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Cell Biology > Lecture 7 - Mechanisms of membrane fusion > Flashcards

Lecture 7 - Mechanisms of membrane fusion Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

What are the mechanisms of membrane fusion?

A
  1. Initial contact
  2. Contact between protein depleted patches (need to overcome protein and electrostatic forces)
  3. Hemifusion stalk
  4. Hemifusion diaphragm
  5. Initial fusion pore
  6. Post fusion conformation
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2
Q

What are the three sequential steps in membrane fusion?

A
  1. Tethering
  2. Docking [formation of the trans-SNARE complex]
  3. Membrane fusion
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3
Q

What does ‘tethering’ require?

A

multi subunit tethering complex ~25nm in length

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4
Q

What protein complex tethers the ER and Golgi?

A

DSL

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5
Q

What protein complex tethers the Golgi stacks?

A

COG

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6
Q

What protein complex tethers the transport vesicles and plasma membrane?

A

EXOCYST

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7
Q

What protein complex tethers the golgi and early endosome?

A

GARP

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8
Q

What protein complex tethers the Early endosome and Late endosome?

A

CORVET

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9
Q

What protein complex tethers the late endosome and lysosome?

A

HOPS

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10
Q

What are the features of the multi-subunit tethering complexes?

A

-made up of subunits
e.g. DSL - SEL1, Tip20, Sec39
-specific to a certain pathway
e.g. DSL - ER to golgi pathway
-specific for a target membrane and vesicle
e.g. DSL - alpha-COP vesicle protein coat
DSL - Sec20, Use1, Ufe (target membrane)
-may use specific SM, SNARE and Rab proteins

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11
Q

What is required for the specificity of membrane tethering?

A
  1. A 4-helical SNARE complex (Qa, Qb, Qc SNARES and R-SNARE)
  2. an Sec/Mon proteins
  3. a Rab protein and effector
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12
Q

Through what experimental techniques was the SNARE hypothesis thought up?

A
  1. Identified NSF (N-ethyl maleimide sensitive factor)
    and αSNAP (soluble NSF attachment protein) attached to NSF
  2. NSF and αSNAP form a 20s complex = SNAP receptor complex on membranes

Experiment
1.blended cow brains in homogeniser
2. incubated with the homogenised cow brains a bead attached to an antibody specific for NSF (/sec18) to which was bound alphaSNAP(/sec17)
Observed:
-in presence of ATP without Mg2+ -> no response
-in presence of ATP with Mg2+ -> NSF hydrolyses ATP and proteins bound to the complex are eluted
-> ran SDS-PAGE and stained with coomassie, removed and sequences proteins
Identified: The SNAP receptor proteins (SNARE)
-VAMP-proteins (synaptobrevin-2)
-SNAP-25
-Syntaxin-B

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13
Q

What are the features of NSF?

A
  • hexamer
  • AAA-ATPase
  • sensitive to NEM
  • equivilent to sec18 in yeast (essential for all intracellular fusion events)
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14
Q

What are the features of αSNAP?

A
  • required for NSF binding to membrane
  • binds and activates NSF-ATPase activity
  • eqivulent to sec17 in yeast (mutants block trafficking)
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15
Q

What are features of the SNAP-receptor complex?

A
  • can be dissociated by hydrolysis of ATP

- thought to be responsible for membrane fusion

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16
Q

What is the SNARE hypothesis?

A

the existance of many SNARE-like proteins which are specific for a vesivle/target membrane
suggests that NSFs and SNAPs are universal componants of a vesicle fusion apparatus (for both regulated and constituative fusion) and that SNAREs help to ensure vesicle-target specificity

17
Q

What are V-SNAREs and T-SNAREs and how do they interact?

A

V-SNAREs = SNAREs on the vesicle
T-SNAREs = SNAREs on the target membrane
Interact with each other
fold up and physically pull two membranes together
Overcome the electrostatic repulsion of membranes
the formation of the trans -SNARE complex leads to fusion and the cis-SNARE complex

18
Q

What is the process of fusion (basic) by V and T-SNAREs?

A

Transport vesicle ‘docks’ through the interaction of its V-SNAREs with the target membranes T-SNAREs, forming a trans-SNARE complex
Membranes coalesce as the SNAREs coil up and bring the two membranes in close assosiation
Membranes fuse and form the cis-SNARE complex

19
Q

What is the SNARE complex structure?

