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Roadmap:
Proteins are translated into the ER, which are translocated into the lumen. They are then
sorted into vesicles which leave the ER for the Golgi, and then out to the plasma
membrane. From the plasma membrane they might go back to the endosomes, and from
the endosomes they might head back to the Golgi.
Vesicle Transport
There are multiple donor and target membranes in the cell, so targeting information is
needed to determine specificity of vesicle targeting. There are two mechanisms to
determine this specificity: the Rab GTPase tethering mechanisms, and the SNARE fusion
machinery.
Cytoskeleton is the tracks of transport for vesicles
Cytoskeleton is made up of protein filaments (actin) and microtubules (α and β tubulin),
and it runs through the cytosol to provide structure to cells. Actin filaments are shorter
and often clustered at the PM; they are highly crosslinked into dynamic networks.
Microtubules are longer, and are usually organized around centrosome near nucleus,
though they can extend into the periphery of the cells. The two are connected to each
other and anchored to PM and organelles.
Transport of vesicles is mediated by motor proteins, which move along the cytoskeleton.
There are two types of motor proteins: myosins move on actin filaments, while dyneins
and kinesins move on microtubules. These motors transport vesicles and other
components along the cytoskeleton, but despite being ATP-dependent (dynein is actually
an AAA protein), they do not specify target.
Rab proteins
Rab proteins are in a large family of monomeric GTPases (same family as the Sar1 and
Arf, which are responsible for vesicle formation) and come in different sets, each
uniquely associated with an organelle and vesicle type in the secretory pathway. The
unique combination gives the targeting specificity of the vesicle.
They have two geranylgeranyl (lipid) groups at the C-termini (to insert into the
membrane); however, Rabs can be removed from the membrane by specific protein
interactions. In general, the GTP-bound state is the active state (GDP-bound is off),
which interacts with various effector proteins and membranes.
The Rab Cycle starts with GDP-bound, inactive Rab
1. The inactive Rab-GDP is bound by GDI (GDP dissociation inhibitor)
a. GDI covers the geranylgeranyls, which makes the inactive Rab-GDP
soluble and not membrane-associated
2. To turn on Rab, GDF (GDP Dissociation Factor) and GEF (GTP Exchange
Factor) on the membrane produce Rab-GTP with exposed geranylgeranyl lipids
that can become anchored to the membrane
3. The activated Rab-GTP works through effector proteins on the vesicle and
acceptor membrane, the interactions of which determine target specificity
4. After targeting has been accomplished GAP (GTPase activating protein) induces
Rab to hydrolyze GTP, and makes it go back to GDP form
5. Rab Escort Proteins and Geranylgeranyl-transferase prepare newly-synthesized
Rabs
Rab Functions in sorting and directing
Rab assists in sorting of cargo receptors into vesicles on donor membranes during
budding. (Rab-GDP’s GDP is taken off by GDF, at the same time that the GEF replaces
the empty GDP slot with the GTP; interactions between Rab and cargo receptors may
help gather cargo into vesicles).
Also, Rab might regulate PI-phosphates by recruiting PI-kinases or PI-phosphatases to
control type of PI-lipids on a membrane. A little evidence that Rab might connect the
vesicles to the cytoskeletal models, though it might not be universal.
A more universal function is that Rabs are able to initiate targeting of vesicle by
connecting the Rab effector proteins via tethering. Tethering involves the approaching of
the vesicle to the acceptor membrane, and the Rabs on the vesicle and on the acceptor
will activate long tethering proteins to help bring the vesicle to the right acceptor. Most
Rab effectors, in fact, are tethers—long proteins that connect the vesicle and acceptor
membranes. Rab/Tether interactions are the primary determinant of vesicle targeting
specificity. Also, the Rab-tether interactions might also prepare the vesicle fusion
machinery (SNARE) on the vesicle and acceptor membranes (they don’t mediate the
fusion, but they help SNAREs mediate the fusion).
More about Tethers: multitasking Coiled-coils, and single-tasking Multisubunits
Multisubunit tethers direct vesicle targeting to the major secretory compartment
membranes. For example, the exocyst (first identified in yeast, but also has parallels in
humans) is a multisubunit tether for vesicle fusion at PM and endosomes. Some
components of the exocyst are carried on vesicles, while others on acceptor membranes.
