Charakterisierung des Zytoskelett-Proteins Villidin und einer dritten Profilin-Isoform in Dictyostelium discoideum

A large number of actin-binding proteins regulates the dynamics of
the actin cytoskeleton. Here we report the identification and
characterization of two new proteins in Dictyostelium discoideum:
the novel cytoskeleton protein villidin and a third profilin
isoform. One goal of the project was to analyze the features of the
villidin sequence in detail and to investigate the protein by
molecular, biochemical and cell biological approaches. Villidin has
a calculated molecular mass of 190,000 Da. Based on the domain
structure of the protein, villidin can be assigned to the
gelsolin/villin-family as well as to the WD-repeat family. In
principal, the group of the WD-repeat proteins includes regulatory
proteins which are involved in signal transduction and other
important cellular processes. The N-terminus of villidin harbours
between four and eight of the specific WD-repeats and forms
probably a β-propeller structure. The WD-domain is followed by an
intervening domain of 400 amino acids that leads to the second
characteristic villidin domain at the C-terminus. This part of the
sequence exhibits a similarity to villin, though the first of the
six villin domains is absent in villidin. However the typical
headpiece is present. Villidin mRNA and protein are expressed in
low amounts during growth and early aggregation, they are increased
during development and reach highest levels at the tipped aggregate
stage. The protein is present in the cytosol as well as in the
cytoskeletal and the membrane fraction. These biochemical results
are in agreement with the immunofluorescence data. The endogenous
villidin is homogeneously distributed throughout the cytosol and
localizes at vesicular structures. Colocalization experiments lead
to the assumption that these structures might belong to a still
unknown population of vesicles. GFP fusion proteins with the villin
homology domains show a similar distribution, whereas GFP fusions
of the N-terminal part encompassing the WD-repeats are present in
F-actin rich regions at the plasma membrane and on internal
membranes during motility, pinocytosis and phagocytosis. Mutants
lacking villidin do not show an aberrant phenotype during growth
and development but are defective in motility and phototaxis in the
multicellular slug stage. Based on this defect and the multidomain
structure of the protein, villidin might be involved in signal
transduction processes leading to phototactic movement. The second
part of the project dealt with a new gene which codes for a third
profilin isoform in D. discoideum. The well-known Dictyostelium
profilin isoforms I and II are able to interact with G-actin, PIP2
and poly-L-prolin. As in the case of profilin I and II, profilin
III is encoded by a single gene. In contrast to profilin I and II,
the transcription of profilin III is not developmentally regulated.
All three isoforms show the typical limited homology at the amino
acid level. The recombinant Profilin III protein cosediments with
poly-L-prolin, inhibits the actin-polymerisation and the PIP2
interaction of profilin III competes with the G-actin affinity. The
low expression of Profilin III mRNA in growing and developing cells
suggests a distinct role of profilin III because a low protein
concentration argues against an actin sequestering function.