In eukaryotic cells a molecular chaperone network associates with translating ribosomes, assisting the maturation of emerging nascent polypeptides. Hsp70 is perhaps the major eukaryotic ...ribosome-associated chaperone and the first reported to bind cotranslationally to nascent chains. However, little is known about the underlying principles and function of this interaction. Here, we use a sensitive and global approach to define the cotranslational substrate specificity of the yeast Hsp70 SSB. We find that SSB binds to a subset of nascent polypeptides whose intrinsic properties and slow translation rates hinder efficient cotranslational folding. The SSB-ribosome cycle and substrate recognition is modulated by its ribosome-bound cochaperone, RAC. Deletion of SSB leads to widespread aggregation of newly synthesized polypeptides. Thus, cotranslationally acting Hsp70 meets the challenge of folding the eukaryotic proteome by stabilizing its longer, more slowly translated, and aggregation-prone nascent polypeptides.
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► Yeast Hsp70 SSB binds cotranslationally to a defined set of nascent polypeptides ► SSB shows specificity for substrates highly challenged in cotranslational folding ► The heterodimeric cochaperone RAC modulates cotranslational SSB specificity ► Loss of SSB function leads to widespread misfolding and aggregation
The Hsp70 molecular chaperone preferentially binds nascent polypeptides susceptible to misfolding and aggregation to cotranslationally promote the synthesis of a functional eukaryotic proteome.
Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the ...functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory “hot spots” that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.
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► We compiled a resource of nearly 200,000 PTMs across 11 eukaryotic species ► Computational methods were developed to assign function to PTMs ► Conservation studies identify regulatory regions within domain families ► Evolutionary analysis suggests that only a fraction of PTMs are functionally important
Analysis of 200,000 PTMs across 11 species in the light of structural and interaction data reveals ground rules for establishing whether a PTM is likely to impact protein function and suggests that only a fraction of modification sites are likely to be functionally important.
Osh6 and Osh7 are lipid transfer proteins (LTPs) that move phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM). High PS levels at the PM are key for many cellular ...functions. Intriguingly, Osh6 and Osh7 localize to ER-PM contact sites, although they lack membrane-targeting motifs, in contrast to multidomain LTPs that both bridge membranes and convey lipids. We show that Osh6 localization to contact sites depends on its interaction with the cytosolic tail of the ER-PM tether Ist2, a homolog of TMEM16 proteins. We identify a motif in the Ist2 tail, conserved in yeasts, as the Osh6-binding region, and we map an Ist2-binding surface on Osh6. Mutations in the Ist2 tail phenocopy
deletion: they decrease cellular PS levels and block PS transport to the PM. Our study unveils an unexpected partnership between a TMEM16-like protein and a soluble LTP, which together mediate lipid transport at contact sites.This article has an associated First Person interview with the first author of the paper.
Yeast cells have a remarkable ability to adapt to nutritional changes in their environment. During adaptation, nutrient-signaling pathways drive the selective endocytosis of nutrient transporters ...present at the cell surface. A current challenge is to understand the mechanistic basis of this regulation. Transporter endocytosis is triggered by their ubiquitylation, which involves the ubiquitin ligase Rsp5 and its adaptors of the arrestin-related family (ART). This step is highly regulated by nutrient availability. For instance, the monocarboxylate transporter Jen1 is ubiquitylated, endocytosed, and degraded upon exposure to glucose. The ART protein Rod1 is required for this overall process; yet Rod1 rather controls Jen1 trafficking later in the endocytic pathway and is almost dispensable for Jen1 internalization. Thus, how glucose triggers Jen1 internalization remains unclear. We report that another ART named Bul1, but not its paralogue Bul2, contributes to Jen1 internalization. Bul1 responds to glucose availability, and preferentially acts at the plasma membrane for Jen1 internalization. Thus, multiple ARTs can act sequentially along the endocytic pathway to control transporter homeostasis. Moreover, Bul1 is in charge of Jen1 endocytosis after cycloheximide treatment, suggesting that the functional redundancy of ARTs may be explained by their ability to interact with multiple cargoes in various conditions.
2-deoxyglucose is a glucose analog that impacts many aspects of cellular physiology. After its uptake and its phosphorylation into 2-deoxyglucose-6-phosphate (2DG6P), it interferes with several ...metabolic pathways including glycolysis and protein N-glycosylation. Despite this systemic effect, resistance can arise through strategies that are only partially understood. In yeast, 2DG resistance is often associated with mutations causing increased activity of the yeast 5’-AMP activated protein kinase (AMPK), Snf1. Here we focus on the contribution of a Snf1 substrate in 2DG resistance, namely the alpha-arrestin Rod1 involved in nutrient transporter endocytosis. We report that 2DG triggers the endocytosis of many plasma membrane proteins, mostly in a Rod1-dependent manner. Rod1 participates in 2DG-induced endocytosis because 2DG, following its phosphorylation by hexokinase Hxk2, triggers changes in Rod1 post-translational modifications and promotes its function in endocytosis. Mechanistically, this is explained by a transient, 2DG-induced inactivation of Snf1/AMPK by protein phosphatase 1 (PP1). We show that 2DG-induced endocytosis is detrimental to cells, and the lack of Rod1 counteracts this process by stabilizing glucose transporters at the plasma membrane. This facilitates glucose uptake, which may help override the metabolic blockade caused by 2DG, and 2DG export—thus terminating the process of 2DG detoxification. Altogether, these results shed a new light on the regulation of AMPK signaling in yeast and highlight a remarkable strategy to bypass 2DG toxicity involving glucose transporter regulation.
