Membrane Fusion

Question 1

What portion of proteins produced in a cell are targeted to membranes or lumen of organelles and what process does this necessitate?

Answer 1

Approximately 1/3 of all proteins produced in a cell are targeted to these locations. This means that they must be secreted into vesicles that are themselves targeted to the proper locations.

Question 2

How is cyclic fibrosis influenced by membrane fusion and what other types of disorders may be associated with transport problems?

Answer 2

Cystic fibrosis results from mis-transport of the mutant proteins.
Neurotransmitter and lysosomal storage diseases may result from mis-transport as well.

Question 3

What are the two primary areas of concern in membrane fusion?

Answer 3

1 How are individual vesicles targeted to their proper destinations.
2 How are the unfavorable thermodynamics of membrane fusion overcome?

Question 4

What are the two thermodynamic issues that must be overcome in a membrane fusion?

Answer 4

Hydration of the hydrophilic lipid heads and electric repulsion of the hydrophilic heads of each membrane.

Question 5

How frequently do membranes spontaneously fuse, and how frequently do they fuse with proper signaling and fusing proteins?

Answer 5

In the absence of fusion processes, membranes spontaneously fuse on the order of minutes to hours. With fusion processes, membranes may fuse in 200 milliseconds or less.

Question 6

What are the three primary SNARE proteins?

Answer 6

Syntaxin - locates on plasma membrane of organelles or cell (1 large amphipathic helix domain)
SNAP-25 - Locates on plasma membrane (2 large amphipathic helix domains)
VAMP - Vesicle Associated Membrane Protein, locates on exterior of vesicles (1 large amphipathic helix domain)

Question 7

How does botulism toxin work?

Answer 7

Botulism toxin inhibits the process of membrane fusion in neurons, inhibits neurotransmitter release. The toxin is a number of proteases that cleave the SNARE proteins that are responsible for neurotransmitter release.

Question 8

Why do coiled coils make very stable structures and how many helixes are involved in the coiled coils that form in membrane fusion?

Answer 8

The coiled coils consist of bound amphipathic helixes that combine to keep their hydrophobic regions interior and hydrophilic regions exterior. Coiled coils may consist of any number of amphipathic helixes, but in membrane fusion there are four helixes to the coiled coils.

Question 9

How do the coiled coils formed by SNARE proteins lead to membrane fusion and what sorts of mutations affect them?

Answer 9

The amphipathic helixes of every SNARE protein are immediately proximal to their transmembrane domains. When the coiled coils form, the membranes are pressed very closely together, overcoming the hydration and electrostatic resistance. Mutations that inhibit amphipathic helixes (such as proline insertion) or that insert peptides between the helix and transmembrane domains diminish or destroy their effectiveness.

Question 10

How are the highly stable coiled coils of SNARE proteins released?

Answer 10

The NSF protein (a homologue of helicase, and a triple-ATPase) forms a hexamer which binds to alpha-SNAP (NOT related to SNAP-25) and “unwinds” the coiled coils, using 6 ATP for each coiled coil complex.

Question 11

Because the unwinding of coiled coils by NSF essentially denatures the SNARE proteins, what returns them to a primed state and what else does this protein do?

Answer 11

n-sec1 refolds syntaxin, functioning like a chaperone. n-sec1 remains attached to syntax inhibiting it from binding with itself or other SNARE proteins until the correct signal comes. The absence of n-sec1 produces constitutively active vesicle fusion in neurons, resulting in uncontrolled firing of neurons.

Question 12

What determines specificity in vesicle fusion?

Answer 12

There are 18 types of syntaxin, 8 VAMP, and 9 SNAP, as well as 8 n-sec1. Different combinations of these proteins produce the necessary specificity to target vesicles to the proper location.

Question 13

What is the only membrane in mammalian cells that does not fuse via SNARE proteins?

Answer 13

Mitochondrial membranes do not use SNARE proteins

Question 14

Three types of envelop viruses, and what is the identifying feature of an envelop virus?

Answer 14

HIV, Influenze, Ebola
Envelop viruses are coated in a plasma membrane derived from the host cell. This requires the envelop virus to undergo membrane fusion, though they lack SNARE proteins.

Question 15

How do envelop viruses achieve membrane fusion without SNARE proteins?

Answer 15

Viral fusogenic proteins are produced in the host cell and present on the plasma membrane that the virus obtains its envelop from. The Fusogenic Peptide (FP) is a small, hydrophobic section of these proteins, usually hidden within the larger coiled coil structure, which is inserted into the cell’s plasma membrane to link the virus and cell.

Question 16

How does an influenza virus use pH to activate its fusogenic proteins?

Answer 16

The virus allows itself to be endocytozed and placed into a lysosome. As the pH drops to about 6, the fusogenic proteins undergo a conformational change to extend the FP domain into the membrane of the lysosome and initiate membrane fusion.

Question 17

How does the HIV virus use receptor binding to activate its fusogenic proteins?

Answer 17

The gp120 protein (a receptor) sits atop the gp41 protein (the fusogenic protein). The gp120 protein undergoes a conformational change when it encounters the CD4 protein on a T-cell, allowing the gp41 protein to expel its FP domain into the plasma membrane of the host cell.

Question 18

How might inhibiting fusion of HIV lead to further infections?

Answer 18

HIV viruses absorbed by dendritic cells can inhibit the formation of lysosomes and remain dormant within the cells for years even if anti-retroviral drugs eliminate all other viruses. Inhibiting fusion in this case ensures the virus’ continued survival, where as encouraging fusion would lead to its degradation by the dendrite.