Q

Separation of proteins found in vesicles isolated from “Torpedo” and mammalian brain membranes led to the discovery of what 2 vesicle proteins?

A

synaptobrevin and synaptotagmin

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2
Q

The molecule NEM (N-ethylmaleimide) is an inhibitor of what process?

A

vesicle trafficking through the Golgi complex

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2
Q

What protein does Tetanus cleave?

A

synaptobrevin

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2
Q

What is the protein “dynamin” used for?

A

the pinching off, or fission, process of a vesicle in endocytosis (in the golgi or surface of the cell membrane)

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3
Q

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?

A

Clathrin

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4
Q

The discovery of what two vesicle proteins led to the immunoprecipitation experiments?

A

synaptobrevin and synaptotagmin

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4
Q

what synaptic vesicle membrane protein senses Ca2+ levels and triggers exocytosis of docked vesicles?

A

Synaptotagmin

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5
Q

What is the function of the membrane associated protein of synaptic vesicles of SV2 ?

A

the function of SV2 is unknown

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5
Q

what synaptic vesicle membrane protein is a GTPase and involved with cellular membrane trafficking?

A

Rab3

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5
Q

What type of enzymes are Botulinum toxins?

A

endopeptidases (cleave the middle)

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5
Q

What do endopeptidases do?

A

cleave the middle of a protein.

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5
Q

What part of a clathrin complex provides flexibility and strength?

A

overlapping arrangement of triskelion

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6
Q

What Synaptic vesicle protein senses Ca2+ concentration and initiates the fusion process?

A

synaptotagmin

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7
Q

what synaptic vesicle membrane proteins have only 1 transmembrane region?

A

Synaptobrevin (VAMP) and Synaptotagmin

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8
Q

Synaptobrevin is cleaved by what proteases?

A

Botulinum B,D,F,G & Tetanus

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10
Q

Formation of Clatherin coated vesicles: What happens following the binding of triskelions to the surface of the membrane?

A

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.

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12
Q

The SNARE complex consists of how many coils?

A

4

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13
Q

Dissolution of a clathrin cage requires:

A

hydrolysis of ATP from cytoplasmic enzymes.

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14
Q

After the crude purification synaptic vesicles, it is further purified by what?

A

permeation chromotography over a controlled-pore glass (CPG) bead column.

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14
Q

vesicle fusion to the presynaptic membrane: What happens following SNARE Complex Disassembly?

A

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.

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14
Q

Where does the coat proteins (clathrin) preassemble to form three-armed “triskelions”?

A

cytoplasm

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15
Q

Immunoprecipitation experiments discovered the proteins on the synaptic terminal membrane such as _________

A

syntaxin

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16
Q

Where are tSNARES located?

A

located on the target membrane of the presynaptic membrane

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17
Q

What are the two types of tSNARES?

A

syntaxin and SNAP-25

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18
Q

List the presynaptic membrane proteins involved with exocytosis:

A

SNAP-25 Syntaxin (n-Sec-1 attached)

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19
Q

What is the order of operations to make a CPG column from a Brain or electric organ homogenate?

A

spin 30,000xg > SUP > spin 100,000xg > Pellet > sucrose gradient is created containing the CPG Column

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20
Q

Formation of Clatherin coated vesicles: The first step:

A

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.

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22
Q

vesicle fusion to the presynaptic membrane: what is the first step (or Initial State) of vesicle fusion to the presynaptic membrane?

A

Vesicles with synaptotagmin and synaptobrevin(v-SNARE or VAMP) move to the nerve terminal membrane which contains syntaxin and SNAP25 ( both tSNARES)

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22
Q

Botulinum A and E cleaves what protein?

A

SNAP25

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24
Q

In Exocytotic release of NT, how many coils does synaptobrevin contribute?

A

one

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25
Q

What two proteins are required to disassociate the SNARE complex to allow the cycle to begin again?

A

NSF and a-SNAP (and ATP)

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26
Q

Following synaptic membrane and vesicle fusion, what proteins bind to the SNARE complex to disassemble it?

A

NSF and a-SNAP (along with ATP)

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27
Q

what synaptic vesicle membrane protein appears to be the molecular switch for exocytotic fusion?

A

Rab3

29
Q

A “triskelion” resembles the adjoining edges of what shape?

A

tetrahedron

30
Q

What mediates the binding between Triskelions and cell membrane receptors?

A

adaptin

31
Q

The specificity for triskelions to bind to particular membrane proteins is conferred by what structure?

A

adaptin

32
Q

In order to purify a synaptic vesicle what general steps need to be performed?

A
  1. differential centrifugation 2. flotation equilibrium in sucrose density gradient
33
Q

What 2 processes is the protein, Clathrin, used for?

A
  1. 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
34
Q

List the membrane associated proteins of synaptic vesicles:

A
  1. 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
36
Q

vesicle fusion to the presynaptic membrane: What happens as the Ternary complex (SNAREs) begin to form? (following Formation of Ternary Complex of SNAREs)

A

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.

37
Q

What protein is inhibited by NEM (N-ethylmaleimide) ?

