Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • Toc and Toc are GTPases that regulate

    2022-07-18

    Toc34 and Toc159 are GTPases that regulate initial steps of preprotein import [3,4,26]. It was shown that GTP hydrolysis of at least one receptor is necessary to initiate the translocation process [23,27]. As reported for other small G-proteins, Toc34 and Toc159 bind their cargo in a nucleotide-dependent manner [[17], [18], [19],23,28]. It is discussed that the nucleotide loading state of the two receptors regulates their ability to form dimers [18,20,29,30], and that they belong to the family of GTPases activated by nucleotide-dependent dimerization (GAD, [20,31]). Surprisingly, the reciprocal activation of Toc34 forming a homodimer could not be proven [20,30,32]. Although an arginine residue of each protomer reaches into the nucleotide binding pocket in trans [30,33,34], it does not function as a catalytic arginine finger known from regulatory GTPase activating proteins (GAPs [35]). It rather seems to be important for stabilization of the homo- or heterodimer as its substitution by an alanine residue keeps Toc34 in a monomeric state [32,33,[36], [37], [38]]. Contrarily, the Toc34 homodimer seems to constitute a GTPase, which is inhibited by dimerization (GID) as nucleotide exchange is significantly reduced [39]. Both GTPases are likely to interact with incoming preproteins as inferred by in vivo crosslink experiments [40], whereby in addition an interplay of both receptors was suggested. The interaction between Toc34 and Toc159 was shown to be important for the insertion of Toc159 into the outer envelope membrane of Amrubicin [18,41]. Pull-down and solid phase binding assays demonstrated that heterologously synthesized G-domains of Toc34 and Toc159 interact in vitro [18,36,[41], [42], [43], [44]]. Their interaction has also been demonstrated in native membranes [20] as well as in vivo [45]. Further, both GTPases have a low intrinsic GTP hydrolysis rate [20,32]. A reciprocal activation of Toc34 and Toc159 was assumed, which might constitute the missing step of the GTP-dependent regulation of preprotein import [30]. The role of the Toc34-Toc159 heterodimer for protein translocation is under debate. Since both GTPases share a high sequence similarity [[32], [33], [34]], previous research mainly focused on the characterization of the homodimer of the G-domain of Toc34. Recently, the homodimer of the G-domain of Toc159 was studied as well [21]. We provide a biochemical dataset describing the special characteristics of the G-domain of Toc159. We demonstrate that both GTPases are regulated differently by their nucleotide loading state. We validate previously described qualitative nucleotide and transit peptide binding data [18,46] by quantitative assays leading to a description of the putative regulatory mode of Toc159. Further, the decreased GTP uptake and transit peptide binding ability of the Toc34 homodimer confirms the hypothesis of Toc34 being a GID. Catalytic rates suggest that the interaction of Toc34 and Toc159 is not sufficient to accelerate their GTPase activity. We discuss a model of the GTPase cycle, which indicates the distinct function of the individual receptors during the initial step of preprotein recognition and explain the necessity of two regulatory GTPases within the TOC.
    Materials and methods
    Results
    Discussion The two G-domain-containing TOC components share a high sequence similarity [53] but the stoichiometry of the components in the TOC complex with Toc34 being more abundant [54] and the more complex domain structure of Toc159 [3,4] suggest distinct properties. In addition, Toc34 homodimerization has been demonstrated in vitro and in isolated outer envelope membrane [20,30,32,33,36], while homodimerization of Toc159 in vivo was not yet confirmed. Moreover, the G-domain of Toc159 does not homodimerize under the conditions used, while heterodimers between the two G-domains of Toc34 and Toc159 are formed with a sufficient dissociation constant (Fig. 1). Even addition of crosslinking reagents to high concentrations of purified Toc159G does not stabilize a homodimeric conformation in a comparable manner to Toc34G, which is in line with the stochiometric models proposed (Fig. 2; [21]). Nevertheless, both proteins are discussed as receptors for the cytosolically synthesized transit peptide containing preproteins at the surface of chloroplasts [4].