scholarly journals Localization and Signaling of GβSubunit Ste4p Are Controlled by a-Factor Receptor and thea-Specific Protein Asg7p

2000 ◽  
Vol 20 (23) ◽  
pp. 8826-8835 ◽  
Author(s):  
Jinah Kim ◽  
Eric Bortz ◽  
Hualin Zhong ◽  
Thomas Leeuw ◽  
Ekkehard Leberer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Subcellular Location ◽  
Regulatory Process ◽  
Yeast Cells ◽  
Specific Gene ◽  
Β Subunit ◽  
Pheromone Signaling ◽  
Receptor Inhibition ◽  
In Cells

ABSTRACT Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein. A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed inMATa cells. Receptor inhibition blocks signaling by inhibiting the activity of the G protein β subunit, Ste4p. To investigate how Ste4p activity is inhibited, its subcellular location was examined. In wild-type cells, α-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections. In cells expressing the a-factor receptor, α-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment. An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor. The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function. ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses. The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates. ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization. These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein β subunit and causes its relocalization within the cell.

scholarly journals Receptor Inhibition of Pheromone Signaling Is Mediated by the Ste4p Gβ Subunit

1999 ◽  
Vol 19 (1) ◽  
pp. 441-449 ◽  
Author(s):  
Jinah Kim ◽  
Andrés Couve ◽  
Jeanne P. Hirsch
Keyword(s):  
G Protein ◽  
Prolonged Exposure ◽  
Receptor Gene ◽  
Specific Gene ◽  
Pheromone Response ◽  
Α Subunit ◽  
A Cells ◽  
Pheromone Signaling ◽  
Receptor Inhibition ◽  
In Cells

ABSTRACT The pheromone response pathway of the yeast Saccharomyces cerevisiae is initiated in MATa cells by binding of α-factor to the α-factor receptor. MATa cells in which the a-factor receptor is inappropriately expressed exhibit reduced pheromone signaling, a phenomenon termed receptor inhibition. In cells undergoing receptor inhibition, activation of the signaling pathway occurs normally at early time points but decreases after prolonged exposure to pheromone. Mutations that suppress the effects of receptor inhibition were obtained in the STE4 gene, which encodes the β-subunit of the G protein that transmits the pheromone response signal. These mutations mapped to the N terminus and second WD repeat of Ste4p in regions that are not part of its Gα binding surface. A STE4 allele containing several of these mutations, called STE4SD13 , reversed the signaling defect seen at late times in cells undergoing receptor inhibition but had no effect on the basal activity of the pathway. Moreover, the signaling properties of STE4SD13 were indistinguishable from those of STE4 in wild-typeMATa and MATα cells. These results demonstrate that the effect of the STE4SD13 allele is specific to the receptor inhibition function of STE4. STE4SD13 suppressed the signaling defect conferred by receptor inhibition in a MATa strain containing a deletion of GPA1, the G protein α-subunit gene; however,STE4SD13 had no effect in a MATαstrain containing a GPA1 deletion. Suppression of receptor inhibition by STE4SD13 in a MATa strain containing a GPA1 deletion was unaffected by deletion of STE2, the α-factor receptor gene. The results presented here are consistent with a model in which an a-specific gene product other than Ste2p detects the presence of the a-factor receptor and blocks signaling by inhibiting the function of Ste4p.

scholarly journals Conformational specificity of opioid receptors is determined by subcellular location irrespective of agonist

2021 ◽  
Author(s):  
Stephanie E. Crilly ◽  
Wooree Ko ◽  
Zara Y. Weinberg ◽  
Manojkumar A. Puthenveedu
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Opioid Receptors ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Subcellular Location ◽  
Unanswered Question ◽  
Receptor Coupling ◽  
Δ Opioid Receptor ◽  
G Protein Coupled

AbstractThe prevailing model for the variety in drug responses is that they stabilize distinct active states of their G protein-coupled receptor (GPCR) targets, allowing coupling to different effectors. However, whether the same ligand can produce different GPCR active states based on the environment of receptors in cells is a fundamental unanswered question. Here we address this question using live cell imaging of conformational biosensors that read out distinct active conformations of the δ-opioid receptor (DOR), a physiologically relevant GPCR localized to Golgi and the surface in neurons. We show that, although Golgi and surface pools of DOR regulated cAMP, the two pools engaged distinct conformational biosensors in response to the same ligand. Further, DOR recruited arrestin on the plasma membrane but not the Golgi. Our results suggest that the same agonist drives different conformations of a GPCR at different locations, allowing receptor coupling to distinct effectors at different locations.

scholarly journals G protein-regulated endocytic trafficking of adenylyl cyclase type 9

2020 ◽  
Author(s):  
André M. Lazar ◽  
Roshanak Irannejad ◽  
Tanya A. Baldwin ◽  
Aparna A. Sundaram ◽  
J. Silvio Gutkind ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Subcellular Location ◽  
Endocytic Pathway ◽  
Heterotrimeric G Proteins ◽  
Camp Production ◽  
Stimulation Of

