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Defining the physiological role of SRP in protein-targeting efficiency and specificity.

ABSTRACT: The signal recognition particle (SRP) enables cotranslational delivery of proteins for translocation into the endoplasmic reticulum (ER), but its full in vivo role remains incompletely explored. We combined rapid auxin-induced SRP degradation with proximity-specific ribosome profiling to define SRP's in vivo function in yeast. Despite the classic view that SRP recognizes amino-terminal signal sequences, we show that SRP was generally essential for targeting transmembrane domains regardless of their position relative to the amino terminus. By contrast, many proteins containing cleavable amino-terminal signal peptides were efficiently cotranslationally targeted in SRP's absence. We also reveal an unanticipated consequence of SRP loss: Transcripts normally targeted to the ER were mistargeted to mitochondria, leading to mitochondrial defects. These results elucidate SRP's essential roles in maintaining the efficiency and specificity of protein targeting.

SUBMITTER: Costa EA

PROVIDER: S-EPMC5970945 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Defining the physiological role of SRP in protein-targeting efficiency and specificity.

Costa Elizabeth A EA Subramanian Kelly K Nunnari Jodi J Weissman Jonathan S JS

Science (New York, N.Y.) 20180118 6376

The signal recognition particle (SRP) enables cotranslational delivery of proteins for translocation into the endoplasmic reticulum (ER), but its full in vivo role remains incompletely explored. We combined rapid auxin-induced SRP degradation with proximity-specific ribosome profiling to define SRP's in vivo function in yeast. Despite the classic view that SRP recognizes amino-terminal signal sequences, we show that SRP was generally essential for targeting transmembrane domains regardless of th ...[more]

PMID: 29348368

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Defining the physiological role of SRP in protein-targeting efficiency and specificity

Project description:The signal recognition particle (SRP) enables cotranslational delivery of proteins for translocation into the endoplasmic reticulum (ER), but its full in vivo role remains incompletely explored. We combined rapid auxin-induced SRP degradation with proximity-specific ribosome profiling to define SRP’s in vivo function in yeast. Despite the classic view that SRP recognizes amino-terminal signal sequences, we show that SRP was generally essential for targeting transmembrane domains regardless of their position relative to the amino-terminus. By contrast, many proteins containing cleavable amino-terminal signal peptides were efficiently cotranslationally targeted in SRP’s absence. We also reveal an unanticipated consequence of SRP loss: Transcripts normally targeted to the ER were mistargeted to mitochondria, leading to mitochondrial defects. These results elucidate SRP’s essential roles in maintaining the efficiency and specificity of protein targeting.

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Defining the physiological role of SRP in protein-targeting efficiency and specificity

Project description:Defining the physiological role of SRP in protein-targeting efficiency and specificity

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Decreased dicer expression enhances SRP-mediated protein targeting.

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Transient tether between the SRP RNA and SRP receptor ensures efficient cargo delivery during cotranslational protein targeting.

Project description:Kinetic control of macromolecular interactions plays key roles in biological regulation. An example of such control occurs in cotranslational protein targeting by the signal recognition particle (SRP), during which the SRP RNA and the cargo both accelerate complex assembly between the SRP and SRP receptor FtsY 10(2)-fold. The molecular mechanism underlying these rate accelerations was unclear. Here we show that a highly conserved basic residue, Lys399, on the lateral surface of FtsY provides a novel RNA tetraloop receptor to mediate the SRP RNA- and cargo-induced acceleration of SRP-FtsY complex assembly. We propose that the SRP RNA, by using its tetraloop to interact with FtsY-Lys399, provides a transient tether to stabilize the early stage and transition state of complex formation; this accelerates the assembly of a stable SRP-FtsY complex and allows the loading of cargo to be efficiently coupled to its membrane delivery. The use of a transient tether to increase the lifetime of collisional intermediates and reduce the dimension of diffusional search represents a novel and effective mechanism to accelerate macromolecular interactions.

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A molecular recognition feature mediates ribosome-induced SRP-receptor assembly during protein targeting.

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Defining the metal specificity of a multifunctional biofilm adhesion protein.

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Lipids trigger a conformational switch that regulates signal recognition particle (SRP)-mediated protein targeting.

Project description:Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.

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Defining the Role of Nucleotide Flipping in Enzyme Specificity Using 19F NMR.

Project description:A broad range of proteins employ nucleotide flipping to recognize specific sites in nucleic acids, including DNA glycosylases, which remove modified nucleobases to initiate base excision repair. Deamination, a pervasive mode of damage, typically generates lesions that are recognized by glycosylases as being foreign to DNA. However, deamination of 5-methylcytosine (mC) generates thymine, a canonical DNA base, presenting a challenge for damage recognition. Nevertheless, repair of mC deamination is important because the resulting G·T mispairs cause C → T transition mutations, and mC is abundant in all three domains of life. Countering this threat are three types of glycosylases that excise thymine from G·T mispairs, including thymine DNA glycosylase (TDG). These enzymes must minimize excision of thymine that is not generated by mC deamination, in A·T pairs and in polymerase-generated G·T mispairs. TDG preferentially removes thymine from DNA contexts in which cytosine methylation is prevalent, including CG and one non-CG site. This remarkable context specificity could be attained through modulation of nucleotide flipping, a reversible step that precedes base excision. We tested this idea using fluorine NMR and DNA containing 2'-fluoro-substituted nucleotides. We find that dT nucleotide flipping depends on DNA context and is efficient only in contexts known to feature cytosine methylation. We also show that a conserved Ala residue limits thymine excision by hindering nucleotide flipping. A linear free energy correlation reveals that TDG attains context specificity for thymine excision through modulation of nucleotide flipping. Our results provide a framework for characterizing nucleotide flipping in nucleic acids using 19F NMR.

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Cotranslational signal-independent SRP preloading during membrane targeting.

Project description:Ribosome-associated factors must properly decode the limited information available in nascent polypeptides to direct them to their correct cellular fate. It is unclear how the low complexity information exposed by the nascent chain suffices for accurate recognition by the many factors competing for the limited surface near the ribosomal exit site. Questions remain even for the well-studied cotranslational targeting cycle to the endoplasmic reticulum, involving recognition of linear hydrophobic signal sequences or transmembrane domains by the signal recognition particle (SRP). Notably, the SRP has low abundance relative to the large number of ribosome-nascent-chain complexes (RNCs), yet it accurately selects those destined for the endoplasmic reticulum. Despite their overlapping specificities, the SRP and the cotranslationally acting Hsp70 display precise mutually exclusive selectivity in vivo for their cognate RNCs. To understand cotranslational nascent chain recognition in vivo, here we investigate the cotranslational membrane-targeting cycle using ribosome profiling in yeast cells coupled with biochemical fractionation of ribosome populations. We show that the SRP preferentially binds secretory RNCs before their targeting signals are translated. Non-coding mRNA elements can promote this signal-independent pre-recruitment of SRP. Our study defines the complex kinetic interaction between elongation in the cytosol and determinants in the polypeptide and mRNA that modulate SRP–substrate selection and membrane targeting.

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