Deborah A. Brown, Ph.D.
Department of Biochemistry and Cell Biology
Life Sciences Building
Stony Brook University
Stony Brook, NY 11794-5215
Office telephone: 631-632-8563
work has given rise to a fundamentally new way of thinking about biological
membranes. Contrary to earlier views, we now know that lipids do not
always mix homogeneously in membranes, but can be organized into domains
in the bilayer. The best-characterized membrane domains, called rafts,
are rich in cholesterol and sphingolipids. The ability of certain proteins
to associate with these domains has profound effects on their function.
This new realization has implications in fields as diverse as signal
transduction, protein and lipid sorting, and cell adhesion and motility.
For instance, following antigen stimulation of T lymphocytes, the T cell
receptor and its signaling partners must cluster together in rafts to initiate
the signal transduction cascade.
Our lab has
developed a model for raft structure, and for how proteins and
lipids associate with rafts, that is now generally accepted in the field. This
model provides a conceptual foundation for further exploration of raft structure
and function. We use a combination of cell biological, biochemical, and biophysical
methods to study rafts in cells and model membranes.
The acyl chains of raft lipids are highly extended
and tightly packed. This arrangement gives raft lipids a high
degree of order. This contrasts with the disordered state of most lipids
in biological membranes. In fact, rafts probably exist in a separate
phase from the rest of the membrane, that has properties similar to those
of the liquid-ordered (lo) phase described in model membranes. Lipids such
as sphingolipids, which have long, saturated acyl chains, partition preferentially
into these ordered domains and are enriched in rafts.
A number of proteins that are modified with
saturated acyl chains (including glycosyl phosphatidylinositol
(GPI)-anchored proteins, myristoylated, and palmitoylated proteins) are
enriched in rafts. These include important signaling proteins, such as Src-family
kinases and heterotrimeric G protein alpha subunits. Association of these proteins
with rafts is likely to be important in function, as has already been
shown for the Src-family kinase Lck in T cells.
of raft structure are a major focus of our lab. How big are rafts?
How are they distributed in membranes? How does clustering of raft proteins
(as can occur during signaling through cell-surface receptors) affect
raft structure and function? How are transmembrane proteins, which might
not be expected to fit well into an ordered lipid environment, targeted to
rafts in the absence of acylation? A close collaboration with the lab of Dr.
Erwin London, a membrane biophysical chemist, allows us to combine complementary
cell biological, biochemical, and biophysical methods. This powerful
approach gives us a unique advantage in studying rafts in both cells and model
Caveolae are 50-100 nm pits in the plasma membrane of many
mammalian cells, that contain the 22 kDa protein caveolin as
a major structural component. Though caveolae were first described more than
30 years ago, their function(s) in endocytosis, cholesterol trafficking, and/or
as signal transduction centers are just now being defined. Rafts have an affinity
for caveolae, although the precise relationship between the two remains an
Caveolin is an unusual protein. Cytoplasmic N- and C-terminal
domains flank a central 33 amino acid hydrophobic domain, which
is presumed to form a tight alpha-helical hairpin that anchors the protein
in the membrane. The protein forms large homo-oligomers, that can be
up to 600 kDa in size. Caveolin may bind directly to a number of signaling
proteins, modulating their function. It also binds tightly to cholesterol,
and has been reported to cycle between the plasma membrane and intracellular
compartments in an unusual manner. Caveolin is also the only protein known
to associate directly with rafts without lipid modification. These and other
unusual properties focus attention on this protein in linking the functions
of rafts and caveolae.
We have purified 100-microgram
amounts of caveolin from recombinant-baculovirus infected
insect cells, and reconstituted it into liposomes for structural studies.
We are also examining the behavior of over-expressed caveolin and of
a number of caveolin mutant proteins in cells. Together, these experiments
will shed new light on this intriguing protein, and how it may function to
organize rafts in cells.
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