Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

THE MOLECULAR MECHANISMS OF MEMBRANE TRANSPORT
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Monomeric GTPases Control Coat Assembly
To balance the vesicle traffic to and from a compartment, coat proteins must
assemble only when and where they are needed. While local production of PIPs
plays a major part in regulating the assembly of clathrin coats on the plasma
membrane and Golgi apparatus, cells superimpose additional ways of regulating
coat formation. Coat-recruitment GTPases, for example, control the assembly
of clathrin coats on endosomes and the COPI and COPII coats on Golgi and ER
membranes.
Many steps in vesicle transport depend on a variety of GTP-binding proteins
that control both the spatial and temporal aspects of vesicle formation and
fusion. As discussed in Chapter 3, GTP-binding proteins regulate most processes
in eukaryotic cells. They act as molecular switches, which flip between an active
state with GTP bound and an inactive state with GDP bound. Two classes of proteins
regulate the flipping: guanine nucleotide exchange factors (GEFs) activate
the proteins by catalyzing the exchange of GDP for GTP, and GTPase-activating
proteins (GAPs) inactivate the proteins by triggering the hydrolysis of the bound
GTP to GDP (see Figures 3–68 and 15–7). Although both monomeric GTP-binding
proteins (monomeric GTPases) and trimeric GTP-binding proteins (G proteins)
have important roles in vesicle transport, the roles of the monomeric GTPases are
better understood, and we focus on them here.
Coat-recruitment GTPases are members of a family of monomeric GTPases.
They include the ARF proteins, which are responsible for the assembly of both
COPI and clathrin coats assembly at Golgi membranes, and the Sar1 protein,
which is responsible for the assembly of COPII coats at the ER membrane.
Coat-recruitment GTPases are usually found in high concentration in the cytosol
in an inactive, GDP-bound state. When a COPII-coated vesicle is to bud from
the ER membrane, for example, a specific Sar1-GEF embedded in the ER membrane
binds to cytosolic Sar1, causing the Sar1 to release its GDP and bind GTP
in its place. (Recall that GTP is present in much higher concentration in the cytosol
than GDP and therefore will spontaneously bind after GDP is released.) In its
GTP-bound state, the Sar1 protein exposes an amphiphilic helix, which inserts
into the cytoplasmic leaflet of the lipid bilayer of the ER membrane. The tightly
bound Sar1 now recruits adaptor coat protein subunits to the ER membrane to
initiate budding (Figure 13–14). Other GEFs and coat-recruitment GTPases operate
in a similar way on other membranes.
The coat-recruitment GTPases also have a role in coat disassembly. The hydrolysis
of bound GTP to GDP causes the GTPase to change its conformation so that
its hydrophobic tail pops out of the membrane, causing the vesicle’s coat to disassemble.
Although it is not known what triggers the GTP hydrolysis, it has been
proposed that the GTPases work like timers, which hydrolyze GTP at slow but predictable
rates, to ensure that vesicle formation is synchronized with the requirements
of the moment. COPII coats accelerate GTP hydrolysis by Sar1, and a fully
formed vesicle will be produced only when bud formation occurs faster than the
timed disassembly process; otherwise, disassembly will be triggered before a
vesicle pinches off, and the process will have to start again, perhaps at a more
appropriate time and place. Once a vesicle pinches off, GTP hydrolysis releases
Sar1, but the sealed coat is sufficiently stabilized through many cooperative interactions,
including binding to the cargo receptors in the membrane, that it may
stay on the vesicle until the vesicle docks at a target membrane. There, a kinase
phosphorylates the coat proteins, which completes coat disassembly and readies
the vesicle for fusion.
Clathrin- and COPI-coated vesicles, by contrast, shed their coat soon after
they pinch off. For COPI vesicles, the curvature of the vesicle membrane serves
as a trigger to begin uncoating. An ARF-GAP is recruited to the COPI coat as it
assembles. It interacts with the membrane, and senses the lipid packing density.
It becomes activated when the curvature of the membrane approaches that of a
transport vesicle. It then inactivates ARF, causing the coat to disassemble.