Conformational changes in the GTPase modules of the signal reception particle and its receptor drive initiation of protein translocation

Abstract

During cotranslational protein targeting, two guanosine triphosphatase (GTPase) in the signal recognition particle (SRP) and its receptor (SR) form a unique complex in which hydrolyses of both guanosine triphosphates (GTP) are activated in a shared active site. It was thought that GTP hydrolysis drives the recycling of SRP and SR, but is not crucial for protein targeting. Here, we examined the translocation efficiency of mutant GTPases that block the interaction between SRP and SR at specific stages. Surprisingly, mutants that allow SRP-SR complex assembly but block GTPase activation severely compromise protein translocation. These mutations map to the highly conserved insertion box domain loops that rearrange upon complex formation to form multiple catalytic interactions with the two GTPs. Thus, although GTP hydrolysis is not required, the molecular rearrangements that lead to GTPase activation are essential for protein targeting. Most importantly, our results show that an elaborate rearrangement within the SRP-SR GTPase complex is required to drive the unloading and initiate translocation of cargo proteins.

Figure 1.

A cotranslational assay to measure protein translocation by bacterial SRP and FtsY. (A) Scheme for the cotranslational targeting assay. (B) SDS-PAGE analysis of the translocation of ³⁵S-labeled pPL. pPL and prolactin indicate the precursor and signal sequence-cleaved form of prolactin, respectively. (C and D) Translocation efficiency is dependent on the concentration of SRP (C) and membrane (D). The data represent the mean of two to three measurements, and the error bars represent the range of values observed.

Figure 2.

Calibration of the sensitivity and dynamic range of the cotranslational targeting assay using mutant GTPases known to block SRP–SR binding. Translocation of pPL was performed in the presence of 150 nM SRP, 4 eq TKRM, and varying concentrations of FtsY as indicated. Representative data for three independent, side-by-side measurements are shown. The FtsY(47–497) construct was used and all the mutant proteins are derived from this construct.

Figure 3.

Class II mutant FtsYs that block reciprocal GTPase activation in the SRP–FtsY complex exhibit severe translocation defects. (A) The FtsY A335W mutant severely blocks protein targeting. The mutant and wild-type proteins are characterized in the context of the FtsY(47–497) construct. (B and C) Quantitation of the translocation defect of class II mutant FtsYs in the context of full-length FtsY (B) or the FtsY(47–497) construct (C). Representative data for three to five independent, side-by-side measurements are shown. (D) Correlation of the translocation activity of each FtsY mutant with its GTP hydrolysis rate from the SRP–FtsY complex. The translocation and GTPase activities of each mutant are normalized to those of the wild-type FtsY(47–497) at saturating protein concentrations and are averaged over three to five parallel measurements. The error bars represent the range of values observed.

Figure 4.

Effect of mutations in the Ffh GTPase site on the reciprocally stimulated GTPase reaction between SRP and FtsY. (A) Mutations in the IBD loop of Ffh substantially compromise reciprocal activation of GTP hydrolysis from the SRP–FtsY complex. Representative data for three independent, side-by-side measurements are shown. (B) Mutants Ffh Q109A and Ffh R194A do not substantially (less than threefold) compromise the formation and activation of the SRP–FtsY complex. Representative data for four independent, side-by-side measurements are shown. (C) Inhibition assay to measure the ability of the mutant SRPs (SRP(mt)) to form a complex with FtsY, as described in the text. (D) Ffh harboring mutations in the IBD loop are strong competitive inhibitors of wild-type SRP. The lines are fits of the data to inhibition curves and give apparent inhibition constants of 0.39, 0.27, and 0.35 μM for Ffh A143W (•), Ffh A144W (▴), and Ffh R141A (▾), respectively. The data are averaged over three independent measurements and the error bars represent the range of values observed.

Figure 5.

Effect of mutations in Ffh on the efficiency of protein translocation. (A) Mutations in the IBD loop of Ffh inhibit translocation of pPL. Representative data for three independent, side-by-side measurements are shown. (B) Mutants Ffh Q109A (▴) and Ffh R194A (▾) exhibit wild-type levels of translocation activity. Representative data for four independent, side-by-side measurements are shown. (C) Correlation of the translocation defect of Ffh mutants with their GTP hydrolysis rate from the SRP–FtsY complex. The translocation and GTPase activities of each mutant are normalized to those of wild-type Ffh at saturating protein concentrations and are averaged over three independent measurements. The error bars represent the range of values observed.

Figure 6.

Nucleotide requirement for protein translocation. (A) Schematics of the posttranslational targeting assay using stalled RNC. (B and C) Effect of nucleotides on the efficiency of protein translocation. T, GTP; D, GDP; N, GMPPNP. The data are the mean of over two independent measurements and the error bars represent the range of values observed.

Figure 7.

Mutant GTPases and GMPPNP block the SRP–SR interaction and the protein targeting reaction at distinct stages. (A) Model for the effects of mutant GTPases and GMPPNP on SRP-SR binding and activation. (1-2) An open-to-closed conformational change in both SRP and SR is required to form a stable SRP–SR complex. This step is specifically inhibited by the class I mutants shown in the blue box. (3) Concerted docking of the IBD loops in both GTPases to form an activated complex. This step is specifically inhibited by the class II mutants shown in the red box. (4) GTP hydrolysis occurs from the activated SRP–SR complex and drives complex disassembly. This step is inhibited by GMPPNP. (B) Model for the effects of mutant GTPases and GMPPNP on the protein targeting reaction. (1 and 1′) An open-to-closed conformational change occurs in SRP and SR upon binding to RNC and to the target membrane, respectively. (2) Complex formation between SRP and SR delivers the RNC to the membrane. This step is depicted as the target of inhibition by the class I mutant GTPases. Alternatively, these mutants could inhibit the open-to-closed conformational change that precedes complex formation (1 and 1′), but for simplicity this alternative scenario is not depicted. (3) Conformational rearrangements in the SRP–SR complex activate GTP hydrolysis and unload the RNC from the SRP to the membrane translocation channel. This step is inhibited by the class II mutants. (4) GTP hydrolysis drives complex disassembly and recycling of the SRP and SR components. This step is inhibited by GMPPNP.