Characterization of the Arabidopsis forminlike protein AFH1 and its interacting protein

A partial cDNA encoding an Arabidopsis thaliana FH (Formin Homology) protein (AFH1) was used as a probe to clone a full length AFH1 cDNA. The deduced protein encoded by the cDNA contains a FH1 domain rich in proline residues and a C-terminal FH2 domain which is highly conserved amongst FH proteins. In contrast to FH proteins of other organisms, the predicted AFH1 protein also contains a putative signal peptide and a transmem-brane domain suggesting its association with membrane. Cell fractionation by differential centrifugation demon-strated the presence of AFH1 in the Triton X-100 insoluble microsomal fraction. An Arabidopsis cDNA library was screened to identify proteins that interact with the C-ter-minal region of AFH1 using yeast two-hybrid assays, and one of the isolated cDNAs encoded a novel protein, FIP2. Experiments using recombinant proteins expressed in E. coli demonstrated that FIP2 interacted directly with AFH1. The amino acid sequence of FIP2 has partial homology to bacterial putative membrane proteins and animal A-type K+ ATPases. AFH1 may form a membrane anchored complex with FIP2, which might be involved in the organization of the actin cytoskeleton. Key words: Actin — Cytoskeleton — FH protein — Membrane protein. The final shape of plant organs is a consequence of cell division and cell expansion. In meristematic tissues, cell division is followed by cell expansion, which determines the shape of individual cells. During cell division, the cytoskeleton is responsible for segregating the chromo-somes and for positioning of the new cell plate (reviewed by Staehelin et al. 1996). The plant cytoskeleton contains microtubules as well as actin microfilaments. Following cell division, longitudinal growth or elongation is associated with the transverse arrangement of cortical microtubules, which are thought to direct the orientation of cellulose Abbreviations: EGTA, ethylene glycol-bis(/?-aminoetyl ether)-N,N,N,N-tetraacetic acid; GST, glutathione-5-transferase; HRP, horse radish peroxidase; IPTG, isopropylthio-/?-D-thiogalacto