In the Toc Regulator Mode Fig recognition of
In the “Toc34 Regulator Mode” (Fig. 6), recognition of the transit peptide by Toc159 monomer (step 4) leads to heterodimer-formation . Subsequently, preprotein interaction with the Toc34G homodimer (step 1) regulates the Toc34G homodimer (step 2, 3). However, this mode requires a parallel induction of Toc34G-GTP hydrolysis by preprotein recognition (Fig. 3) and Toc159G-Toc34G complex formation.
Irrespective of the mode, our model considers the formation of a heterodimeric state of both receptors loaded in the GTP state (Fig. 6; step 5; ). In the absence of a clear biochemical evidence, we propose that the heterodimeric complex interacts with an additional TOC component to initiate the translocation (step 6). This notion is in line with the interaction of the preprotein with Toc75 at an early translocation state as shown by chemical crosslinking .
Next, the transit peptide is transferred by GTP hydrolysis. Although we cannot exclude that hydrolysis of Toc34 precedes the one of Toc159 (Fig. 6; transparent path), the opposite order is more likely for the following reasons: (i) Preprotein recognition by Toc159 G-domain induces GTP hydrolysis (Fig. 3). (ii) GTP hydrolysis of Toc34 monomer occurs without initial activation due to the low Vincristine sulfate sale barrier (Table 2). (iii) The GDP loaded Toc159 shows a very low affinity for GTP loaded Toc34 . (iv) Toc34 recognizes the C-terminal region of the transit peptide with higher affinity, while Toc159 recognizes the N-terminus of the transit peptide with higher affinity (Table 1; ). Considering that the N-terminus needs to be translocated first, an early release from Toc159 would allow the initiation of translocation.
Thus, in the absence of alternative evidence we favor that Toc159 is hydrolyzing GTP (Fig. 6, step 7) leading to its dissociation at least from the transit peptide and Toc34G (step 8; ). Finally, the N-terminal segment of the transit peptide likely enters the pore formed by Toc75  and dissociates from Toc34 (step 11). It is likely that Toc34 hydrolyses GTP, either during its association with the other TOC unit (step 10, step 11) or after dissociation (step 11, step 10). Nevertheless, hydrolyses of GTP bound to Toc34 is likely the mechanism to reach the ground state because the dissociation rate for GTP is rather low (Table 4).
To reach the ground state, the GDP loaded Toc34 G-domains homodimerize (Fig. 6; step 12) and Toc159 undergoes nucleotide exchange. The latter is consistent with the high GDP dissociation and GTP association rate of Toc159G (Table 4) as well as with the low dissociation constant for GTP (Table 4).
Thus, our biochemical analysis allows the formulation of a hypothesis concerning the action of the GTPases within the TOC complex. We provide evidence that the heterodimer of the G-domains in the GTP state is likely the transition point between events during preprotein recognition and preprotein translocation. Moreover, while GDP-loaded Toc34 likely represents the ground state before preprotein recognition, Toc159 is GTP loaded in its ground state. In future, this hypothesis needs to be challenged by e.g. incorporation of additional domains of the TOC complex to fully reconstitute the molecular events during recognition and translocation.
Introduction Small GTPase family is a GTP-binding protein family which can be commonly found in eukaryotic cells . It is a kind of GTPases can achieve mutual transformation between GTP and GDP in cytoplasm . These small GTPases act as molecular switches in cells, affecting almost all cellular processes . The most prominent member of small GTPase family is the Ras GTPase, thus the family is also called the Ras superfamily , .
Structure and action mechanism of small GTPases
Function of small GTPase and its role in nano delivery system Small GTPase can regulate various vital movements of cells, including cell growth, differentiation, cell movement, lipid vesicle trafficking, etc. , . In term of function, Ras subfamily regulates the gene expression; Rho subfamily regulates cytoskeleton reorganization, cell wall synthesis, cell cycle progression, and MAP kinase signal transduction; Rab subfamily and Sar/Arf subfamily regulate the vesicle trafficking and clathrin formation; Ran subfamily regulates karyoplasm transport, microtubule formation, formation of mitotic spindle apparatus and the assembly of karyotheca after cell division. At present, more and more researches suggest the important role of small GTPase in the nanoparticle transport , , , .