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Journal of Biomedical Science (2007) 14:505–508 DOI 10.1007/s11373-007-9166-2

Toc GTPases Hsou-min Li*, Muppuru M. Kesavulu, Pai-Hsiang Su, Yi-Hung Yeh & Chwan-Deng Hsiao* Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, 115, ROC Received 31 January 2007; accepted 8 March 2007 Ó 2007 National Science Council, Taipei

Key words: chloroplast, GTPase, GAP, GEF, protein translocon, Tic, Toc

Abstract Chloroplasts import more than 90% of their protein constituents from the cytosol. The import is mediated by translocon complexes located in the chloroplast envelope and the stroma. This review focuses on the two GTPases in the Toc (translocon at the outer envelope membrane of chloroplasts) complex. Hypotheses are presented about gating across the outer membrane and the possible functional states of the GTPases.

Introduction Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol as precursors with N-terminal targeting signals called transit peptides. The machinery for importing these precursor proteins consists of components from the outer membrane (the Toc proteins), the inner membranes (the Tic proteins, or translocon at the inner envelope membrane of chloroplasts) and several chaperone and co-chaperone proteins in the stroma [1–3]. Three Toc proteins, Toc159, Toc75 and Toc34, form the core of the Toc complex [4]. Toc75 most likely forms the major part of the protein-conducting channel across the outer membrane [5]. Toc34 has a cytosolically-exposed GTPase domain (G domain) followed by a short membrane anchor. Toc159 has an acidic N-terminal domain, followed by a GTPase domain homologous to the Toc34 G domain, and a membrane-protected C-terminal domain. Non-hydrolyzable GTP ana*To whom correspondence should be addressed. Hsou-min Li Fax: +886-2-2782-6085; E-mail: [email protected], Chwan-Deng Hsiao; Fax: +886-2-2782-6085; E-mail: hsiao@ gate.sinica.edu.tw This article is for the Special Issue of IMB 20th anniversary.

logues severely inhibit precursor binding to chloroplasts [6, 7], and evidence indicates that GTP hydrolysis by both Toc GTPases are required for stable precursor binding to chloroplasts [6–9]. However, the exact functions of these two GTPase are still in dispute. At least two models have been proposed. In the ‘‘targeting model’’, Toc159 is proposed to function like the signal recognition particle (SRP) in protein import into the endoplasmic reticulum, by bringing in precursors from the cytosol to chloroplasts. Toc34, similar to the SRP receptor (SR), functions as the docking site for Toc159. In the ‘‘motor’’ model, Toc34 is the initial receptor for precursors on the chloroplast surface. Precursors are then transferred to Toc159 and pushed through the Toc75 channel by conformational changes in Toc159 resulted from GTP hydrolysis [9–11].

Toc34 structure and GTPase activity Crystal structure analysis of pea Toc34 G domain revealed that motifs and residues involved in GTP binding and hydrolysis are uniquely arranged and differs in sequences from the Ras class of GTPases [12]. From crystal structure it was also found the G domain crystallizes as dimers in which

506 residues from one monomer interacts with residues in the GTP binding pocket of the other monomer. One monomer also provides the catalytic arginine finger to the other monomer [12]. Hence, it was proposed that within the Toc34 dimer, each monomer functions as the other monomerÕs GTPase activation protein (GAP). This proposal was cast into doubt by work of Weibel et al. [13] who showed that when the residue that is supposed to form the arginine finger in the major Arabidopsis Toc34 homologue, atToc33, was mutated into alanine, the mutation did not have an effect in atToc33 GTP hydrolysis and only reduced the ability of atToc33 to form dimers. However, our recent results show that, in freshly prepared atToc33, dimer formation correlates positively with GTPase activity (unpublished results).

Is there a GTPase gate? The chloroplast outer membrane is porous and can allow diffusion of molecules as large as 7 to 13 kD [14]. Therefore it does not seem necessary to have the Toc75 channel gated. Indeed when Toc75 was reconstituted into planar lipid bilayer, electrophysiological studies indicated that the Toc75 stays in an open state in the absence of a membrane potential, which is likely to be the state of the outer membrane considering its porous nature. However, conformational changes of Toc34 and Toc159 coupled with opening and closing of the Toc75 channel, or plugging of Toc75 by Toc159, is nevertheless depicted in various models [1, 2, 10, 15]. Here we consider three hypotheses concerning the gating of Toc75 by Toc34 or Toc159. 1. The inner membrane channel may be tightly linked to Toc75 and therefore Toc75 has to be sealed to prevent leakage of the stroma content. Large conformational changes in Toc34 or Toc159 may shield and un-shield the Toc75 channel. This large conformational change can result from monomer–dimer transition and/or through shifting of the hinge region between the G domain and the membrane anchor. 2. Toc75 shows channel closing when there is a membrane potential greater than ±75 mV across the lipid bilayer [5]. It is possible that

