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.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Ludwig-Maximilians-Universität

Year

2001

Author dc:creator

• Gloss, Annika

Contributors dc:contributor

• Siegert, Florian

Repository record source_url

https://edoc.ub.uni-muenchen.de/37/

OAI identifier oai:identifier

oai:edoc.ub.uni-muenchen.de:37