The NG domain of the prokaryotic signal recognition particle receptor, FtsY, is fully functional when fused to an unrelated integral membrane polypeptide. Proc Natl Acad Sci USA

Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/1997; 94(12):6025-9. DOI: 10.1073/pnas.94.12.6025
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Recent studies have revealed that Escherichia coli possesses an essential targeting system for integral membrane proteins, similar to the mammalian signal recognition particle (SRP) machinery. One essential protein in this system is FtsY, a homologue of the alpha-subunit of the mammalian SRP-receptor (SR-alpha). However, E. coli does not possess a close homologue of the integral membrane protein SR-beta, which anchors SR-alpha to the membrane. Moreover, although FtsY can be found as a peripheral membrane protein, the majority is found soluble in the cytoplasm. In this study, we obtained genetic and biochemical evidence that FtsY must be targeted to the membrane for proper function. We demonstrate that the essential membrane targeting activity of FtsY is mediated by a 198-residue-long acidic N-terminal domain. This domain can be functionally replaced by unrelated integral membrane polypeptides, thus avoiding the need for specific FtsY membrane targeting factors. Therefore, the N terminus of FtsY constitutes an independent domain, which is required only for the targeting of the C-terminal NG domain of FtsY to the membrane.

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Available from: Andrei Seluanov, Oct 07, 2015
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    • "This question depends on whether or not FtsY needs to be targeted to membranes co-translationally for efficient biological activity. In any case, this and other studies disfavor the possibility that soluble FtsY plays a critical role in the targeting pathway [35] [111] [112]. "
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    ABSTRACT: All living cells have co-translational pathways for targeting membrane proteins. Co-translation pathways for secretory proteins also exist but mostly in eukaryotes. Unlike secretory proteins, the biosynthetic pathway of most membrane proteins is conserved through evolution and these proteins are usually synthesized by membrane-bound ribosomes. Translation on the membrane requires that both the ribosomes and the mRNAs be properly localized. Theoretically, this can be achieved by several means. (i) The current view is that the targeting of cytosolic mRNA-ribosome-nascent chain complexes (RNCs) to the membrane is initiated by information in the emerging hydrophobic nascent polypeptides. (ii) The alternative model suggests that ribosomes may be targeted to the membrane also constitutively, whereas the appropriate mRNAs may be carried on small ribosomal subunits or targeted by other cellular factors to the membrane-bound ribosomes. Importantly, the available experimental data do not rule out the possibility that cells may also utilize both pathways in parallel. In any case, it is well documented that a major player in the targeting pathway is the signal recognition particle (SRP) system composed of the SRP and its receptor (SR). Although the functional core of the SRP system is evolutionarily conserved, its composition and biological practice come with different flavors in various organisms. This review is dedicated mainly to the Escherichia (E.) coli SRP, where the biochemical and structural properties of components of the SRP system have been relatively characterized, yielding essential information about various aspects of the pathway. In addition, several cellular interactions of the SRP and its receptor have been described in E. coli, providing insights into their spatial function. Collectively, these in vitro studies have led to the current view of the targeting pathway [see (i) above]. Interestingly, however, in vivo studies of the role of the SRP and its receptor, with emphasis on the temporal progress of the pathway, elicited an alternative hypothesis [see (ii) above]. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
    Biochimica et Biophysica Acta 03/2011; 1808(3):841-50. DOI:10.1016/j.bbamem.2010.07.025 · 4.66 Impact Factor
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    • "Archaea FtsY shares its conserved NG region with NG of SRP54, including the I-box, but differs from SRP54 with respect to several short amino acid stretches as revealed by the alignment of 95 archaea FtsY sequences (Figure 3, Supplementary Material 1). The NG regions are symmetrically arranged in three dimensions to constitute the structural and functional core of signal sequence release and nascent polypeptide delivery into the cell membrane (Figure 4) by mutually catalyzing the hydrolysis of GTP [63] [64] [65]. As has been observed within the bacterial genomes [66] [67] several archaea FtsY sequences consist only of the NG domain and lack an N-terminal acidic (A) domain. "
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    ABSTRACT: Archaea SRP is composed of an SRP RNA molecule and two bound proteins named SRP19 and SRP54. Regulated by the binding and hydrolysis of guanosine triphosphates, the RNA-bound SRP54 protein transiently associates not only with the hydrophobic signal sequence as it emerges from the ribosomal exit tunnel, but also interacts with the membrane-associated SRP receptor (FtsY). Comparative analyses of the archaea genomes and their SRP component sequences, combined with structural and biochemical data, support a prominent role of the SRP RNA in the assembly and function of the archaea SRP. The 5e motif, which in eukaryotes binds a 72 kilodalton protein, is preserved in most archaea SRP RNAs despite the lack of an archaea SRP72 homolog. The primary function of the 5e region may be to serve as a hinge, strategically positioned between the small and large SRP domain, allowing the elongated SRP to bind simultaneously to distant ribosomal sites. SRP19, required in eukaryotes for initiating SRP assembly, appears to play a subordinate role in the archaea SRP or may be defunct. The N-terminal A region and a novel C-terminal R region of the archaea SRP receptor (FtsY) are strikingly diverse or absent even among the members of a taxonomic subgroup.
    Archaea 06/2010; 2010:485051. DOI:10.1155/2010/485051 · 2.71 Impact Factor
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    • "SDS-PAGE was conducted according to Laemmli [26]. Western blotting was performed as described previously [27] using rabbit affinity-purified antibodies to alkaline phosphatase (Rockland) or to SecA and goat anti-L9 antibodies (lab collection). Affinity-purified antibodies to FtsY and Ffh were prepared in the course of this study, using NTA-purified 6-His tagged proteins. "
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    ABSTRACT: The Escherichia coli version of the mammalian signal recognition particle (SRP) system is required for biogenesis of membrane proteins and contains two essential proteins: the SRP subunit Ffh and the SRP-receptor FtsY. Scattered in vivo studies have raised the possibility that expression of membrane proteins is inhibited in cells depleted of FtsY, whereas Ffh-depletion only affects their assembly. These differential results are surprising in light of the proposed model that FtsY and Ffh play a role in the same pathway of ribosome targeting to the membrane. Therefore, we decided to evaluate these unexpected results systematically. We characterized the following aspects of membrane protein biogenesis under conditions of either FtsY- or Ffh-depletion: (i) Protein expression, stability and localization; (ii) mRNA levels; (iii) folding and activity. With FtsY, we show that it is specifically required for expression of membrane proteins. Since no changes in mRNA levels or membrane protein stability were detected in cells depleted of FtsY, we propose that its depletion may lead to specific inhibition of translation of membrane proteins. Surprisingly, although FtsY and Ffh function in the same pathway, depletion of Ffh did not affect membrane protein expression or localization. Our results suggest that indeed, while FtsY-depletion affects earlier steps in the pathway (possibly translation), Ffh-depletion disrupts membrane protein biogenesis later during the targeting pathway by preventing their functional assembly in the membrane.
    PLoS ONE 02/2010; 5(2):e9130. DOI:10.1371/journal.pone.0009130 · 3.23 Impact Factor
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