An interesting question is why RhoF has such the

An interesting question is why RhoF has such the slow rate of GDP dissociation. The amino mek inhibitors mutations in a presumable GDP-binding site of RhoF had only moderate effects on the GDP dissociation (Fig. 3C), which suggests a mechanism involving the global amino acid sequence of RhoF, rather than the local amino acid sequence surrounding the GDP-binding site. Our structural analysis using NMR and DLS clearly showed that RhoF adopts a unique multimeric structure, which has not been reported in other RAS superfamily GTPases. This multimeric structure might stabilize the GDP-binding of RhoF by (1) increasing intersubunit interactions between GDP and other RhoF subunits or by (2) decreasing solvent accessibility to bound GDP. It should be emphasis that the multimeric RhoF has a normal GTPase activity (Fig. 2) and a secondary structure very similar to K-ras (Fig. 1C), ruling out the possibility of protein misfolding. The unique N-terminal 16-residues extension of RhoF might contribute to the assembly of the multimeric structure [3], which should be further investigated in future studies. The multimeric structure of RhoF can switch between different conformations depending on the GTP/GDP-bound state (Fig. 4E). This finding is of biological significance, because it provides the first direct evidence for RhoF to function as a molecular switch. Consistent with the typical RAS superfamily GTPases [1], the global folding of RhoF was maintained during the switching, but only a local region undergoes a structural rearrangement, as represented by the peak shifts in the NMR spectra (Fig. 4A). This indicates that RhoF has the classical Ras-like switching mechanism by assembling into a multimeric structure.
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Competing financial interests
Acknowledgments This study was supported by a grant from the Yasuda Medical Foundation (to R.H.).
Introduction Protein translocation across membranes is a highly regulated process [[1], [2], [3], [4]]. Proteinaceous, oligomeric complexes in the cytoplasm or residing in the target-membranes catalyze transport to, perception at and translocation across the membrane. In general, the regulation of these complexes is linked to the intracellular signaling and metabolite state sensing. Moreover, many complexes involved in these translocation events are driven by ATPases and regulated by GTPases [[4], [5], [6]]. The translocon of the outer envelope membrane of chloroplasts (TOC) enables the import of post-translationally targeted proteins into chloroplasts [3,4]. The majority of these nucleus-encoded proteins is synthesized as precursor proteins (preproteins) and contain an N-terminal transit peptide thought to serve at least in parts as targeting sequence [7,8]. The targeting sequence is recognized by proteins, such as chaperones, residing in the cytosol, the intermembrane space (IMS) and the stroma of chloroplasts [9]. Currently it is discussed that chaperones of the Hsp70, Hsp90 and Hsp100 family guide preproteins to their subcellular destination and constitute the energy providing units for the translocation process [[9], [10], [11], [12], [13]]. Despite the chaperones, and maybe 14–3-3 proteins [14], cytosolic factors for proteins with N-terminal transit peptides are yet unknown. Moreover, the transit peptides of several preproteins are phosphorylated by cytoplasmic kinases [15], which have been discussed to be regulatory for recognition by targeting and transport factors [e.g. 4]. At the membrane, the transit peptide is recognized by receptors of the TOC, namely Toc34 and Toc159 [[16], [17], [18], [19], [20]]. Recently, a possible transit peptide binding site of Toc159 was described [21], but the exact mechanism and binding sites remain to be confirmed. The preproteins are translocated across the outer envelope membrane through the β-barrel pore forming subunit Toc75 [22,23] and interact with subunits of the translocon of the inner envelope membrane of chloroplasts (TIC) for guidance to their final sub-organellar destination [24,25].