SummaryGPCRs are increasingly recognized to initiate signaling via heterotrimeric G proteins as they move through the endocytic network, but little is known about how relevant G protein effectors are localized. Here we report dynamic trafficking of adenylyl cyclase type 9 (AC9) from the plasma membrane to endosomes, while adenylyl cyclase type 1 (AC1) remains in the plasma membrane, and stimulation of AC9 trafficking by ligand-induced activation of Gs-coupled GPCRs or Gs. AC9 transits a similar dynamin-dependent early endocytic pathway as activated GPCRs but, in contrast to GPCR trafficking which is regulated by β-arrestin but not Gs, AC9 trafficking is regulated by Gs but not β-arrestin. We also show that AC9, but not AC1, contributes to cAMP production from endosomes. These results reveal dynamic and isoform-specific trafficking of adenylyl cyclase in the endocytic network, and a discrete role of a heterotrimeric G protein in controlling subcellular location of a relevant effector.

scholarly journals A G-Protein β Subunit Required for Sexual and Vegetative Development and Maintenance of Normal Gα Protein Levels in Neurospora crassa

2002 ◽  
Vol 1 (3) ◽  
pp. 378-390 ◽  
Author(s):  
Qi Yang ◽  
Sheven I. Poole ◽  
Katherine A. Borkovich
Keyword(s):  
Neurospora Crassa ◽  
G Protein ◽  
Single Gene ◽  
Cryphonectria Parasitica ◽  
Female Sterility ◽  
Specific Gene ◽  
Extension Rate ◽  
Β Subunit ◽  
Submerged Cultures ◽  
Protein Levels

ABSTRACT The genome of the filamentous fungus Neurospora crassa contains a single gene encoding a heterotrimeric G-protein β subunit, gnb-1. The predicted GNB-1 protein sequence is most identical to Gβ proteins from the filamentous fungi Cryphonectria parasitica and Aspergillus nidulans. N. crassa GNB-1 is also 65% identical to the human GNB-1 protein but only 38 and 45% identical to Gβ proteins from budding and fission yeasts. Previous studies in animal and fungal systems have elucidated phenotypes of Gβ null mutants, but little is known about the effects of Gβ loss on Gα levels. In this study, we analyzed a gnb-1 deletion mutant for cellular phenotypes and levels of the three Gα proteins. Δgnb-1 strains are female-sterile, with production of aberrant fertilized reproductive structures. Δgnb-1 strains conidiate more profusely and have altered mass on solid medium. Loss of gnb-1 leads to inappropriate conidiation and expression of a conidiation-specific gene during growth in submerged culture. Intracellular cyclic AMP levels are reduced by 60% in vegetative plate cultures of Δgnb-1 mutants. Loss of gnb-1 leads to lower levels of the three Gα proteins under a variety of conditions. Analysis of transcript levels for the gna-1 and gna-2 Gα genes in submerged cultures indicates that regulation of Gα protein levels by gnb-1 is posttranscriptional. The results suggest that GNB-1 directly regulates apical extension rate and mass accumulation. In contrast, many other Δgnb-1 phenotypes, including female sterility and defective conidiation, can be explained by altered levels of the three N. crassa Gα proteins.

scholarly journals Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.

1984 ◽  
Vol 99 (3) ◽  
pp. 1101-1109 ◽  
Author(s):  
A A Rogalski ◽  
J E Bergmann ◽  
S J Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
G Protein ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Temperature Shift ◽  
Surface Expression ◽  
Microtubule Assembly ◽  
In Cells

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.

scholarly journals Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

2000 ◽  
Vol 11 (3) ◽  
pp. 957-968 ◽  
Author(s):  
Carol L. Manahan ◽  
Madhavi Patnana ◽  
Kendall J. Blumer ◽  
Maurine E. Linder
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Yeast Cells ◽  
Pheromone Response ◽  
Guanine Nucleotide Binding Protein ◽  
Α Subunit ◽  
Protein Activation ◽  
Pheromone Response Pathway ◽  
G Protein Activation

To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide–binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein γ subunit (Ste18p) is unusual among Gγsubunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the Gγsubunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of Gβγafter receptor-stimulated release from Gα. The G protein α subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway.

scholarly journals Feedback Phosphorylation of the Yeast a-Factor Receptor Requires Activation of the Downstream Signaling Pathway from G Protein through Mitogen-Activated Protein Kinase

2000 ◽  
Vol 20 (2) ◽  
pp. 563-574 ◽  
Author(s):  
Ying Feng ◽  
Nicholas G. Davis
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Signaling Pathway ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Striking Difference ◽  
Signaling Mechanism ◽  
Pheromone Signaling ◽  
Mitogen Activated Protein

