Domänenanalyse des Cyclase-assoziierten Proteins (CAP) und Charakterisierung von Filactin, einem neuartigen actinähnlichen Protein aus Dictyostelium discoideum

The cyclase-associated protein (CAP) from Dictyostelium discoideum
was studied in detail regarding its structure and function
relationships. The second part of the thesis describes the
characterization of the novel actin-related protein filactin from
D. discoideum. CAP homologs are multifunctional proteins: they are
involved in signal dependent changes in the actin cytoskeleton, in
vesicle transport and cell development. The binding of monomeric
actin through the C-terminal domain represents a common feature of
all CAPs. From four highly conserved regions in this domain the
verprolin homology region of the D. discoideum CAP was analyzed in
this work. Loss of this region led to a clear decrease, but not
suppression of the actin-sequestering activity. In agreement to
these data, stable complexes of the modified CAP-C with G-actin
could be identified in chemical crosslinking experiments, and it
could be shown that CAP-C is able to dimerize. Considering the high
conservation of cyclase-associated proteins and their importance
for cell biological processes it is remarkable that no structure of
this protein has been elucidated so far. Therefore, in the frame of
this thesis the structure of the membrane-associated N-terminal
domain of D. discoideum CAP was to be determined in cooperation
with the group of T. Holak at the MPI f. Biochemistry
(Martinsried). For this purpose numerous constructs from the
N-terminal domain had to be cloned and expressed, to be purified
and examined for their stability and threedimensional folding. It
turned out that the stable core of the aminoterminal domain covers
the amino acids 51-226. The structure was determined by nuclear
magnetic resonance spectroscopy and X-ray crystallography. Six
antiparallel a-helices are connected by loop elements and form a
cylindrical core domain which can form a dimer in the crystal,
dimerization occurs through Mg2+ ions. The clarification of the
N-terminal structure of D. discoideum CAP will now simplify the
research on its interaction with phospholipids and CAP binding
proteins The second part of this work dealt with the
characterization of filactin. The unique 105 kDa protein contains
two filamin homologous regions in its N-terminal, and a clearly
actin-related domain within the C-terminal part. While in resting
cells the endogenous filactin shows cytoplasmic distribution and
interaction with protein aggregates as well, the GFP construct of
the actin-related domain displays an actin-like behavior during
cell movement or phagocytosis. A stimulus-induced colocalization of
actin and filactin was observed in experiments with chemotactically
stimulated cells. The alignment of its C-terminal amino acid
sequence with the structure of muscle actin predicts a globular,
actin-related structure containing all residues that are important
for ATP/ADP binding.