A

-NORMALLY anchored in the membrane
-NORMALLY through type II membrane proteins which have the N-terminus in the cytosol
-NORMALLY transmembrane
-ALWAYS 4 helices:
Normally from four individual proteins, however occasionally SNAP helices are 2 helices e.g. SNAP-25-C and SNAP-25-N (two α helical coils, normally palmitylated) with syntaxin and VAMP
-NORMALLY on helic from one membrane and 3 from another
-zipping up of SNAREs pulls membranes close together, overcoming electrostatic repulsion of phospholipids and leading to membrane fusion
-SNARE structure normally basic but when fusion occurs the SNAREs coil up into the SNARE complex structure
(strong and hard to break)

20
Q

What are features of the SNARE complex

A

-inside the 4 helical structure it is very hydrophobic
-large number of hydrogen binding and salt bridges
-normally a glutamine(Q)/arginine(R) which contribute to the central layer:
Q-SNARE: [target membrane SNARE]
-Qa (e.g. syntaxin)
-Qb (e.g. SNAP-N)
-Qc (e.g.SNAP-C)
R-SNARE: e.g. VAMP (vesicle assosiated membrane protein)

21
Q

Give an example of how SNAREs provide specificity to membrane fusion events

A

SNAP23 used for recycling endosome and secretory granule

VAMP8 used for secretory granule and between late endosome and lysosome

22
Q

What did experimental work on the ‘combinational SNARE complexes with AMP7/8 define late endocytic fusion events’ show?

A 
Late endosomes can fuse with either homotypic or heterotypic fusion, dependent on altering the RSNARE to give specificity
Late endosome Q-SNAREs:
Syntaxin 7, syntaxin 8, Vti1b
Homotypic fusion with another late endosome
Late endosome R-SNAREs:
VAMP-8
Heterotypic fusion  with a lysosome
Lysosome R-SNARE: 
VAMP 7

Vamp8 - late endosome
Vamp7-lysosome

23
Q

How was it shown that only SNAREs are required for membrane fusion?

A

SNARe proteins were flipped around so that instead of being internal on the cytosolic portion of a vesicle, the SNARE complex forming section was on the outside of a cell

  • one set of cells where V-SNARE (R-SNARE)[VAMP] was on the outside of cell and cytosol dyed red
  • one set of cells where T-SNAREs (Q-SNAREs)[Syntaxin H3, SNAP-25] were on the outside of the cell and labelled with CFP-NLS so the nucleus fluoresced blue
  • put two populations of cells together
  • fused to each other
  • showed that the minimum requirement for fusing is SNARE proteins
24
Q

How do NSF and αSNAP proteins allow SNARE proteins to be recyled?

A
  1. Vesicle with R-SNARE (VAMP) nucleates through the action of Rab causing n-Sec1 to be lost from Syntaxin on the taget membrane
  2. Along with Ca2+ this allows the formations of the trans-SNARE complex containing VAMP, Snap-25 and syntaxin, pulling the membranes in close apposition through the ‘zippering’ of the complex
  3. membranes fuse and cis-SNARE complex is held together strongly. NSF bound to αSNAP binds to the complex and through the ATPase action of NSF allows the dissociation of the SNARE complex, along with the rebinding of n-sec1
25
Q

Why is the recycling of SNARE proteins necessary?

A

-recycled SNAREs need to be targeted to particular membrane (R-SNAREs need to be recovered to vesicles)

26
Q

How do Sec1/Mun18 proteins regulate SNARE complex assembly?

A
  • N-teminal extension of Syntaxin is normally bound back upon the coil (forms SNARE) with n-sec-1 , making the amino acids involved in binding SNARE proteins unavailable (n-Sec-1-syntaxin complex) cannot be broken by other SNAREs as intramolecular bonds are stronger than intermolecular bonds
  • therefore the sec-1 proteins regulate when SNAREs come together
27
Q

What are the different models for the way SM proteins regulate membrane fusion?

A
  • permenant model
  • transient model
  • non-funcitonal SNARE complex

SM complex regulates SNARE fusion

28
Q

What are Rab proteins?

A
  • small GTPases with small lipid anchor
  • family of 60 genes in humans
  • interact reversibly with membranes and effectors in GTP depenendent manner
  • prime membranes for fusion by associating with the membrane and allowing it to associate with sec proteins and tethers through the hydrolysis of GTP to GDP
  • regulate the cycle of pulling membrane in and fusion
29
Q

How is Viral fusion akin to SNARE fusion events, what is the process?

A
  1. Interaction between viral envelope protein and target cell receptor causing the hose protease to cleave viral protein
  2. Viral protein then releases fusion protein and inserts a peptide into the host membrane
  3. hinge area then pulls the two membranes together (akin to coiling up of SNARE proteins) allowing membrane fusion
30
Q

What process of viral fusion allows influenxa entry?

A

Once internalised into an endosome, the change in pH from 7 to 5-5.5 causes a confirmational change in the viral protein
-releases viral peptide allowing it to insert into the vesicle membrane, fuse and escape into the cell

Cell Biology (15 decks)

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