Differential Regulation of G Protein α Subunit Trafficking by Mono- and Polyubiquitination

Wang, Yuqi; Marotti, Louis A.; Lee, Michael J.; Dohlman, Henrik G.

doi:10.1074/jbc.m411624200

Cited by 35 publications

(63 citation statements)

References 61 publications

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“…⌬UD lacks a domain (UD, residues 129 -236) that is required for ubiquitination by SCF as well as phosphorylation by Elm1 (4,21,22). The Gpa1 ubiquitination domain cannot be the only target of phosphorylation, however, because mutation of all 15 serine and threonine residues within this region failed to diminish phosphorylation in vitro (data not shown) or ubiquitination in vivo (supplemental Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, we asked whether Elm1 phosphorylation promotes Gpa1 polyubiquitination. We compared Gpa1 polyubiquitination in the presence and absence of ELM1 using a proteasome-deficient yeast strain, cim3-1 (22,23). For these experiments, Gpa1 was overexpressed to allow detection of the minor ubiquitinated species.…”

Section: Gpa1mentioning

confidence: 99%

“…In Vivo Ubiquitination Assays-Gpa1 polyubiquitination was detected by constitutive (ADH1 promoter) expression of Gpa1 in yeast harboring a temperature-sensitive proteasome mutation (cim3-1) or by coexpression of Gpa1 and Myc-ubiquitin as described previously (22). For cim3-1 and isogenic wild type cells, log-phase cultures were grown at the permissive temperature (25°C) to an A 600 nm of 0.5-0.6, followed by transition to the restrictive temperature (37°C) for 3 h. Inducible Mycubiquitin strains were grown at 30°C to an A 600 nm of 0.5-0.6, followed by addition of CuSO 4 to 100 M for 3 h at 30°C as described previously (29).…”

Section: S200ementioning

confidence: 99%

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Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein α Subunit

Torres

Clement

Cappell

et al. 2011

Journal of Biological Chemistry

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A diverse array of external stimuli, including most hormones and neurotransmitters, bind to cell surface receptors that activate G proteins. Mating pheromones in yeast Saccharomyces cerevisiae activate G protein-coupled receptors and initiate events leading to cell cycle arrest in G 1 phase. Here, we show that the G␣ subunit (Gpa1) is phosphorylated and ubiquitinated in response to changes in the cell cycle. We systematically screened 109 gene deletion strains representing the non-essential yeast kinome and identified a single kinase gene, ELM1, as necessary and sufficient for Gpa1 phosphorylation. Elm1 is expressed in a cell cycle-dependent manner, primarily at S and G 2 /M. Accordingly, phosphorylation of Gpa1 in G 2 /M phase leads to polyubiquitination in G 1 phase. These findings demonstrate that Gpa1 is dynamically regulated. More broadly, they reveal how G proteins can simultaneously regulate, and become regulated by, progression through the cell cycle.

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Section: Discussionmentioning

confidence: 99%

Section: Gpa1mentioning

confidence: 99%

Section: S200ementioning

confidence: 99%

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Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein α Subunit

Torres

Clement

Cappell

et al. 2011

Journal of Biological Chemistry

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“…Pheromone pathway signaling is attenuated by the pheromone-dependent monoubiquitination and endocytosis of the G protein-coupled receptor Ste2 (16). As with the receptor, monoubiquitination leads to the endocytosis and eventual vacuolar degradation of Gpa1 (25). In contrast to Ste2, monoubiquitination of Gpa1 is not dependent on pheromone stimulation.…”

mentioning

confidence: 99%

“…Site-directed mutagenesis was used to introduce a silent mutation at bp 181 to generate an AflII site for integration. Construction of the pRS406-GPA1-GFP, pRS406-GPA1 ⌬UD -GFP, pRS423-FUS1-lacZ, and pADM4-GPA1 vectors has been described previously (25,33,34).Bacterial Protein Expression and Purification-Generating the quantities of pure Gpa1 necessary to complete biophysical studies has been a barrier to progress both because of low initial protein expression and impurities, necessitating additional purification steps. We optimized the process using small-scale batch purification and TEV protease-cleavage to release protein from the nickel beads (Table 1).…”

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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cry1 and GPA1 signaling genetically interact in hook opening and anthocyanin synthesis in Arabidopsis

et al. 2012

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While studying blue light-independent effects of cryptochrome 1 (cry1) photoreceptor, we observed premature opening of the hook in cry1 mutants grown in complete darkness, a phenotype that resembles the one described for the heterotrimeric G-protein α subunit (GPA1) null mutant gpa1. Both cry1 and gpa1 also showed reduced accumulation of anthocyanin under blue light. These convergent gpa1 and cry1 phenotypes required the presence of sucrose in the growth media and were not additive in the cry1 gpa1 double mutant, suggesting context-dependent signaling convergence between cry1 and GPA1 signaling pathways. Both, gpa1 and cry1 mutants showed reduced GTP-binding activity. The cry1 mutant showed wild-type levels of GPA1 mRNA or GPA1 protein. However, an anti-transducin antibody (AS/7) typically used for plant Gα proteins, recognized a 54 kDa band in the wild type but not in gpa1 and cry1 mutants. We propose a model where cry1-mediated post-translational modification of GPA1 alters its GTP-binding activity.

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Differential Regulation of G Protein α Subunit Trafficking by Mono- and Polyubiquitination

Cited by 35 publications

References 61 publications

Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein α Subunit

Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein α Subunit

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

cry1 and GPA1 signaling genetically interact in hook opening and anthocyanin synthesis in Arabidopsis

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