Polarization of the Yeast Pheromone Receptor Requires Its Internalization but Not Actin-dependent Secretion

Suchkov, Dmitry V.; DeFlorio, Reagan; Draper, Edward; Ismael, Amber; Sukumar, Madhushalini; Arkowitz, Robert A.; Stone, David E.

doi:10.1091/mbc.e09-08-0706

Cited by 42 publications

(82 citation statements)

References 69 publications

(90 reference statements)

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“…In addition to its role in recovery from pheromone, receptor endocytosis may contribute to generating the receptor distribution for the polarized chemotropic growth that occurs during mating (51). Most importantly, our work answers previously unresolved questions about how Ste2 is recognized by Rsp5 to promote both its basal and ligand-induced endocytosis.…”

Section: Discussionmentioning

confidence: 63%

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Specific α-Arrestins Negatively Regulate Saccharomyces cerevisiae Pheromone Response by Down-Modulating the G-Protein-Coupled Receptor Ste2

Alvaro

O’Donnell

Prosser

et al. 2014

Molecular and Cellular Biology

159

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e G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate responses to extracellular stimuli by mediating ligand-dependent activation of cognate heterotrimeric G proteins. In yeast, occupancy of GPCR Ste2 by peptide pheromone ␣-factor initiates signaling by releasing a stimulatory G␤␥ complex (Ste4-Ste18) from its inhibitory G␣ subunit (Gpa1). Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G 1 arrest, including upregulation of the expression of an ␣-factor-degrading protease (Bar1), a regulator of G-protein signaling protein (Sst2) that stimulates Gpa1-GTP hydrolysis, and Gpa1 itself. Ste2 is also downregulated by endocytosis, both constitutive and ligand induced. Ste2 internalization requires its phosphorylation and subsequent ubiquitinylation by membrane-localized protein kinases (Yck1 and Yck2) and a ubiquitin ligase (Rsp5). Here, we demonstrate that three different members of the ␣-arrestin family (Ldb19/Art1, Rod1/Art4, and Rog3/ Art7) contribute to Ste2 desensitization and internalization, and they do so by discrete mechanisms. We provide genetic and biochemical evidence that Ldb19 and Rod1 recruit Rsp5 to Ste2 via PPXY motifs in their C-terminal regions; in contrast, the arrestin fold domain at the N terminus of Rog3 is sufficient to promote adaptation. Finally, we show that Rod1 function requires calcineurin-dependent dephosphorylation.

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Section: Discussionmentioning

confidence: 63%

“…Subsequent studies have provided more detailed analysis of spatiotemporal aspects of Ste2 (50,51) and Ste3 (52,53) internalization and better delineated the phosphorylation and ubiquitinylation sites (54,55). However, how Rsp5 recognizes and is recruited to these GPCRs to catalyze ubiquitinylation remained unresolved.…”

mentioning

confidence: 99%

Specific α-Arrestins Negatively Regulate Saccharomyces cerevisiae Pheromone Response by Down-Modulating the G-Protein-Coupled Receptor Ste2

Alvaro

O’Donnell

Prosser

et al. 2014

Molecular and Cellular Biology

159

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“…Gpa1 was recently shown to concentrate to the bud neck during G 2 /M phase, where Elm1 is located (58).…”

Section: Discussionmentioning

confidence: 99%

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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“…To determine whether receptor polarity influences the tendency of Gb P2 bud1D cells to form proximal mating projections, we constructed Gb P2 bud1D and Gb bud1D strains expressing the Ste2 7XR/GPAAD mutant form of the receptor. Ste2 7XR/GPAAD cannot be internalized (Ballon et al, 2006) and therefore does not polarize in response to pheromone (Suchkov et al, 2010). Although cells expressing Ste2 7XR/GPAAD also formed blunt shmoos, preventing receptor polarization suppressed the formation of proximal projections by Gb P2 bud1D cells (Fig.…”

Section: Gbmentioning

confidence: 93%

“…Pheromone triggers polarization of the receptor and G-protein to the mating projection (Suchkov et al, 2010) -the same area where the growth site wanders in Gb P2 bud1D cells. This confined wandering might underlie the formation of proximal projections.…”

Section: Gbmentioning

confidence: 99%

Gβ phosphorylation is critical for efficient chemotropism in yeast

DeFlorio¹,

Brett²,

Waszczak³

et al. 2013

Journal of Cell Science

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SummaryMating yeast cells interpret complex pheromone gradients and polarize their growth in the direction of the closest partner. Chemotropic growth depends on both the pheromone receptor and its associated G-protein. Upon activation by the receptor, Ga dissociates from Gbc and Gb is subsequently phosphorylated. Free Gbc signals to the nucleus via a MAPK cascade and recruits Far1-Cdc24 to the incipient growth site. It is not clear how the cell establishes and stabilizes the axis of polarity, but this process is thought to require local signal amplification via the Gbc-Far1-Cdc24 chemotropic complex, as well as communication between this complex and the activated receptor. Here we show that a mutant form of Gb that cannot be phosphorylated confers defects in directional sensing and chemotropic growth. Our data suggest that phosphorylation of Gb plays a role in localized signal amplification and in the dynamic communication between the receptor and the chemotropic complex, which underlie growth site selection and maintenance.

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Polarization of the Yeast Pheromone Receptor Requires Its Internalization but Not Actin-dependent Secretion

Abstract: The data presented in this paper suggest that pheromone-induced receptor phosphorylation and internalization, but not actin-dependent directed secretion, are required to establish receptor polarity.

Cited by 42 publications

References 69 publications

Specific α-Arrestins Negatively Regulate Saccharomyces cerevisiae Pheromone Response by Down-Modulating the G-Protein-Coupled Receptor Ste2

Specific α-Arrestins Negatively Regulate Saccharomyces cerevisiae Pheromone Response by Down-Modulating the G-Protein-Coupled Receptor Ste2

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

Gβ phosphorylation is critical for efficient chemotropism in yeast

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