ABSTRACT The two yeast pheromone receptors, the a and α-factor receptors, share many functional similarities: both G protein-coupled receptors couple to the same downstream signal transduction pathway, and both receptors undergo feedback regulation involving increased phosphorylation on their C-terminal domains in response to ligand challenge. The present work, which focuses on the signaling mechanism controlling this feedback phosphorylation, indicates one striking difference. While the α-factor-induced phosphorylation of the α-factor receptor does not require activation of the downstream G protein-directed signaling pathway (B. Zanolari, S. Raths, B. Singer-Kruger, and H. Riezman, Cell 71:755–763, 1992), the a-factor-induced phosphorylation of the a-factor receptor (Ste3p) clearly does. Induced Ste3p phosphorylation was blocked in cells with disruptions of various components of the pheromone response pathway, indicating a requirement of pathway components extending from the G protein down through the mitogen-activated protein kinase (MAPK). Furthermore, Ste3p phosphorylation can be induced in the absence of the a-factor ligand when the signaling pathway is artificially activated, indicating that the liganded receptor is not required as a substrate for induced phosphorylation. While the activation of signaling is critical for the feedback phosphorylation of Ste3p, pheromone-induced gene transcription, one of the major outcomes of pheromone signaling, appears not to be required. This conclusion is indicated by three results. First,ste12Δ cells differ from cells with disruptions of the upstream signaling elements (e.g., ste4Δ,ste20Δ, ste5Δ, ste11Δ,ste7Δ, or fus3Δ kss1Δ cells) in that they clearly retain some capacity for inducing Ste3p phosphorylation. Second, while activated alleles of STE11 andSTE12 induce a strong transcriptional response, they fail to induce a-factor receptor phosphorylation. Third, blocking of new pheromone-induced protein synthesis with cycloheximide fails to block phosphorylation. These findings are discussed within the context of a recently proposed model for pheromone signaling (P. M. Pryciak and F. A. Huntress, Genes Dev. 12:2684–2697, 1998): a key step of this model is the activation of the MAPK Fus3p through the Gβγ-dependent relocalization of the Ste5p-MAPK cascade to the plasma membrane. Ste3p phosphorylation may involve activated MAPK Fus3p feeding back upon plasma membrane targets.

scholarly journals Mating yeast cells use an intrinsic polarity site to assemble a pheromone-gradient tracking machine

2019 ◽  
Vol 218 (11) ◽  
pp. 3730-3752 ◽  
Author(s):  
Xin Wang ◽  
Wei Tian ◽  
Bryan T. Banh ◽  
Bethanie-Michelle Statler ◽  
Jie Liang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
G Protein ◽  
Yeast Cells ◽  
Mating Types ◽  
Gradient Sensing ◽  
Feedback Mechanisms ◽  
Growth Site ◽  
Endocytic Machinery ◽  
Mating Projection

The mating of budding yeast depends on chemotropism, a fundamental cellular process. The two yeast mating types secrete peptide pheromones that bind to GPCRs on cells of the opposite type. Cells find and contact a partner by determining the direction of the pheromone source and polarizing their growth toward it. Actin-directed secretion to the chemotropic growth site (CS) generates a mating projection. When pheromone-stimulated cells are unable to sense a gradient, they form mating projections where they would have budded in the next cell cycle, at a position called the default polarity site (DS). Numerous models have been proposed to explain yeast gradient sensing, but none address how cells reliably switch from the intrinsically determined DS to the gradient-aligned CS, despite a weak spatial signal. Here we demonstrate that, in mating cells, the initially uniform receptor and G protein first polarize to the DS, then redistribute along the plasma membrane until they reach the CS. Our data indicate that signaling, polarity, and trafficking proteins localize to the DS during assembly of what we call the gradient tracking machine (GTM). Differential activation of the receptor triggers feedback mechanisms that bias exocytosis upgradient and endocytosis downgradient, thus enabling redistribution of the GTM toward the pheromone source. The GTM stabilizes when the receptor peak centers at the CS and the endocytic machinery surrounds it. A computational model simulates GTM tracking and stabilization and correctly predicts that its assembly at a single site contributes to mating fidelity.

Heterogeneity of Size and Distribution of Intramembrane Particles in the Plasma Membrane of Yeast

1977 ◽  
Vol 35 ◽  
pp. 596-597
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Synthetic Medium ◽  
Freeze Fracture ◽  
Translational Diffusion ◽  
Yeast Cells ◽  
Distilled Water ◽  
Intramembrane Particles ◽  
The Matrix ◽  
Water Cell

The matrix of biological membranes consists of a lipid bilayer into which proteins or protein aggregates are intercalated. Freeze-fracture techni- ques permit these proteins, perhaps in association with lipids, to be visualized in the hydrophobic regions of the membrane. Thus, numerous intramembrane particles (IMP) have been found on the fracture faces of membranes from a wide variety of cells (1-3). A recognized property of IMP is their tendency to form aggregates in response to changes in experi- mental conditions (4,5), perhaps as a result of translational diffusion through the viscous plane of the membrane. The purpose of this communica- tion is to describe the distribution and size of IMP in the plasma membrane of yeast (Candida utilis).Yeast cells (ATCC 8205) were grown in synthetic medium (6), and then harvested after 16 hours of culture, and washed twice in distilled water. Cell pellets were suspended in growth medium supplemented with 30% glycerol and incubated for 30 minutes at 0°C, centrifuged, and prepared for freeze-fracture, as described earlier (2,3).

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