ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

vascular tone, protect neurons and muscles from ischemia,
and are responsive to leptin. K ATP channels have
an unusual octameric stoichiometry consisting of four
pore-lining inward rectifier a α subunits (Kir6.1/6.2;
two transmembrane segments) like other K+ channels,
but also contain four regulatory sulphonylureabinding
ß subunits (SUR1/2A/2B; probably seventeen
transmembrane segments) that belong to the ATPbinding
cassette (ABC) family of proteins.
We found that only octameric K ATP channel complexes
were capable of expressing on the cell surface, implying
that quality control mechanisms must exist to prevent
monomers and partial complexes from expressing
on the cell surface. Surprisingly, we found that
the primary quality control mechanism during K ATP
assembly did not involve ER degradation or ER chaperones,
but rather the exposure of an ER retention/
retrieval signal (RKR) present in cytosolic domains
of each subunit. Mutating the retention sequences did
not affect protein levels, but allowed surface expression
of monomers and partially assembled complexes,
including improperly gated channel combinations. We
further showed that the RKR trafficking signal was
functional in a variety of eukaryotic cells including
yeast, mammalian cells, and Xenopus oocytes. Interestingly,
this RKR motif did not require proximity
to the N- or C-terminus like all other known ER
retention/retrieval signals and may, therefore, be more
common. In conclusion, quality control during K ATP
assembly is mediated by a short trafficking signal
whose exposure reflects the assembly state of the
channel. These results provide a clear example of how
a trafficking checkpoint serves as an important quality
control mechanism during the assembly of an ion
channel complex. Furthermore, they identify a new
ER retention/retrieval motif that could explain transient
or permanent ER localization of many proteins.
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Our current projects are designed to better define this
new class of ER retention/retrieval signals, to extend
the analysis of their role in assembly-dependent trafficking
using K ATP channels as a model system, and
to identify and characterize the molecular machinery
that recognizes this class of signals.
PUBLICATIONS
Schwappach B., Stobrawa S., Hechenberger M., Steinmeyer
K., and Jentsch T.J. (1998). Golgi Localization
and functionally important domains in the NH 2 and
COOH terminus of the yeast CLC putative chloride
channel Gef1p. J. Biol. Chem. 273, 15110–15118.
Zerangue N.*, Schwappach B.*, Jan Y.N., and Jan
L.Y. (1999). A new ER trafficking signal regulates
the subunit stoichiometry of plasma membrane K ATP
channels. Neuron 22, 537-548 (*these authors contributed
equally).
Schwappach, B.*, Zerangue, N.* Jan Y.N., and Jan
L.Y. (2000). Molecular basis for K ATP assembly: transmembrane
interactions mediate association of a K +
channel with an ABC transporter. Neuron 26, 155-167
(*these authors contributed equally).
STRUCTURE OF THE GROUP*
E-mail: b.schwappach@zmbh.uni-heidelberg.de
Group leader Schwappach, Blanche, Dr.*
Ph.D. student Yuan, Hebao, M.A.*
Techn. assistant Metz, Jutta*
* since July/August 2000
Dominique Soldati
Cell and Molecular Biology of the Obligate
Intracellular Parasite Toxoplasma
gondii
Apicomplexan parasites are of enormous medical
and veterinary significance, being responsible for a
wide variety of diseases including malaria, toxoplasmosis,
coccidiosis and cryptosporidiosis. Successful
attachment and invasion of the host cells are key to
the survival of these obligate intracellular parasites.
T. gondii, the most ubiquitous of the Apicomplexa,
has developed a remarkable ability to actively penetrate
almost any nucleated cells from virtually all
warm-blooded animals. Most invasive forms of the
Apicomplexa share a common set of apical structures
and exhibit an unusual form of substrate-dependent
gliding motility as an adaptative mechanism to
actively penetrate host cells. In absence of locomotive
organelles such as cilia or flagella, the basic
engine for gliding locomotion is the actin cytoskeleton
and involves myosin(s) to generate the mechanochemical
force along the actin filaments, allowing
parasites to move at rates from 1 to 10 micrometers
per second.
Successive exocytosis of regulated compartments
including rhoptries and micronemes is part of the
invasion process. Micronemal proteins are apparently
used for host-cell recognition, binding, and possibly
motility, while rhoptry and dense granule proteins
contribute to the parasitophorous vacuole formation
and its remodeling into a metabolically active compartment.
Release by micronemes occurs in the earliest
phase of invasion, upon contact with the host cells
and raise in intracellular Ca 2+ .
Molecular motors, adhesins and proteases are among
the distinct classes of genes coding for invasion factors.
These genes are anticipated to be essential for
the survival of these parasites and therefore the ability
to conditionally turn on or off their expression is
prerequisite for their in vivo studies.
I. Myosins: molecular motors in Apicomplexa
parasites
Five myosins of T. gondii and three myosins of P. falciparum
have been identified so far. These unconventional
myosins are the founders of a novel phylogenetic
and structural class of myosin (XIV), restricted
to the Apicomplexa. Members of this class are small
with molecular weights ranging between 90 and 125
kDa and exhibit unusual structural features.
Myosins have been implicated either genetically, biochemically
or by cytolocalization in a variety of cellular
functions. Three distinct forms of gliding characterize
T. gondii motility: circular gliding, upright
twirling, and helical rotation. Inhibitors of actin filaments
(cytochalasin D) and myosin ATPase (butanedione
monoxime) disrupted all three forms of motility.
When applied on intracellular parasites, these
inhibitors also interfere with proper parasite division.
To determine whether one of these myosins functions
as a motor in actin-based gliding motility and
plays a role in cell replication, we have examined
their stage-specific pattern of expression, subcellular
distribution and biochemical properties.
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