Vesicle Fusion

Front 1

Intra-organelletransport of proteins: Vesicles

Back 1

•Docking a Fusion are two separate events, withdifferent sets of proteins and mechanisms.

•Docking determines specificity. Where the vesicles goes.

•Fusionovercomes the energetically unfavorable process of fusing two plasma membranes.Some specificity is determined by fusion proteins (SNARES) but specificity isprimarily determined by docking machinery. •Together,Docking and Fusion machinery work together to make sure cargo get’s were it issupposed to go

Front 2

VesicleDocking and Fusion at the Molecular Level (the SNARE hypothesis)

Back 2

Fusion, but not docking, ismediated by proteins called SNARES. V-SNAREs on the vesicle bind T-SNAREs onthe target membrane, forming a complex with another SNARE, SNAP-25.

Front 3

SNAREproteins bring membranes together so they can fuse(theSNARE hypothesis) II

Back 3

•SNARE complexes are made up of three proteins (one V-SNARE, one T-SNARE, and one Snap25)

•The energy release from the coilingtogether of SNARE proteinsis used to overcome the energetically unfavorable process of fusing membranes.

•Ifthe SNARE complex is so stable, what problem does that present to the cell

Front 4

Snare Structure

Back 4

SNAREs coil into this structure,releasing a ton of energyto form this highly stable complex. In the process of forming this complex agreat amount of energy is released and the membrane are forced so closetogether that fusion occurs. What do you notice about the two transmembrane (TM) membranes? Note, in the structureabove, only the parts of the protein that will crystalize are shown. Thenon-structured “floppy” parts are omitted.

Front 5

NSF complex pulls SNARE complexes apart

Back 5

The SNARE complex is taken apart byalpha-SNAP and NSF, two protein complexes that use the energy from ATPhydrolysis to unwind the SNARE complex into its individual components.SNARE proteins are recycled this way and usedagain and again.

Front 6

Regulation of vesicle fusion

Back 6

Vesicles can remained docked at atarget membrane for a long time. So howdo you prevent the SNARE proteins from interacting and triggering fusion andhow do get vesicles to fuse at the right moment?

Front 7

Fusion can be regulated by calcium

Back 7

Some neurons can fire almost 1000 times persecond. How can such a fast rate of firing be supported. Manyvesicles are docked at a synapse and are “primed”. When an action potentialarrives, it triggers the opening of a Calcium channel near the synaptic cleftcausing and increase in the Calcium concentration. This triggers aconformational change in a protein called snyptotagmin that triggers membrane fusion.Typically, only a few vesicles fuse each time an action potential arrives

Front 8

SNARE Diversity

Back 8

hella snares. Specific t-snare for your v-snare

Front 9

Where would you expect the t-SNAREfor synaptic vesicles to localize?

Back 9

Surprisingly, t-SNAREs can be found in a large areaaround the cell, not just in the synapse where the vesicle fuses. This suggestthat the SNARES are not the primary means of determining specificity

Front 10

Where would you expect tethers forsynaptic vesicles to localize?

Back 10

Tethering systems that drivedocking account for most of the specificity of vesicles for target membranes.Since docking MUST precede fusion, vesicles cannot fuse withthe incorrect target membrane, even if the correct SNARE is located there!

Front 11

Vesicle Docking and Fusion at theMolecular Level

Back 11

•What does the GTP analogue experiment suggest? What type of proteindo you think Rab is?

•Docking workswhen tethering proteins on the vesicle bind tethering proteins on the targetmembrane. This interaction only occurs when the correct rab, asmall GTPase thatmust be in the active GTP-bound state on the vesicle, is present. Upon fusion,hydrolysis of GTP by the rab,stimulated by target membrane GAPs, results in disassembly of tetheringproteins.Since vesicle tethering proteins are peripheral membrane proteins recruited tovesicle by active Rabs,they are released from the target membrane.

Front 12

Rabsare Small GTPases

Back 12

•GTPases again. The molecular switchof the cell.

•There aremany purposes of rab invesicle trafficking beyond just tethering. As all small GTPases, they bindtarget proteins that mediate their cellular functions only in the GTP-boundstate and are switched on and off like other small GTPases.

•There are a huge number of Rab GTPases (number in the 60s).

The Rab GTPase cycle. Like the other GTPases we havetalked about, the Rab protein cycle between two differentstates. In the GTP bound state, they are associated with a membrane andinteract with Rab effector proteins. When they are in theGDP bound state, they bind Rab escort proteins (REP) or GDPdissociation inhibitor (GDI) proteins. REP and GDI proteins are involved in recycling the Rabproteins back to their original membrane and keeping them soluble in thecytosol. REP and GDI proteins both have protein domains that can bind and“cover” the hydrophobic portion of Rab proteins, making them soluble again.

Front 13

There is a Rabprotein for every membrane

Back 13

Hereis how the general cycle would work. A new Rab protein is snythesized in the cytoplasm by a ribosomes. Afterbeing modified and having a hydrophobic tail added on, the Rabprotein is delivered to its source membrane (the trans Golgi network forexample). Typically, there is a Rab GEF in the source membrane triggersexchange of GDP for GTP and the Rab becomes activated. Once activated, itstarts to recruit Rab effectors that do all the work (help invesicle budding and targeting). Once the vesicle (with its Rab)arrive at the destination membrane, a Rab GAP deactivates the Rabprotein and it is bound by a REP or GDI and it disassociates from the membraneand is recycled back to its source membrane.

Front 14

Rab GTPases work with Rab effectors todetermine target specificity

Back 14

The cytosol of a cell is a complex placewith a huge diversity of membranes/organelles. A vesicles is likely to bumpinto a lot of other vesicles and membranes. So how does it know which one to goto? Specificity is determine by proteins found on the surface of the vesicleand the target membrane. Two sets of proteins are responsible for this job. Rabproteins and Rab effectors. The distribution of Rab proteins is highlyspecific. They are ideal for marking specific membrane compartments. Likecoat-recruitment GTPases (think of Sar1 from last lecture), they float aroundthe cytoplasm in an inactive GDP-bound state. They are often bound by a Rab-GDPdissociation inhibitor (GDI) that keeps them soluble and inactive. When theyare activated, they associate with the target membrane or vesicle membranethrough an exposed lipid anchor. Once activated, Rab-GTPases recruite Rab effector proteins. Rab effectorproteins are very diverse. Some will interact with motors that move thevesicles in a certain direction along actin filaments or microtubles. Some are tethering proteins that actlike arms that reach out for vesicles. Rab proteins can be found on thevesicle, target membrane, or both. Rab effectors can also interact with SNAREcomponents. Often Rab proteins will have feedback loop to build a Rab domain ona membrane. For example, once a Rab protein is activated, one of the Rabeffectors will recruit more Rab-GEF. This has the effect of brining more Rab tothat region of the membrane. As more Rab is recruited, you build a domain of ofRab and Rab effectors on the membrane that helps establish the function andidentity of a specific membrane enclosed compartment. Changes in the Rabproteins can lead to changes in the organelle.

Front 15

Rab cascades can change the identity ofan organelle

Back 15

In the example above, RabA recruits several rabeffectors. One of these effectors is a RabB GEF. This has the effect of recruiting RabBto the membrane. Once RabB is activated, one of the RabBeffectos is a RabA-GAP and deactivates RabA.This forms a feedback loop that makes this process uniderectional.