Efficient interaction between two GTPases allows the chloroplast SRP pathway to bypass the requirement for an SRP RNA - PubMed (original) (raw)

Efficient interaction between two GTPases allows the chloroplast SRP pathway to bypass the requirement for an SRP RNA

Peera Jaru-Ampornpan et al. Mol Biol Cell. 2007 Jul.

Abstract

Cotranslational protein targeting to membranes is regulated by two GTPases in the signal recognition particle (SRP) and the SRP receptor; association between the two GTPases is slow and is accelerated 400-fold by the SRP RNA. Intriguingly, the otherwise universally conserved SRP RNA is missing in a novel chloroplast SRP pathway. We found that even in the absence of an SRP RNA, the chloroplast SRP and receptor GTPases can interact efficiently with one another; the kinetics of interaction between the chloroplast GTPases is 400-fold faster than their bacterial homologues, and matches the rate at which the bacterial SRP and receptor interact with the help of SRP RNA. Biochemical analyses further suggest that the chloroplast SRP receptor is pre-organized in a conformation that allows optimal interaction with its binding partner, so that conformational changes during complex formation are minimized. Our results highlight intriguing differences between the classical and chloroplast SRP and SRP receptor GTPases, and help explain how the chloroplast SRP pathway can mediate efficient targeting of proteins to the thylakoid membrane in the absence of the SRP RNA, which plays an indispensable role in all the other SRP pathways.

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Figures

Figure 1.

Figure 1.

GTPase cycles of cpSRP54 (blue) and cpFtsY (green). The triangular cycles on the top left and right depict the basal GTPase cycles of cpSRP54 and cpFtsY, respectively. Binding of GTP to cpSRP54 (or cpFtsY) is characterized by the association rate constant _k_1 (or _k_1′), dissociation rate constant _k_−1 (or _k_−1′), and equilibrium dissociation constant _K_1 (or _K_1′). GTP hydrolyzes from cpSRP54 and cpFtsY with rate constants of _k_2 and _k_2′, respectively. Binding of GDP to cpSRP54 (or cpFtsY) is characterized by the association rate constant _k_3 (or _k_3′), dissociation rate constant _k_−3 (or _k_−3′), and equilibrium dissociation constant _K_3 (or _K_3′). Complex formation between cpSRP54 and cpFtsY is characterized by the association rate constant _k_4 and dissociation rate constant _k_−4. The two bound GTPs are hydrolyzed from the GTP·cpSRP54•cpFtsY·GTP complex, represented collectively by the rate constant _k_5. The GDP·cpSRP54•cpFtsY·GDP complex then dissociates with a rate constant _k_6.

Figure 2.

Figure 2.

Basal GTPase reactions of cpSRP54 (A) and cpFtsY (B). The data were fit to Eq. 1 in Materials and Methods and gave a _k_max of 0.017 min−1 and _K_1/2 of 2.8 μM for cpSRP54, and a _k_max of 0.0045 min−1 and _K_1/2 of 2.1 μM for cpFtsY.

Figure 3.

Figure 3.

Interaction of nucleotides with cpSRP54 and cpFtsY. (A and B) Fluorescence emission spectra of mant-GTP (A) or mant-GDP (B) in the absence of protein (○) and in the presence of 5 μM cpSRP54 (•) or cpFtsY(♦). (C and D) Titration of the fluorescence changes of mant-GTP (C) and mant-GDP (D) in the presence of cpSRP54 (■) or cpFtsY (•). The data were fit to Eq. 5 in Materials and Methods, and the _K_d values are summarized in Table 2. (E and F) Dissociation of mant-GTP (E) and mant-GDP (F) from cpFtsY. The data were fit to single exponential rate equations and gave dissociation rate constants of 5.4 and 8.1 s−1 for mant-GTP and mant-GDP, respectively.

Figure 4.

Figure 4.

