Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

Abstract

The ribosome exit site is a crowded environment where numerous factors contact nascent polypeptides to influence their folding, localization, and quality control. Timely and accurate selection of nascent polypeptides into the correct pathway is essential for proper protein biogenesis. To understand how this is accomplished, we probe the mechanism by which nascent polypeptides are accurately sorted between the major cotranslational chaperone trigger factor (TF) and the essential cotranslational targeting machinery, signal recognition particle (SRP). We show that TF regulates SRP function at three distinct stages, including binding of the translating ribosome, membrane targeting via recruitment of the SRP receptor, and rejection of ribosome-bound nascent polypeptides beyond a critical length. Together, these mechanisms enhance the specificity of substrate selection into both pathways. Our results reveal a multilayered mechanism of molecular interplay at the ribosome exit site, and provide a conceptual framework to understand how proteins are selected among distinct biogenesis machineries in this crowded environment.signal recognition particle | trigger factor | ribosome | protein biogenesis | GTPases P roper protein biogenesis is a prerequisite for the maintenance of a functional proteome. Accumulating data indicate that this process begins at the ribosome exit site, where many protein biogenesis machineries can interact and gain access to the nascent polypeptide. This includes chaperones (1-5) such as trigger factor (TF) (1, 4, 6, 7), Hsp70, and the nascent polypeptide-associated complex (8-13); modification enzymes (10, 14-16) such as N-acetyl transferase, methionine aminopeptidase, and arginyl transferase; protein-targeting and translocation machineries such as signal recognition particle (SRP) (17-20), SecA (21), the SecYEG (or Sec61p) (22, 23) and YidC translocases (24,25), and the ribosome-bound quality control complex (26)(27)(28)(29)(30). Engagement of these factors with nascent polypeptides influences their folding, assembly, localization, processing, and quality control. Within seconds after the nascent polypeptide emerges from the ribosomal exit tunnel, it must engage the correct set of factors and thus commit to the proper biogenesis pathway. How this is accomplished in the crowded environment at the ribosome exit site is an emerging question. In this work, we address this question by deciphering how nascent proteins are selected between two major protein biogenesis machineries in bacteria, SRP and TF.SRP is a universally conserved ribonucleoprotein complex responsible for the cotranslational targeting of proteins to the eukaryotic endoplasmic reticulum (ER), or the bacterial plasma membrane (31). SRP recognizes ribosome-nascent chain complexes (termed RNC or cargo) carrying strong signal sequences and delivers them to the SecYEG or YidC translocation machinery on the target membrane. SRP binds RNC via two interactions: a helical N domain in the SRP54 protein (called Ffh in bacteria) binds the ribosoma...

SecA mediates cotranslational targeting and translocation of an inner membrane protein

“…1 C ); this affinity is ∼900-fold higher than that of SecA for empty ribosomes ( Huber et al, 2011 ), suggesting additional interactions of SecA with the RodZ nascent chain. As other ribosome-associated protein biogenesis factors such as SRP and trigger factor (TF) could compete for binding to the ribosome and RodZ nascent chain under physiological conditions ( Ariosa et al, 2015 ; Gamerdinger et al, 2015 ), we further tested whether the SecA–RNC RodZ interaction survives the presence of these factors. Equilibrium titrations in the presence of near-physiological concentrations of SRP (400 nM) or TF (2 µM) showed that the SecA–RNC RodZ interaction was weakened by these factors but remained strong, with K d values of ∼19 nM and ∼55 nM, respectively ( Figs.…”

“…Through the interaction between SRP and the SRP receptor (SR; termed FtsY in bacteria), the nascent protein is delivered to the SecYEG (or Sec61p) protein translocation machinery on the bacterial inner membrane (or the eukaryotic endoplasmic reticulum; Zhang et al, 2010 ). SRP-dependent targeting is complete before ∼130 amino acids of the nascent polypeptide C-terminal to the signal sequence or TMD is translated ( Siegel and Walter, 1988 ; Ariosa et al, 2015 ), and releasing nascent proteins from the ribosome abolishes the targeting of SRP-dependent substrates ( Kuruma et al, 2005 ). In bacteria, SRP is generally thought to mediate the targeted delivery of the majority of inner membrane proteins and several periplasmic secretory proteins that contain highly hydrophobic signal sequences ( Luirink and Sinning, 2004 ; Schibich et al, 2016 ).…”

Formyl-methionine as a degradation signal at the N-termini of bacterial proteins

“…A ribosome-associated chaperone called trigger factor (TF) interacts with proteins emerging from the tunnel 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 . The signal recognition particle (SRP) also binds to some nascent proteins, recognizing specific sequence motifs (signal sequences) and directing SRP-associated proteins for translocation through the inner membrane 42 43 44 45 .…”

ATPase and GTPase Tangos Drive Intracellular Protein Transport

“…In contrast, the processes mediated by the SIMIBI family of GTPases and ATPases are often constitutive and must occur rapidly. For example, co-translational protein targeting must occur before the nascent polypeptide reaches a critical length, which imposes a time window of 3–6 seconds for SRP and SR to complete each targeting cycle[72, 73]. The ability of SRP•SR and Get3 to directly respond to effector molecules in their respective pathways without the need to recruit extrinsic regulatory factors may be especially beneficial for vectorial processes that must occur efficiently.…”