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the molecular basis for a GTPase-driven kinetic proofreading mechanism (Sato and
Nakano
2007
).
Individual steps in the formation and uncoating of COPII-coated vesicles are
depicted in Fig.
9.2
.
9.5 Conclusions and Perspectives
A unifying hallmark in the process of transport by coated vesicles is the multitask-
ing of a small GTPase: Arf and Sar1p have various roles in the cycle of coated
carriers. They catalyse steps that require hydrolysis of GTP (cargo uptake and
uncoating) and steps independent of GTP hydrolysis (coat recruitment and scis-
sion). Whereas the mechanism of GTP hydrolysis dependent uptake of cargo is still
elusive, our knowledge on the mechanisms of coat recruitment and coat release is
more advanced, as outlined above.
From investigations on both the COPI and the COPII systems a simple mecha-
nistic model emerges for the step of membrane separation, i.e. the scission of the
vesicle membrane from the donor membrane (Beck et al.
2011
; Faini et al.
2013
).
Membrane scission relies upon the membrane curvature potentiating activity of the
activated small GTPase. Shallow insertion of the G protein's amphiphilic helix into
the cytoplasmic leaflet stabilises positive curvature of the bilayer (in liposomes with
transmembrane proteins lacking, shallow insertion may even convert the bilayer
into tubules, a read-out for membrane curvature potentiating activity). It is then the
coat protein recruited by the small GTPase that sculpts the membrane into the shape
of a bud. As the bud matures, a neck is formed between bud and donor membrane,
with an area of negative curvature. Accommodation in negatively curved layers is
energetically highly unfavourable for an amphipatic helix (McMahon and Gallop
2005
). Therefore, when forced to stay in such areas of negative curvature by
multiple interactions of the small GTPase with the covering inner layer of the
coat, the high-energy state in these zones is released by fusion of the closely
juxtapositioned membranes in the neck (Beck et al.
2011
).
Such a mechanism could hold for all scission steps of vesicles that operate with
small GTPases, COPI, COPII, and CCVs with AP1, AP3, and GGA1-3, as well as
clathrin-independent AP4 vesicles. The model differs from those for endocytosis
based on CCVs with AP2, where the large GTPase dynamin plays a role, and where
hydrolysis of GTP is thought to be prerequisite for the scission step proper.
Although our insight is quite advanced into molecular mechanisms that involve
small GTPases in vesicular transport, there are fascinating questions left, mecha-
nistic ones and more general ones, e.g. how is GTP hydrolysis mechanistically
linked to the uptake of cargo? How is the soluble, non-aggregated state of coatomer
regained after uncoating? What are the specific roles of the various ArfGAPs? Are
all proteins actively sorted into the secretory pathway by interaction of motifs with
receptors? What is the role of the isoforms of COPs? What is the role of the various
Arf family members? Do isoforms of coat proteins create uniformly coated
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