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Dynamin, Schmid now believes, is a much more sophisticated
molecule. "Dynamin is not just the brawn," Schmid says. "It's
part of the brains." She thinks that it is integrating the
process of endocytosis with other events in the cell. It may
be rearranging the actin cytoskeleton of the cell, monitoring
what is entering cell-wide, and inducing a stress response.
A Powerful Assay
The biochemical assay that the Schmid laboratory developed
involves purifying plasma membranes, stripping them of all
their materials, and reconstituting the machinery to generate
coated vesicles and recreate each of the steps leading to
endocytosis in the test tube. This assay has allowed her to
study and understand the action of dynamin and to identify
the other cellular machinery that carries out vesicle formation.
"It is different than assays we have used in the past, which
use a single receptor and a single ligand," says Schmid. "Now
we can look at any receptor we want."
One of the things these studies have revealed is that the
regulation of receptor-mediated endocytosis is a highly sophisticated
interaction between the cargo molecules, the receptors, dynamin
and other enzymes, and the clathrin coat.
In addition to having binding sites outside the cell that
recognize and collect the cargo molecules, receptors have
binding sites on their portions inside the cell that are used
for binding as well—to the clathrin molecules that form
the cage around the budding vesicle.
However, clathrin does not recognize the receptors directly.
The cell employs "adaptor" proteins that recognize the sorting
motifs on the receptors and then "adapt" the cargo molecules
to the coat. Like the familiar three-pronged to two-pronged
converters people use to plug their toaster ovens into old
outlets, adaptor molecules fit the receptors to the clathrin
scaffold during vesicle formation.
Moreover, the adaptors exert control over endocytosis because
they trigger assembly of the clathrin coat, and they group
the receptors together, thus concentrating the cargo. And
to make the situation even more elaborate, the adaptors are,
in turn, controlled by other parts of the cellular machinery.
About a year ago, the Schmid laboratory discovered a new
"kinase" enzyme that is involved in the regulation of cargo
selection by controlling the adaptors. The kinase that Schmid
found attaches a phosphate group to adaptor molecules and
thereby regulates cargo selection by altering the adaptor.
The adaptor molecule is called AP-2, and Schmid's new kinase
binds to it and attaches a phosphate group to the portion
of AP-2 that is responsible for recognizing the receptor molecule.
Schmid found that when the kinase attaches the phosphate to
the adaptor molecule the affinity for the cargo molecules
increases 25-fold.
"That's a huge difference," she says. "Kinases usually have
a 2- to 3-fold effect."
Into the Cell
However, when Schmid and her colleagues went beyond the
biochemical assay and designed a series of mutant cells that
would allow them to see what happens when they tinker with
the kinase, they were surprised.
"We learned things we never could have anticipated from
what we had done in the test tube," she says.
What they anticipated was that by overexpressing the kinase,
they would be overphosphorylating the AP-2 adaptor molecules.
They reasoned that the overphosphorylated adaptors would be
randomly distributed and unable to cluster.
Then, since adaptor clustering leads to clathrin clustering,
Schmid and her laboratory thought that overexpressing the
kinase would shut down vesicle formation and endocytosis.
However it did not.
In fact, to their surprise, they found that the clathrin
distribution was not affected by essentially knocking out
the function of AP-2. Other adaptors, they concluded may be
working independently of AP-2 to accomplish the same goal.
"AP-2 may be just another cargo-specific adaptor,"
she concludes.
In general, she adds, receptor-mediated endocytosis seems
to be more sophisticatedly regulated than was ever previously
thought.
"In retrospect this makes sense," says Schmid. "Cells communicate
between themselves and with their environment through the
plasma membrane. Endocytosis plays a critical role in regulating
this communication."
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Yellow LDL particles that carry cholesterol
in the bloodstream are captured by receptors on the cell surface.
Three-legged, clathrin triskelions (red) assemble into soccer-ball-like
lattices to pinch off a piece of the membrane-carrying receptors
and cargo into the cell. Electron micrographs (at 100,000X
magnification) show flat and deeply curved clathrin coated
pits on the inside of the cell. Another protein, dynamin,
forms a spiral "collar" around the coated pit and
is required, like a purse-string, for sealing the neck and
detaching the coated vesicle. Green virus particles can, like
Trojan Horses, gain access to the cell through this entryway.
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