Co-translational Folding Intermediate Dictates Membrane Targeting of the Signal Recognition Particle Receptor

https://doi.org/10.1016/j.jmb.2018.04.017Get rights and content

Highlights

  • Our work suggest that during translation of the SRP receptor FtsY, its N-domain (helices N2–4) assumes a distinct functional folding intermediate, which then disappears after completion of the receptor translation. This is based on new crystal structures and various biochemical results.

  • Our work presents evidence that upon completion of FtsY translation and its release from the ribosome, residues at the C-terminus of the G-domain facilitate the formation of a stable, three-helical bundle state of N2–4.

  • The conformational switch of N2–4 might separate between its role in cotranslational targeting of FtsY and ribosomes to the cytoplasmic membrane and its role in the assembly and downstream functions of the mature receptor.

Abstract

Much of our knowledge on the function of proteins is deduced from their mature, folded states. However, it is unknown whether partially synthesized nascent protein segments can execute biological functions during translation and whether their premature folding states matter. A recent observation that a nascent chain performs a distinct function, co-translational targeting in vivo, has been made with the Escherichia coli signal recognition particle receptor FtsY, a major player in the conserved pathway of membrane protein biogenesis. FtsY functions as a membrane-associated entity, but very little is known about the mode of its targeting to the membrane. Here we investigated the underlying structural mechanism of the co-translational FtsY targeting to the membrane. Our results show that helices N2–4, which mediate membrane targeting, form a stable folding intermediate co-translationally that greatly differs from its fold in the mature FtsY. These results thus resolve a long-standing mystery of how the receptor targets the membrane even when deleted of its alleged membrane targeting sequence. The structurally distinct targeting determinant of FtsY exists only co-translationally. Our studies will facilitate further efforts to seek cellular factors required for proper targeting and association of FtsY with the membrane. Moreover, the results offer a hallmark example for how co-translational nascent intermediates may dictate biological functions.

Introduction

Folding is usually required to achieve the three-dimensional structure of proteins and therefore shapes their function [1], [2]. It is well-established that individual domains of multi-domain proteins can already reach their “final” folding state during protein synthesis at the ribosome [3], [4], [5]. However, co-translational folding of a sub-domains might differ from how they finally fold in the mature state. Such bistable folding intermediates could execute distinct functions during translation and other functions or none after translation when the proteins mature. If this was true, as we propose here for the signal recognition particle (SRP) receptor, it offers an additional layer of biological regulation.

The SRP receptor is a major player in the pathway of membrane protein biogenesis in all living cells [6], [7], [8], [9]. Its functional interactions with the SRP [10], [11] and the translocon [12], [13], [14] and its association with membrane-bound ribosomes [15], [16] underscore its central role in ribosome targeting and biogenesis of membrane proteins. The Escherichia coli receptor FtsY contains three domains, termed A, N and G [17], [18] (Fig. 1a). The A domain is not essential for bacterial growth [19], although it was proposed that this long acidic domain participates in regulatory aspects of the pathway [14], [20]. The G domain is responsible for GTP binding and it interacts with SRP [21], [22] through the homologous domain of the SRP protein, Ffh [24]. The structure of a functional NG + 1 [19] core of FtsY (Fig. 1B) [23] shows that the N-domain is composed of four well-defined α-helices (termed N1–4), oriented in an anti-parallel manner. A short helical segment that precedes helix N1 mediates the essential regulation of FtsY by anionic phospholipids at the inner membrane [23], [25], [26], [27], [28]. Proper formation of this amphipathic element is essential for the receptor function in vivo [19], through its lipid-responsive effect on the GTPase activity of the SRP/SRP receptor complex [25]. Its ability to interact with lipids and its similarity to the most C-terminal membrane targeting segment of MinD [29] have led to the suggestion that this short helix is the primary membrane targeting sequence (MTS) of FtsY [30]. However, we have shown that NG, a construct defective in forming this short helical structure, is also able to target the membrane in vivo [25]. Moreover, our recent studies with translation intermediates (TIs) lacking this helix, further emphasized the question what mediates targeting of FtsY to the membrane [31]. Based on these and other studies, we proposed that the short helix is not required for membrane targeting but rather serves as a lipid-responsive element (LRE). The work described here offers an interesting structural view on how FtsY targeting to the membrane occurs in the absence of the LRE and helix N1.