Interaction of cpSRP54 and cpFtsY is much more efficient than that of their E. coli homologues. Rates of the stimulated GTPase reaction were determined for 100 nM cpSRP54 and cpFtsY (•) or for 100 nM E. coli Ffh and E. coli FtsY with (▾) and without (▴) 4.5S SRP RNA. The data were fit to Eq. 2 in Materials and Methods and gave a _k_cat value of 50 min−1 and a _K_m value of 0.97 μM for the chloroplast GTPases, and _k_cat values of 49 and 4.8 min−1 and _K_m values of 0.76 and 18 μM for the E. coli GTPases with and without the SRP RNA, respectively.

Figure 5.

Figure 5.

cpFtsY preferentially binds and hydrolyzes its cognate nucleotide. (A and B) Basal GTPase (A) and XTPase (B) reactions of wild-type cpFtsY (•) and mutant cpFtsY(D283N) (■). The data were fit to Eq. 1 and gave a _k_max value of 0.0045 min−1 and a _K_1/2 value of 2.1 μM for GTP hydrolysis by wild-type cpFtsY, and a _k_max value of 0.0022 min−1 and a _K_1/2 value of 2.2 μM for XTP hydrolysis by mutant cpFtsY(D283N). (C) GppNHp binds more strongly to wild-type cpFtsY (•) than to mutant cpFtsY(D283N) (■). (D) XppNHp binds more strongly to mutant cpFtsY(D283N) (■) than to wild-type cpFtsY (•). The _K_i values are reported in Table 2. (E and F) Titration of the change in fluorescence of mant-GTP (•) and mant-XTP (■) upon binding to wild-type cpFtsY (E) and mutant cpFtsY(D283N) (F). The data were fit to Eq. 5, and the _K_d values are summarized in Table 2.

Figure 6.

Figure 6.

Nucleotide hydrolyses from the cpSRP54•cpFtsY(D283N) complex are asymmetric. (A) Stimulation of the GTPase reaction of cpSRP54 by cpFtsY(D283N), determined as described in Materials and Methods using 0.2 μM cpSRP54 and 20 μM XTP. The data were fit to Eq. 1 and gave a maximal rate constant of 0.037 min−1. (B) Stimulation of the XTPase reaction of cpFtsY(D283N) by cpSRP54, determined as described in Materials and Methods using 0.2 μM cpFtsY(D283N) and 20 μM GTP. The data were fit to Eq. 1 and gave a maximal rate constant of 0.30 min−1. (C) Time courses for GTP and XTP hydrolyses from the GTP·cpSRP54•cpFtsY(D283N)·XTP complex, determined as described in Materials and Methods. The data were fit to single exponential rate equations and gave rate constants of 0.86 and 3.7 min−1 for the GTPase (•) and XTPase (■) reactions, respectively.

Figure 7.

Figure 7.

cpFtsY(D283N) prefers GTP over XTP when it forms a complex with cpSRP54. (A) GTP hydrolysis rates when 100–500 nM cpSRP54 interacts with wild-type cpFtsY (•), cpFtsY(D283N) bound to GTP (♦) and cpFtsY(D283N) bound to XTP (■). The following nucleotide concentrations were used: 100 μM GTP for reaction with wild-type cpFtsY, 200 μM GTP for reaction with cpFtsY(D283N) bound to GTP, and 20 μM GTP and 50 μM XTP for reaction with cpFtsY(D283N) bound to XTP. The data were fit to Eq. 2, which gave _k_cat values of 50 (•) and 39 min−1 (♦). (B) XTP inhibits the ability of GTP-bound cpFtsY(D283N) to stimulate GTP hydrolysis by cpSRP54. Reactions were carried out in the presence of 500 nM cpSRP54, 2 μM cpFtsY(D283N) and 200 μM GTP, as described in Materials and Methods. The data were fit to Eq. 3 and gave an apparent inhibition constant of 9.0 μM.

Figure 8.

Figure 8.

Model for the interactions of cpSRP54 with the side chain of cpFtsY Asp283 or with GTP. (A–C) Proposed interactions between the side chain of residue 283 with a hydrogen bond donor from cpSRP54 (-AH) for the wild-type cpSRP54•cpFtsY complex (A) and the cpSRP54•cpFtsY(D283N) complex with XTP (B) or GTP (C) bound to cpFtsY(D283N). (D) The GTP bound to cpFtsY interacts with a hydrogen bond acceptor (-B:) from cpSRP54.

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