The two parts come together to form large octameric complexes attached to Rabs. A
similar protein to Rab is Rho, which is a regulator on the PM and could form the
octameric complex tethers with Rabs.
Another type of tether is the Coiled-coil tethers, which are special dimeric tethers with
long coiled-coil structures (pairs of α-helices wrapped around each other) that connect
vesicles to Golgi membranes. The Coiled-coil tethers are also responsible for the shape of
the Golgi stack in its layered form (second function). On the other hand, they are not
related to the multisubunit tethers, and attach to membranes via anchors or interactions
with Rab and Arf.
Some Rab effectors (other than tethers) have GEF or PI kinase/phosphatase activities.
This means that when an activated Rab turn on an effector, the effector is actually able to
turn on another Rab (through GEF activity) within proximity to it. This positive feedback
loop (Rab-GTP producing more Rab-GTP in a localized spot in the membrane)
contributes to the clustering of tethers in the site of fusion. Also, the PI-phosphates
phosphorylates other PIs, which provide additional binding sites for Rab effectors such as
tethers.
Targeting and Fusion: SNAREs also determine specificity
SNARE proteins are a family of related membrane proteins. Specific v-SNAREs on
vesicles recognize specific partner t-SNAREs on target membranes. The SNAREs
function after tethering and are the second determinant of targeting specificity. Formation
of SNARE complexes between the vesicles and acceptor membranes mediates fusion.
V-Snares are monomers with single TM domains. T-SNAREs are trimers with one TM
domain and two peripheral subunits. The correct set of v- and t-SNAREs form a stable
tetramer, 4 tightly wound α-helices in a bundle, the formation energy of which drives
fusion.
Fusion Mechanism
SNARE complex structure (coiled-coil interactions) resembles certain viral coat proteins.
For viruses, they fuse their membranes with host cell PM during infection. The
mechanism of fusion is mediated by the coat proteins—which bind to the host membrane,
then change conformation in helical domains to cause fusion. This gave rise to SNARE
hypothesis: vesicle fusion was triggered by conformational changes in SNAREs, the
same way that viral coat proteins change conformation in helical domains to fuse.
The mechanism of SNARE fusion is as follows:
1. V-SNARE monomer, before fusion, is not stably folded. The majority of its
cytosolic domain is flopping around. However, it folds into helical bundles when
brought together with t-SNAREs (which are also originally unstable, but since
they have two peripheral subunits, they are somewhat more stable than vSNAREs)
2. The base of the v-SNARE eventually becomes close with the base of the tSNARE. Thus, the folding process pulls the membranes close together and
generates physical strain (like a spring).
a. This is NOT dependent on ATP/GTP!
3. The membranes are held together in such a way that water is excluded from the
area
a. This exclusion allows hydrophobic contacts between lipids of membranes
i. Outer leaflet (outer layer of lipids) joining = hemifusion; this is an
unstable intermediate
ii. Inner leaflet fusion (inner layer of lipids) joining = fusion; this is
the stable final product, which relieves strain from the SNARE
complex
SNARE Dissociation
After fusion, the SNARE complex has had all of its strain relieved, and is consequently
stable and inactive. However, the v- and t-SNAREs are still hooked up together on the
acceptor membrane; what separates them?
NSF is an AAA-family ATPase protein that separates and recycles v- and t-SNAREs on
the acceptor protein, which is essential for continuation of vesicle traffic. (Note the theme
of AAA: their favorite pastimes seems to be pulling things apart—pulling apart ER
lumen proteins, proteasomal proteins, and now pulling apart SNAREs). With accessory
proteins and ATP, it rips the v-t complex apart. The t-SNAREs are active again, and the
v-SNAREs are recycled back to their donor membranes by other vesicles.
Homotypic Fusion: when donor and target membranes are the same
Examples of homotypic fusion are when COP-II vesicles make the VTC (vesiculartubular cluster), or the re-formation of organelles after cell division. In these cases, both
membranes have identical v- and t-SNAREs. Therefore, in these cases, SNAREs mut be
separated by NSF to allow fusion. (So the last step becomes the first step).
1. t- and v-SNAREs are separated on vesicles by NSF
2. NSF dissociates and the two vesicles fuse by their respective t-v-SNAREs in
homotypic fusion
3. Now there are two sets of v-t-SNARE complexes on one vesicle