Clearly, as outlined above, FtsY executes its essential function at the membrane, but only little is known about the underlying mechanisms that ensure its productive membrane localization. Previously, we hypothesized that FtsY might target the membrane co-translationally [6], [32], [33] and recent studies with FtsY TIs demonstrated feasibility [31]. Moreover, this work also showed that exposing helices N2–4 of the N-domain at the ribosome during translation (TI-N2–4) is necessary and sufficient for membrane targeting. However, it remains a mystery how these helices (N2–4) can target the stalled ribosomes to membranes in vivo, especially since N1 and the LRE, which were previously assigned to mediate functional membrane lipid interactions, are absent in TI-N2–4. In addition, helices N2–4 do not contain a bona fide transmembrane segment [31], suggesting that they interact with the membrane peripherally, maybe through membrane-associated proteins. All together, these observations suggested that helices N2–4 (i) are not involved in the interaction with lipids, but possibly with non-lipid components at the membrane [34]; (ii) exhibit a specific conformational state in the context of TI-N2–4; and (iii) undergo a conformational rearrangement in the context of the fully translated G-domain of FtsY. If true, such a co-translational folding intermediate could carry the information for targeting of the SRP receptor to its destination(s) at the membrane.

To challenge these ideas, we performed a series of experiments that strongly support the notion that N2–4 of FtsY adopts two distinct conformational states: an elongated helix and a three-helical bundle. The elongated helical state represents an intermediate that exists only throughout the translation of FtsY. Upon completion of FtsY synthesis, residues at the C-terminus of the G-domain facilitate the formation of a stable, three-helical bundle state of N2–4. The conformational switch of N2–4 might separate between its role in co-translational targeting of FtsY and ribosomes to the cytoplasmic membrane and its role in the assembly and downstream functions of the mature receptor. We propose that the underlying mechanistic basis for this behavior of N2–4 is “conformational bistability.” We speculate that FtsY presents a hallmark example for other, yet unknown proteins with bistable folding states that execute distinct biological functions during and after their translation.

Section snippets

N2–4 localizes with the membrane fraction and does not co-migrate with cytoplasmic ribosomes

We previously showed that exposing helices N2–4 of the N-domain at the ribosome during translation (TI-N2–4) is necessary and sufficient for membrane targeting in vivo [31]. These results suggested to us that N2–4 performs an individual function, independently of the other FtsY domains, co-translationally. To investigate whether N2–4 can target the membrane also in the absence of the ribosome, we analyzed the subcellular distribution of N2–4 as an independently expressed protein. Cell

Discussion

The E. coli SRP receptor FtsY performs its biological role at the membrane together with SRP and the translocon(s) [37], [38]. A poorly answered question is how FtsY functionally targets and binds the membrane? Previous work identified a short amphipathic helix at the N-terminus of N1 in the N-domain, capable of interacting with anionic phospholipids. This amphipathic element is essential for FtsY function because it is necessary for the functional response of the receptor to anionic lipids [23]

E. coli strains, plasmids and growth conditions

E. coli TOP10 was used for propagation and preparation of various plasmid constructs. E. coli BL21(DE-03) (Novagen) served in cell fractionation studies and protein purifications. E. coli IY28 [28], which contains a chromosomal ftsY gene under the arabinose promoter, was used for FtsY-complementation experiments. E. coli UT5600 ΔssrAsmpB was used for expression of TIs. Typically, cells were cultured in LB medium supplemented with the appropriate antibiotics (kanamycin was used at 30 μg/mL and

Acknowledgments

We thank members of the E.B. and G.B. laboratories for helpful discussions. We thank E. Michel and G. Stier for plasmids pEM(GB1) and pET(6His-TEV), respectively. This work was supported by a grant from the Israel Science foundation (ISF 600/11) and the Erica Drake Fund to E.B., and the LOEWE excellence initiative of the state of Hessen, Germany to G.B. D.M. was supported by the Peter and Traudl Engelhorn foundation. We acknowledge great support provided by the ESRF, Grenoble, France. G.B.

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      This phenomenon is clearly enhanced by synthetically lengthening the half-life of the nascent chains in the FtsY translation intermediates. Such non-specific interactions are not surprising, because of the assumed significant solvent accessibility of the elongated helical N2–439 that might amplify such non-functional interactions.45 Therefore, and in light of the preliminary results (Figure S1), we focused our analyses on the likely relevant identifications of soluble and membrane proteins that are components of the membrane-targeting and insertion/translocation machinery (Table 1).

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    A.K. and D.M. contributed equally to this work.

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