GTPases become molecular switches to control many cellular processes including signalling protein translation and targeting. functions of which as well as the Volasertib elements regulating this event are largely unknown. In the present study we show that homodimerization reduces the rate of nucleotide exchange which Volasertib is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously but to control the nucleotide-loading state. We discuss this novel regulatory ATN1 mode and its impact on the current models of protein import into the chloroplast. Toc33) and its orthologue Toc34) revealed a homodimeric conversation [10 11 Indeed homodimerization of Toc33 at the chloroplast membrane was demonstrated by copper-induced cross-linking [12]. Moreover mutations reducing the affinity of homodimerization affect the import efficiency in plants [13]. This suggests that Toc33 is usually a GTPase regulated by dimerization [14]. The structural observations led to the proposal that GTP-regulated dimerization constitutes an element of the GTPase regulation within the translocon. Alternatively it was proposed that this Toc33 homodimer mimics a heterodimeric GTPase complex between Toc33 and the highly homologous G domain name of Toc159 a complex thought to be vital for translocation. Thus the precise role of the Toc33 dimerization in the translocation reaction has not yet been defined. One potential role of dimerization is usually to provide a reciprocal GAP function either in the homo- or the hetero-dimeric context. This hypothesis was based on the geometry of the active site as a conserved arginine residue from one protomer was inserted into the active site of the other at a position near the [16] (see below). Furthermore a stable conversation between Toc33 and a precursor protein in the context of the TOC complex could be detected by label-transfer experiments only in the presence of GDP but not GTP [18]. In turn reports have described an influence of the precursor protein on GTP hydrolysis of Toc33 [17 19 an observation that cannot be reconciled directly with the GDP dependence of the label transfer. This prompted us to investigate a possible physiological function of Toc33 homodimers with respect to nucleotide exchange and precursor-protein recognition. For the biochemical analyses in the present study Toc33 was expressed in as a truncated version (pSSU (precursor of the small subunit of Rubisco where Rubisco is usually ribulose-1 5 carboxylase/oxygenase)] MVAPFTGLKSAA-S(PO3)-FPVSRKQNLDITSC [20]; and C (control peptide) SKS-MTEIEVTDVDMPC. Analytical ultracentrifugation of Information Resource; http://www.arabidopsis.org) accession number AT1G02280; (see above). Heterologously produced grey circles) the rates were reduced by increasing protein concentrations consistent with the results obtained for -imido] triphosphate a non-hydrolysable GTP analogue)-bound forms [5]. In addition our present (Physique 1) and previous [19] results indicate that pre-protein binding modulates receptor GTPase activity by stimulating nucleotide exchange. This prompted us to analyse the influence of peptides representing the pSSU transit sequence on dimerization and nucleotide exchange by and [12 13 In contrast with GADs described so far [14] homodimerization had not been found to become GTP-dependent and will not straight donate to GTP hydrolysis (for instance discover [10 17 increasing the question where functional system homodimerization plays a part in the legislation of proteins translocation. Our present outcomes reveal that dimerization decelerates the speed of GTP binding (Body 2) and GDP discharge (Body 3) by recording the nucleotide within a cage on the dimer user interface. Regarding monomeric Ras-like GTPases an analogous function Volasertib is certainly satisfied by GDIs (GDP-dissociation inhibitors; for instance discover [26 28 29 that have to become replaced with Volasertib a GDF (GDI-displacement aspect; for example discover [30 31 ahead of nucleotide exchange. Hence a function equivalent with this of the GDF is certainly achieved by the receptor substrate the transit peptide from the pre-protein. The peptide alters the kinetic properties of homodimerization as well as the affinity of the response outcomes in an improved GDP discharge (Body 3). The transit peptide will not induce the Volasertib discharge from the destined GDP through the monomeric receptor straight and.