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    • We observed that the Toc G domain recognizes the N

    2022-01-24

    We observed that the Toc159G domain recognizes the N-terminal portion of the transit peptide of pSSU with a dissociation constant of 70 μM (Fig. 2; Table 1), while a tenfold higher KD was obtained for the phosphorylated C-terminal region. In turn, the homodimer of Toc34G recognizes the phosphorylated C-terminal portion with a KD of 150 μM, while the dissociation constant for the N-terminus is four fold higher (Fig. 2; Table 1). The values for the dissociation constants of Toc34G/CTPP [16], Toc159G/NTP and Toc34G-Toc34G/CTPP complexes are in the same range. However, the Toc34G homodimer and the Toc34G-Toc159G heterodimer show the highest crosslinking efficiency to the full-length transit peptide (Fig. 2). This is in line with the observed enhanced heterodimer formation in the presence of pSSU [18]. The importance of Toc34 and Toc159 for protein translocation is established, but the impact of their GTPase function is not fully understood, especially as they show distinct GTPase properties. The Toc34G monomer shows a high autocatalytic GTPase hydrolysis with a low activation energy, which is enhanced by dimerization (Fig. 3; Table 2; [20,39]). The Toc159G monomer shows a high activation energy (Table 2). Initial GTP turnover experiments show an activation of the GTPase activity of Toc159G by addition of the transit peptide of pSSU (Fig. 3; [18]), which might hint at a reduction of the activation energy. Further, Toc34 and Toc159 are distinct with respect to their nucleotide affinity. Toc34G has the highest affinity for GDP and Toc159 for GTP based on the dissociation constants and the nucleotide Ferrostatin-1 determined by crosslinking (Fig. 4; Table 3). The selectivity of Toc159G for GTP is defined by lower dissociation rate of GTP than for GDP, while the association rate is comparable (Fig. 5; Table 4). Note, the high dissociation rate for GDP suggests that a GEF is not required for nucleotide exchange. In turn, Toc34G has a general faster association and dissociation of GDP than GTP, which in turn explains the more comparable dissociation constant. Homodimerization of Toc34G particularly affects the association rate of GTP, which is reduced by at least one order of magnitude (Fig. 5). This is in line with the reduced binding efficiency of the homodimer to radioactively labelled GTP (Fig. 4). The heterodimer shows an intermediate behavior between the Toc34G and Toc159G monomer (Fig. 4; Table 3), while GTP binding appears to be distinct between Toc34G and Toc159G. Here, crosslinking of radioactive GTP to Toc159G appears not to be affected in the heterodimer, while the GTP crosslinking to Toc34G appears to be reduced in comparison to the respective monomer (Fig. 4). This suggests that the GTP binding pocket of Toc159 is not blocked by Toc34. This latter is in line with the observed GDP dissociation constant of the heterodimer, which is comparable to that of Toc159G and the observation that heterodimerization manipulates the association and dissociation of GDP in general (Fig. 5; Table 3). As a working hypothesis, we formulated a model of the interactions during translocation (Fig. 6). Most of the events are independent of the question whether Toc34 (“Toc34 Receptor Mode”) or Toc159 (“Toc34 Regulator Mode”) is the initial receptor, especially as the lowest dissociation constant for transit peptide interaction of 150 μM and 70 μM (Table 1), respectively, is rather comparable. We assume that Toc34 forms a homodimer in the GDP state (Fig. 3; Table 3, Table 4; [20,39]) before transit peptide recognition, which is consistent with the higher affinity of homodimerization of Toc34 in the GDP state [20,30]. Toc159 is initially in the GTP bound state stabilized by the high activation energy for GTP hydrolysis and the lower dissociation rate of GTP when compared to GDP (Fig. 3; Table 1, Table 2, Table 3, Table 4). According to the “Toc34 Receptor Mode “(Fig. 6), recognition of the transit peptide by Toc34 homodimer (step 1) leads to dissociation of the dimer (step 2) and nucleotide exchange (step 3; Table 4; [20,39]). As the two receptors Toc159 and Toc34 recognize distinct regions of the transit peptide (Fig. 2; Table 1), Toc159 can recognize the transit peptide while it is bound to Toc34 (Fig. 6; step 4; [18]).