Journal of Molecular Biology
Co-translational Folding Intermediate Dictates Membrane Targeting of the Signal Recognition Particle Receptor
Graphical Abstract
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.
References (64)
A pause for thought along the co-translational folding pathway
Trends Biochem. Sci.
(2009)Early targeting events during membrane protein biogenesis in Escherichia coli
Biochim. Biophys. Acta
(2011)- et al.
Escherichia coli signal recognition particle receptor FtsY contains an essential and autonomous membrane-binding amphipathic helix
J. Biol. Chem.
(2007) - et al.
Structural basis for conserved regulation and adaptation of the signal recognition particle targeting complex
J. Mol. Biol.
(2016) - et al.
Membrane targeting of ribosomes and their release require distinct and separable functions of FtsY
J. Biol. Chem.
(2007) - et al.
Two cooperating helices constitute the lipid-binding domain of the bacterial SRP receptor
J. Mol. Biol.
(2009) - et al.
Genetic evidence for functional interaction of the Escherichia coli signal recognition particle receptor with acidic lipids in vivo
J. Biol. Chem.
(2010) - et al.
The MinD membrane targeting sequence is a transplantable lipid-binding helix
J. Biol. Chem.
(2003) - et al.
Lipids trigger a conformational switch that regulates signal recognition particle (SRP)-mediated protein targeting
J. Biol. Chem.
(2011) Is there a twist in the Escherichia coli signal recognition particle pathway?
Trends Biochem. Sci.
(2012)
FtsY binds to the Escherichia coli inner membrane via interactions with phosphatidylethanolamine and membrane proteins
J. Biol. Chem.
A site-specific, membrane-dependent cleavage event defines the membrane binding domain of FtsY
J. Biol. Chem.
The signal recognition particle-targeting pathway does not necessarily deliver proteins to the sec-translocase in Escherichia coli
J. Biol. Chem.
Applications of the restriction free (RF) cloning procedure for molecular manipulations and protein expression
J. Struct. Biol.
Exploration of the arrest peptide sequence space reveals arrest-enhanced variants
J. Biol. Chem.
Folding of newly translated proteins in vivo: the role of molecular chaperones
Annu. Rev. Biochem.
Molecular chaperones in protein folding and proteostasis
Nature
Cotranslational folding of spectrin domains via partially structured states
Nat. Struct. Mol. Biol.
Insights into cotranslational nascent protein behavior from computer simulations
Annu. Rev. Biophys.
Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane
Annu. Rev. Cell Biol.
Protein targeting by the signal recognition particle
Biol. Chem.
Defining the physiological role of SRP in protein-targeting efficiency and specificity
Science
Signal-sequence-independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle
Mol. Biol. Cell
Interaction of E. coli Ffh/4.5S ribonucleoprotein and FtsY mimics that of mammalian signal recognition particle and its receptor
Nature
FtsY, the bacterial signal-recognition particle receptor, interacts functionally and physically with the SecYEG translocon
EMBO Rep.
The bacterial SRP receptor, FtsY, is activated on binding to the translocon
Mol. Microbiol.
Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon
J. Cell Biol.
Accumulation of endoplasmic membranes and novel membrane-bound ribosome-signal recognition particle receptor complexes in Escherichia coli
J. Cell Biol.
Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum
J. Cell Biol.
Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle
Nature
Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains
Nature
The core Escherichia coli signal recognition particle receptor contains only the N and G domains of FtsY
J. Bacteriol.
Cited by (9)
Coping with stress: How bacteria fine-tune protein synthesis and protein transport
2023, Journal of Biological ChemistryCo-Translational Membrane Targeting and Holo-Translocon Docking of Ribosomes Translating the SRP Receptor
2022, Journal of Molecular BiologyCitation Excerpt :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).
Structural basis for (p)ppGpp-mediated inhibition of the GTPase RbgA
2018, Journal of Biological Chemistry
- †
A.K. and D.M. contributed equally to this work.