A

NEM-sensitive protein or NSF

39
Q

After permeation chromotography, purified synaptic vesicles are monitored by absorbance at 310nm what does this do?

A

measures the total light scattering material.

40
Q

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)

A

Fusion and Exocytosis: the entry of Ca2+ and its binding to synaptotagmin triggers the fusion event.

41
Q

Where are the vSNARES located?

A

located on the vesicles in the presynaptic membrane

42
Q

In Exocytotic release of NT, how many coils does syntaxin contribute?

A

one

43
Q

What type of complex is created with the winding of synaptobrevin around the syntaxin/SNAP25 complex?

A

Ternary complex

44
Q

vesicle fusion to the presynaptic membrane: What happens following Ca2+ entry into the presynaptic terminal and fusion has been triggered (after Fusion and Exocytosis)?

A

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.

45
Q

What protein was discovered by immunoprecipitation experiments and is located on the synaptic terminal membrane that interacts with previously discovered synaptobrevin and synaptotagmin?

A

syntaxin

47
Q

Isolation of synaptic vesicles was used for what purpose?

A

isolation is used to identify vesicle proteins involved in exocytotic fusion

48
Q

what synaptic vesicle membrane protein is may mediate the attachment of the vesicle to the actin cytoskeleton? (anchor)

A

Synapsin

49
Q

Tetanus toxin cleaves what protein?

A

synaptobrevin

50
Q

What provides the “fuel” for synaptic exocytotic fusion?

A

ATP

51
Q

Are “triskelions” planar?

A

no

52
Q

What protein is required in endocytosis, for the internalization of membrane from the cell surface?

A

Clathrin

54
Q

syntaxin is cleaved by what proteases?

A

Botulinum C1

54
Q

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

a GTP-binding protein called dynamin

56
Q

what synaptic vesicle membrane protein is a v-SNARE and is essential for transmitter release?

A

Synaptobrevin (VAMP)

57
Q

“SNAP” In SNAP-25 stands for?.

A

syNaptic associated proteins!

58
Q

What protein was discovered by the addition of an inhibitor called NEM (N-ethylmaleimide) ?

A

NEM-sensitive protein or NSF

59
Q

When you monitor a pool of synaptic vesicle proteins by absorbance at 310nm, how are you able to find the “vesicle peak”?

A

the vesicle peak is identified by its high ATP content. (i.e. where there is the most concentrated absorbance)

60
Q

What are the 2 types of SNAREs?

A

vSNAREs and tSNAREs

61
Q

vesicle fusion to the presynaptic membrane, when is the ternary complex created?

A

with the winding of synaptobrevin around the syntaxin/SNAP25 complex.

62
Q

Botulinum C1 cleaves what protein?

A

syntaxin

63
Q

At the presynaptic membrane, what 3 protein structures are involved in a coiled-coil structure?

A

Synaptobrevin VAMP syntaxin SNAP-25

64
Q

The end of each triskelion leg turns inward so that..

A

the amino-terminal domain forms an intermediate shell.

65
Q

Botulinum B, D, F, and G cleaves what protein?

A

synaptobrevin

66
Q

After you identify the vesicle peak by its high ATP content, how is the particular protein identified?

A

you must compare the absorbance concentration to a Western blot , when a match is found the protein is identified.

67
Q

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?

A

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.

68
Q

In Exocytotic release of NT, how many coils does SNAP-25 contribute?

A

two

69
Q

what synaptic vesicle membrane protein is part of a complex that drives exocytotic fusion?

A

Synaptobrevin (VAMP - Vesicle associated membrane protein)

71
Q

what synaptic vesicle membrane protein plays a role in the formation of a fusion pore?

A

Synaptophysin

72
Q

SNAP25 is cleaved by what proteases?

A

Botulinum A,E

73
Q

What are adapter proteins that link the membrane and protein tails to the triskelion scaffold?

A

adaptin

74
Q

what synaptic vesicle membrane protein has two external repetitive domains that appear to mediate binding of Ca2+ ?

A

Synaptotagmin

75
Q

clathrin coats surrounding vesicles have how many pentagons?

A

12

77
Q

Before fusion of vesicles can begin in the presynaptic terminal, what peripheral protein must release from syntaxin?

A

n-Sec-1

78
Q

Formation of Clathrin coated vesicles: What happens after the fission or pinching off of the coated vesicle?

A

The coated vesicle remains coated until removed by the hydrolysis of ATP creating an uncoated vesicle. The triskelions and adaptins are recycled.

79
Q

Each triskelion is composed of what?

A

3 clathrin light chains and 3 clathrin heavy chains

80
Q

What protein(s) does Botulinum cleave?

A

synaptobrevin, SNAP25, and syntaxin

81
Q

Proteins that interact with SNAPs are called..

A

SNAREs (SNAP receptors)

82
Q

dynamin was discovered how?

A

dynamin was identified through temperature dependent drosophila mutant

83
Q

what synaptic vesicle membrane protein is the synaptic vesicle Ca2+ receptor?

A

Synaptotagmin

84
Q

proteins that interact with NSF are called..

A

SNAPs (for Soluble NSF Attachment Proteins)

PHPY 301 (23 decks)

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