Molecular chaperones assist cellular protein folding as well as oligomeric complex assembly. In eukaryotic cells, several chaperones termed chaperones linked to protein synthesis (CLIPS) are ...transcriptionally and physically linked to ribosomes and are implicated in protein biosynthesis. In this study, we show that a CLIPS network comprising two ribosome-anchored J-proteins, Jjj1 and Zuo1, function together with their partner Hsp70 proteins to mediate the biogenesis of ribosomes themselves. Jjj1 and Zuo1 have overlapping but distinct functions in this complex process involving the coordinated assembly and remodeling of dozens of proteins on the ribosomal RNA (rRNA). Both Jjj1 and Zuo1 associate with nuclear 60S ribosomal biogenesis intermediates and play an important role in nuclear rRNA processing, leading to mature 25S rRNA. In addition, Zuo1, acting together with its Hsp70 partner, SSB (stress 70 B), also participates in maturation of the 35S rRNA. Our results demonstrate that, in addition to their known cytoplasmic roles in de novo protein folding, some ribosome-anchored CLIPS chaperones play a critical role in nuclear steps of ribosome biogenesis.
Nutrient availability controls the landscape of nutrient transporters present at the plasma membrane, notably by regulating their ubiquitylation and subsequent endocytosis. In yeast, this involves ...the Nedd4 ubiquitin ligase Rsp5 and arrestin-related trafficking adaptors (ARTs). ARTs are targeted by signaling pathways and warrant that cargo ubiquitylation and endocytosis appropriately respond to nutritional inputs. Here, we show that glucose deprivation regulates the ART protein Csr2/Art8 at multiple levels to trigger high-affinity glucose transporter endocytosis. Csr2 is transcriptionally induced in these conditions through the AMPK orthologue Snf1 and downstream transcriptional repressors. Upon synthesis, Csr2 becomes activated by ubiquitylation. In contrast, glucose replenishment induces
transcriptional shutdown and switches Csr2 to an inactive, deubiquitylated form. This glucose-induced deubiquitylation of Csr2 correlates with its phospho-dependent association with 14-3-3 proteins and involves protein kinase A. Thus, two glucose signaling pathways converge onto Csr2 to regulate hexose transporter endocytosis by glucose availability. These data illustrate novel mechanisms by which nutrients modulate ART activity and endocytosis.
The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the ...experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called “Ubiquiton” system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.
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•Ubiquiton affords inducible substrate-specific M1-, K48-, or K63-polyubiquitylation•Ubiquiton combines custom linkage-specific E3s with cognate modification sites•The K48-Ubiquiton acts as a rapamycin-inducible degron in yeast and human cells•K63-polyubiquitylation is sufficient for endocytosis of a plasma membrane protein
Analytical tools for investigating ubiquitin signaling are abundant, but methods to induce linkage-specific polyubiquitylation of a protein of interest have been missing. Renz et al. have now developed rapamycin-inducible E3s specific for three major linkages and validated them for use in vitro in budding yeast and in mammalian cells.
Molecular chaperones assist the folding of newly translated and stress-denatured proteins. In prokaryotes, overlapping sets of chaperones mediate both processes. In contrast, we find that eukaryotes ...evolved distinct chaperone networks to carry out these functions. Genomic and functional analyses indicate that in addition to stress-inducible chaperones that protect the cellular proteome from stress, eukaryotes contain a stress-repressed chaperone network that is dedicated to protein biogenesis. These stress-repressed chaperones are transcriptionally, functionally, and physically linked to the translational apparatus and associate with nascent polypeptides emerging from the ribosome. Consistent with a function in de novo protein folding, impairment of the translation-linked chaperone network renders cells sensitive to misfolding in the context of protein synthesis but not in the context of environmental stress. The emergence of a translation-linked chaperone network likely underlies the elaborate cotranslational folding process necessary for the evolution of larger multidomain proteins characteristic of eukaryotic cells.
Background Information
Comprehensive libraries of plasmids for SARS‐CoV‐2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential ...protein–protein interactions between the SARS‐CoV‐2 virus and host proteins.
Results
We present here a large library of SARS CoV‐2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS‐2B), as well as in budding yeast. The localisation of a few SARS‐CoV‐2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria.
Conclusions and Significance
This library should facilitate further cellular investigations, notably by imaging techniques.
Research article: Comprehensive proteomic studies have suggested numerous interactions between SARS‐CoV‐2 and host cellular proteins.
We prepared SARS‐CoV‐2 GFP or mCherry fusion constructs and analysed their cellular localisation.
The localisation a few SARS‐CoV‐2 fluorescent proteins matches their proposed molecular interactions.
These include Nsp13 at the centrosome, Orf3a on late endosomes and Orf9b at mitochondria.
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