Lipid rafts in cell physiology and disease--Kai Simons

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

For a long time the prevailing view of the lipid bilayer in cell membranes was that
it constituted a boring solvent for the membrane proteins.. Considering that cell
membranes are known to contain a complex lipid composition of close to a
thousand different lipid species this complexity could not be just fortuitous. One
function is the in-built capability of dynamic sub-compartmentalization and this is
where lipid rafts come into the picture. Rafts can form as dynamic platforms with
a key role in regulating membrane functions. These are made up of
sphingolipids and cholesterol, associating in the extra-cytoplasmic leaflet of the
bilayer and organizing the underlying cytoplasmic leaflet such that a linked
assembly is formed. Our present concept of lipid rafts is that they are dynamic
assemblies of sphingolipids, cholesterol and raft proteins that dissociate and
associate on a rapid timescale. These assemblies can be induced to coalesce
into raft clusters and these are the platforms that can be visualized by
microscopy.



The raft concept came from our studies on MDCK cells. The enrichment of
sphingolipids in the apical membrane, together with their propensity to associate
with cholesterol to form lipid rafts, led to the concept that rafts preferentially traffic
to the apical membrane after intracellular assembly. Since certain proteins
associate with rafts during apical transport, rafts could act as apical sorting
platforms in the Golgi.

Raft sorting also occurs in yeast. We have shown that sphingolipid-ergosterol
rafts play a role in surface delivery in yeast. Recently, we finished a genomic
screen that was designed to identify proteins involved in raft protein delivery to
the cell surface. We identified several enzymes involved in sphingolipid and
ergosterol synthesis in addition to other proteins, which are candidates for
machinery. This result confirmed in a non-biased manner that the delivery of a
raft-associated protein from the Golgi to the plasma membrane could be
hindered by mutants affecting raft lipid metabolism. We have now worked out
methods to isolate the membrane vesicles that deliver the raft marker protein to
the cell surface in yeast from the TGN. Our results using novel mass
spectrometric methods demonstrate that these vesicles are enriched in raft lipids.

Another example of raft clustering comes from our work on the amyloid
precursor protein (APP) that plays a crucial role in the pathogenesis of
Alzheimer's disease. During intracellular transport APP undergoes a series of
proteolytic cleavages that lead to the release of an amyloidogenic fragment, β-
amyloid (Aβ). It is Aβ that accumulates in the brain lesions that are a hallmark of
the disease. While inside raft clusters seems to be cleaved by β-secretase, APP
outside rafts undergoes cleavage by alpha-secretase. Thus, access of alpha-
and beta-secretase to APP, and therefore Aβ generation, may be determined by
dynamic interactions of APP with lipid rafts.


We have also demonstrated that β-cleavage is occurring in early endosomes
and we have recently obtained evidence that a fraction of Aβ leaves the cell
associated with exosomes. We are now reconstituting amyloidogenic processing
in liposomes. We have established a novel in vitro assay to study how the
membrane lipid composition influences beta-cleavage of APP. For this, we
expressed and purified APP and BACE and reconstituted them into lipid bilayers
of specific lipid composition. Our data show

the enzyme is activated by sphingolipids, cholesterol and acidic phospholipids in
vitro. We have also shown that BACE partitions with liquid-order phases in giant
unilamellar vesicles and that this partitioning is augmented by raft clustering. All
these results support our model for APP processing.