Newly synthesized proteins are triaged between biosynthesis and degradation to maintain cellular homeostasis, but the decision-making mechanisms are unclear. Here, we reconstitute the core reactions for membrane targeting and ubiquitination of nascent tail-anchored membrane proteins to understand how their fate is determined. The central six-component triage system is divided into an uncommitted client-SGTA complex, a self-sufficient targeting module, and an embedded but self-sufficient quality control module. Client-SGTA engagement of the targeting module induces rapid, private, and committed client transfer to TRC40 for successful biosynthesis. Commitment to ubiquitination is dictated primarily by comparatively slower client dissociation from SGTA and non-private capture by the Bag6 subunit of the quality control module. This provides a paradigm for how priority and time are encoded within a multi-chaperone triage system.Protein biosynthesis and quality control pathways are precisely balanced to provide nascent proteins an initial opportunity to mature, while favoring degradation over time (1-3). Deviations from this balance lead to premature degradation of normal maturation intermediates or persistence of misfolded proteins, either of which can cause disease (2,4,5). Although accurate triage between biosynthesis and degradation is essential for maintaining protein homeostasis, a mechanistic understanding of protein triage for any pathway has been hampered by the lack of a fully reconstituted system that faithfully recapitulates both the biosynthetic and quality control outcomes of a nascent protein.To achieve this goal, we chose the pathway of tail-anchored (TA) membrane protein insertion as a model. TA proteins engage a conserved and well-defined pathway for targeting and insertion at the ER (6-8), which in mammals is monitored by an embedded quality control (QC) pathway (9, 10) to degrade products that fail targeting ( fig. S1). Studies on the yeast TA targeting pathway (7,(11)(12)(13) and the mammalian TA targeting (6, 14-16) and ubiquitination (9, 10) reactions suggest that six core factors constitute a minimal mammalian triage system amenable to complete reconstitution.Three of the factors (SGTA, Bag6, and TRC40) can recognize and shield the transmembrane domain (TMD) of a TA protein client (6,(16)(17)(18) Analyses with purified factors rigorously established the sufficiency of these modules. In these experiments, radiolabeled TA protein is synthesized with purified E. coli translation factors supplemented with the desired TMD-binding chaperone ( fig. S5). This orthogonal system produces homogeneous TA-chaperone complexes that can be used for downstream functional assays (24). Isolated TA-TRC40 complex was competent for ER targeting and insertion ( Fig. 2A), but not ubiquitination by RNF126 ( fig. S6). By contrast, the TA-Bag6 complex could not mediate insertion (data not shown), but allowed ubiquitination via RNF126 recruitment to Bag6's N-terminal UBL domain (Fig. 2B).The TA-SGTA complex in iso...
Background: Pediatric cardiomyopathies are a clinically and genetically heterogeneous group of heart muscle disorders associated with high morbidity and mortality. Although knowledge of the genetic basis of pediatric cardiomyopathy has improved considerably, the underlying cause remains elusive in a substantial proportion of cases. Methods: Exome sequencing was used to screen for the causative genetic defect in a pair of siblings with rapidly progressive dilated cardiomyopathy and death in early infancy. Protein expression was assessed in patient samples, followed by an in vitro tail-anchored protein insertion assay and functional analyses in zebrafish. Results: We identified compound heterozygous variants in the highly conserved ASNA1 gene (arsA arsenite transporter, ATP-binding, homolog), which encodes an ATPase required for post-translational membrane insertion of tail-anchored proteins. The c.913C>T variant on the paternal allele is predicted to result in a premature stop codon p.(Gln305*), and likely explains the decreased protein expression observed in myocardial tissue and skin fibroblasts. The c.488T>C variant on the maternal allele results in a valine to alanine substitution at residue 163 (p.Val163Ala). Functional studies showed that this variant leads to protein misfolding as well as less effective tail-anchored protein insertion. Loss of asna1 in zebrafish resulted in reduced cardiac contractility and early lethality. In contrast to wild-type mRNA, injection of either mutant mRNA failed to rescue this phenotype. Conclusions: Biallelic variants in ASNA1 cause severe pediatric cardiomyopathy and early death. Our findings point toward a critical role of the tail-anchored membrane protein insertion pathway in vertebrate cardiac function and disease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.