there still could be a membrane potential across the outer membrane, established through some yet unknown mechanism. Toc75 can be self-gated through its own conformational changes in response to changes in the membrane potential. 3. There is no gating across the outer membrane. Toc75 is always open and Toc159 and Toc34 only regulate the binding and release of precursors and do not plug the Toc75 pore. The inner-membrane seal can be maintained by gating of the Tic channel. The opening of the Tic channel can be induced by transit peptides emerging from the Toc75 channel. Or it can be gated by other translocon components located in the intermembrane space or in the stroma.

Homodimer or heterodimer? GTP or GDP? Toc159 and Toc34 have homologous G domains. Drawing analogy from the SRP-SR interaction, it is plausible to envision that Toc159 and Toc34 may form heterodimers. Indeed, many data, using in-vitro-translated radioactive amounts of Arabidopsis Toc159 (atToc159) and recombinant lg amounts of atToc33 (or vice versa), support that atToc33 may function as the docking site for atToc159 [11, 13, 16–18]. However, we have not been able to observe heterodimers of Toc34Toc159 when the G domains of the two proteins were mixed in equal molar amount in vitro, or when chloroplast membranes were solubilized with detergents (unpublished results). On the other hand, homodimers of Toc34 have been observed in vivo (unpublished results) and in vitro [12, 13], and pea Toc159 G-domain homodimers have also been observed in vitro (unpublished results). These data at least suggest that homodimers of the Toc GTPases is a stable structure. Heterodimers may be a more transient state in vivo. It is not clear what the nucleotide states of the Toc GTPases are when they interact with precursors in vivo. In vitro, atToc159 binds to precursors equally well when it is bound with GTP, GDP or GMP-PNP [19]. Toc34, on the other hand, seems to bind transit peptides in its GTP state [20, 21]. So far no experimental data are available to indicate which component might

507

Figure 1. Hypothetical model for dimerization and nucleotide states of Toc159 and Toc34 during precursor binding and translocation across the outer envelope membrane. A, G, and M indicate the various domains of Toc159 and Toc34. D/D and T/T stand for the nucleotide states of Toc159 and Toc34 homodimers. See text for details.

function as the GTP/GDP exchange factor (GEF) for the two Toc GTPases. Interestingly the GTPase activity of Toc34 could be stimulated up to 30-folds by incubation with precursors [22, 23], and the GTPase activity of proteoliposomes containing reconstituted Toc complex could be stimulated up to 100-folds by precursors [9]. This kind of large stimulation is usually brought about by the presence of both GAP and GEF. Since each monomer in the Toc34 dimer functions as the

other monomerÕs GAP, we hypothesize that precursor binding could be a possible trigger for nucleotide exchange. Based on these discussions, we propose another model modified from both the ‘‘motor’’ and the ‘‘targeting’’ hypotheses (Figure 1). We hypothesize that there are stable binding sites for precursors in Toc159 bound with GDP, the resting homodimeric state of Toc159. Precursor induces the exchange of GDP to GTP on Toc159, which in turn induces

508 the GDP to GTP exchange in the Toc34-GDP homodimer. This may be effected through interactions between Toc159-Toc34 or even among Toc159-Toc75-Toc34. The two Toc GTPases then interact in their GTP state (heterodimer). Toc34GTP may interact with precursors at this stage. The two Toc GTPases stimulate each otherÕs GTP hydrolysis. The energy of GTP hydrolysis is used to dissociate precursors from the Toc GTPases and transfer precursors to a stable binding site on the trans side of Toc75 or to another binding protein in the intermembrane space. If GTP hydrolysis is prevented by non-hydrolyzable analogues, precursor may only dissociate but fail to be translocated across Toc75. Clearly more experiments are needed to understand the interactions among the Toc components and their interactions with precursors. Other possibilities, like whether the membrane anchors or even the G domains are actually required for oligomerization of the Toc complex, or required for correct folding of Toc75, also need to be considered. The model depicting the mechanism of SR-SRP interaction is still being modified to this date. Our model is only meant to provide a hypothesis and serve as foundation for future modifications.

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