G Protein Mono-ubiquitination by the Rsp5 Ubiquitin Ligase

Torres, Matthew P.; Lee, Michael J.; Ding, Feng; Purbeck, Carrie; Kuhlman, Brian; Dokholyan, Nikolay V.; Dohlman, Henrik G.

doi:10.1074/jbc.m809058200

Cited by 27 publications

(31 citation statements)

References 63 publications

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“…Third, Rsp5 is capable of ubiquitinating Ste4 in vitro. Notably, earlier studies showed that Rsp5 is also responsible for the ubiquitination of Ste2 (receptor) (44) and the monoubiquitination (but not the polyubiquitination) of Gpa1 (G␣) (43). Thus, Ste4 joins a growing list of signaling proteins that serve as substrates of Rsp5.…”

Section: Discussionmentioning

confidence: 99%

“…This is in marked contrast with both Ste2 and Gpa1. In both cases, ubiquitination by Rsp5 serves to target the proteins for internalization and eventual degradation in the vacuole (43,44).…”

Section: Discussionmentioning

confidence: 99%

“…Monoubiquitination of many plasma membrane-associated proteins, including Ste2 and Gpa1, is catalyzed by Rsp5, a HECT-type E3 ubiquitin ligase (43,44). One unique feature of Rsp5 is that it can form strong interactions with its substrates (45).…”

Section: Ubiquitination Of Ste4 Is Dependent On the E3 Ligase Rsp5-mentioning

confidence: 99%

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Pheromone- and RSP5-dependent Ubiquitination of the G Protein β Subunit Ste4 in Yeast

Zhu

Torres

Kelley

et al. 2011

Journal of Biological Chemistry

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Ste4 is the ␤ subunit of a heterotrimeric G protein that mediates mating responses in Saccharomyces cerevisiae. Here we show that Ste4 undergoes ubiquitination in response to pheromone stimulation. Ubiquitination of Ste4 is dependent on the E3 ligase Rsp5. Disrupting the activity of Rsp5 abolishes ubiquitination of Ste4 in vivo, and recombinant Rsp5 is capable of ubiquitinating Ste4 in vitro. We find also that Lys-340 is a major ubiquitination site on Ste4, as pheromone-induced ubiquitination of the protein is prevented when this residue is mutated to an arginine. Functionally, ubiquitination does not appear to regulate the stability of Ste4, as blocking ubiquitination has no apparent effect on either the abundance or the half-life of the protein. However, when presented with a concentration gradient of pheromone, Ste4 K340R mutant cells polarize significantly faster than wild-type cells, indicating that ubiquitination limits pheromone-directed polarized growth. Together, these findings reveal a novel stimulus-dependent posttranslational modification of a G␤ subunit, establish Ste4 as a new substrate of the E3 ligase Rsp5, and demonstrate a role for G protein ubiquitination in cell polarization.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Pheromone- and RSP5-dependent Ubiquitination of the G Protein β Subunit Ste4 in Yeast

Zhu

Torres

Kelley

et al. 2011

Journal of Biological Chemistry

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“…However, the UD functions as a site of phosphorylation, polyubiquitination (for targeting to the proteasome), and monoubiquitination (for trafficking to the vacuole) (25, 26, 28 -31). Gpa1 is monoubiquitinated by the Rsp5 ubiquitin ligase (26). However, the proteins involved in Gpa1 endocytosis and specifically those that recognize monoubiquitin within the UD are not known.…”

Section: Structural Contributions Of the Ubiquitination Domain Tomentioning

confidence: 99%

“…In contrast to Ste2, monoubiquitination of Gpa1 is not dependent on pheromone stimulation. Both proteins are monoubiquitinated by the same ubiquitin ligase, Rsp5 (26,27).…”

mentioning

confidence: 99%

Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast

Dixit¹,

Baker²,

Sacks³

et al. 2014

Journal of Biological Chemistry

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Background:The yeast G␣ protein contains a unique domain that is monoubiquitinated, leading to vacuolar degradation. Results: A gene deletion screen reveals ubiquitin binding domain proteins necessary for G␣ trafficking. Loss of the ubiquitination domain impedes cellular morphogenesis and mating. Conclusion: Proper endocytosis of G␣ is required for sustained morphogenesis and efficient mating. Significance: G␣ endocytosis promotes signaling. Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins.There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein ␣ subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the ␣-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.G␣ proteins are enzymatic switches that are part of a multicomponent signaling complex. The complex typically consists of a seven-transmembrane G protein-coupled receptor, a guanine nucleotide-binding protein (G␣), and an associated dimer consisting of ␤ and ␥ subunits (G␤␥) (1). Signaling is turned on and off based on receptor activation, which in turn dictates the nucleotide-bound state of the G␣ protein. When G␣ is GDPbound, G␤␥ is sequestered, and signaling pathways are off (1). When G␣ releases GDP and binds GTP, G␤␥ dissociates, and the signaling pathways are turned on. Subsequent GTP hydrolysis is accelerated by regulators of G protein signaling (RGS 3 proteins) (2, 3). Large G␣ proteins contain a Ras-like domain as well as an independently folded ␣-helical domain (1). Within this group of proteins there is a well established role for the Ras-like domain in specifying interactions with G␤␥, effectors, and RGS proteins (1). However, recent evidence has shown that the ␣-helical domain is also important for signa...

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Prefusion spike protein stabilization through computational mutagenesis

2020

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A novel severe acute respiratory syndrome (SARS)‐like coronavirus (SARS‐CoV‐2) has emerged as a human pathogen, causing global pandemic and resulting in over 400 000 deaths worldwide. The surface spike protein of SARS‐CoV‐2 mediates the process of coronavirus entry into human cells by binding angiotensin‐converting enzyme 2 (ACE2). Due to the critical role in viral‐host interaction and the exposure of spike protein, it has been a focus of most vaccines' developments. However, the structural and biochemical studies of the spike protein are challenging because it is thermodynamically metastable. Here, we develop a new pipeline that automatically identifies mutants that thermodynamically stabilize the spike protein. Our pipeline integrates bioinformatics analysis of conserved residues, motion dynamics from molecular dynamics simulations, and other structural analysis to identify residues that significantly contribute to the thermodynamic stability of the spike protein. We then utilize our previously developed protein design tool, Eris, to predict thermodynamically stabilizing mutations in proteins. We validate the ability of our pipeline to identify protein stabilization mutants through known prefusion spike protein mutants. We finally utilize the pipeline to identify new prefusion spike protein stabilization mutants.

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G Protein Mono-ubiquitination by the Rsp5 Ubiquitin Ligase

Cited by 27 publications

References 63 publications

Pheromone- and RSP5-dependent Ubiquitination of the G Protein β Subunit Ste4 in Yeast

Pheromone- and RSP5-dependent Ubiquitination of the G Protein β Subunit Ste4 in Yeast

Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast

Prefusion spike protein stabilization through computational mutagenesis

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