An official website of the United States government
Abstract Protein biogenesis is essential in all cells and initiates when a nascent polypeptide emerges from the ribosome exit tunnel, where multiple ribosome-associated protein biogenesis factors (RPBs) direct nascent proteins to distinct fates. How distinct RPBs spatiotemporally coordinate with one another to affect accurate protein biogenesis is an emerging question. Here, we address this question by studying the role of a cotranslational chaperone, nascent polypeptide-associated complex (NAC), in regulating substrate selection by signal recognition particle (SRP), a universally conserved protein targeting machine. We show that mammalian SRP and SRP receptors (SR) are insufficient to generate the biologically required specificity for protein targeting to the endoplasmic reticulum. NAC co-binds with and remodels the conformational landscape of SRP on the ribosome to regulate its interaction kinetics with SR, thereby reducing the nonspecific targeting of signalless ribosomes and pre-emptive targeting of ribosomes with short nascent chains. Mathematical modeling demonstrates that the NAC-induced regulations of SRP activity are essential for the fidelity of cotranslational protein targeting. Our work establishes a molecular model for how NAC acts as a triage factor to prevent protein mislocalization, and demonstrates how the macromolecular crowding of RPBs at the ribosome exit site enhances the fidelity of substrate selection into individual protein biogenesis pathways.
more »
« less
NAC guides a ribosomal multienzyme complex for nascent protein processing
Approximately 40% of the mammalian proteome undergoes N-terminal methionine excision and acetylation, mediated sequentially by methionine aminopeptidase (MetAP) and N-acetyltransferase A (NatA), respectively1. Both modifications are strictly cotranslational and essential in higher eukaryotic organisms1. The interaction, activity, and regulation of these enzymes on translating ribosomes are poorly understood. Here, biochemical, structural, and in vivo studies show that the nascent polypeptide-associated complex (NAC)2,3 orchestrates the action of these enzymes. NAC assembles a multienzyme complex with MetAP1 and NatA early during translation and pre-positions both enzyme active sites for timely sequential processing of the nascent protein. NAC further releases the inhibitory interactions from the NatA regulatory protein Huntingtin-Yeast-two-hybrid-Protein-K (HYPK)4,5 to activate NatA on the ribosome, enforcing cotranslational N-terminal acetylation. Our results provide a mechanistic model for the cotranslational processing of proteins in eukaryotic cells.
more »
« less
- Award ID(s):
- 2219287
- PAR ID:
- 10580618
- Publisher / Repository:
- Nature
- Date Published:
- Journal Name:
- Nature
- Volume:
- 633
- Issue:
- 8030
- ISSN:
- 0028-0836
- Page Range / eLocation ID:
- 718 to 724
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins’ sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins’ sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily.more » « less
-
Rationale: NAA15 (N-alpha-acetyltransferase 15) is a component of the NatA (N-terminal acetyltransferase complex). The mechanism by which NAA15 haploinsufficiency causes congenital heart disease remains unknown. To better understand molecular processes by which NAA15 haploinsufficiency perturbs cardiac development, we introduced NAA15 variants into human induced pluripotent stem cells (iPSCs) and assessed the consequences of these mutations on RNA and protein expression. Objective: We aim to understand the role of NAA15 haploinsufficiency in cardiac development by investigating proteomic effects on NatA complex activity and identifying proteins dependent upon a full amount of NAA15. Methods and Results: We introduced heterozygous loss of function, compound heterozygous, and missense residues (R276W) in iPSCs using CRISPR/Cas9. Haploinsufficient NAA15 iPSCs differentiate into cardiomyocytes, unlike NAA15 -null iPSCs, presumably due to altered composition of NatA. Mass spectrometry analyses reveal ≈80% of identified iPSC NatA targeted proteins displayed partial or complete N-terminal acetylation. Between null and haploinsufficient NAA15 cells, N-terminal acetylation levels of 32 and 9 NatA-specific targeted proteins were reduced, respectively. Similar acetylation loss in few proteins occurred in NAA15 R276W induced pluripotent stem cells. In addition, steady-state protein levels of 562 proteins were altered in both null and haploinsufficient NAA15 cells; 18 were ribosomal-associated proteins. At least 4 proteins were encoded by genes known to cause autosomal dominant congenital heart disease. Conclusions: These studies define a set of human proteins that requires a full NAA15 complement for normal synthesis and development. A 50% reduction in the amount of NAA15 alters levels of at least 562 proteins and N-terminal acetylation of only 9 proteins. One or more modulated proteins are likely responsible for NAA15-haploinsufficiency mediated congenital heart disease. Additionally, genetically engineered induced pluripotent stem cells provide a platform for evaluating the consequences of amino acid sequence variants of unknown significance on NAA15 function.more » « less
-
The extreme N‐terminal residues of the mitochondrial ribosomal bL27m proteins reside within the ribosomal peptidyl transferase center (PTC) and are conserved from their bacterial ancestors. Mutation or truncation of the N‐terminal region of the yeast Mrp7/bL27m protein did not inhibit protein synthesis but significantly impacted the efficacy of the mitochondrial translational process with respect to yielding proteins competent to assemble into functional oxidative phosphorylation enzymes. The requirement for the N‐terminal residues of Mrp7/bL27m to support normal mitotranslation was more apparent under respiratory growth. We demonstrate that the N‐terminal region of Mrp7/bL27m impacts the environment of the PTC and speculate the bL27m proteins serve to fine‐tune and optimize mitoribosomal activity with respect to the downstream fate of the nascent chain.more » « less
-
During the last few decades, the ribosome has been regarded primarily as a major cell player devoted to the catalysis of protein biosynthesis during translation [1-5]. It is therefore not surprising that several processes related to translation exploit the ribosome as a central hub. For instance, it is well-known that many events related to translational regulation are mediated by interactions between the ribosome and initiation, elongation or termination factors [6-9]. In addition, the ribosome is involved in mRNA-code recognition and proofreading [10-12] as well as in the control of translation rates via interactions with mRNA codons bearing high- and lowfrequency [13-15] and associated with variable tRNA abundance within the translation machinery [16-18]. Interestingly, the ribosome also assists de novo protein structure formation by minimizing cotranslational aggregation, thus increasing the yield of native-protein production [19,20]. The latter event, however, has not been shown to require -- or even involve -- direct interactions between the ribosome and the nascent protein chain. A notable exception is that of nascent chains bearing either N-terminal signal sequences or translational-arrest tags. These proteins are known to establish short- or long-term contacts with various regions of the ribosome during translation [21-25]. In summary, until recently very little knowledge has been available about direct contacts between the ribosome and nascent polypeptides and proteins that do not carry signal or arrest sequences. Studies based on fluorescence depolarization in the frequency domain [26] and NMR spectroscopy [27-30] provided interesting data that are consistent with, but do not unequivocally establish, the presence of these interactions.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s41586-024-07846-7
