scholarly journals The KlGpa1 Gene Encodes a G-Protein α Subunit That Is a Positive Control Element in the Mating Pathway of the Budding Yeast Kluyveromyces lactis

2001 ◽  
Vol 183 (1) ◽  
pp. 229-234 ◽  
Author(s):  
Alma L. Saviñón-Tejeda ◽  
Laura Ongay-Larios ◽  
Julián Valdés-Rodrı́guez ◽  
Roberto Coria
Keyword(s):  
G Protein ◽  
Stop Codon ◽  
Kluyveromyces Lactis ◽  
Control Element ◽  
Wild Type ◽  
Pheromone Response ◽  
Α Subunit ◽  
Pheromone Response Pathway ◽  
G Protein Α Subunit ◽  
Mating Pathway

ABSTRACT The cloning of the gene encoding the KlGpa1p subunit was achieved by standard PCR techniques and by screening a Kluyveromyces lactis genomic library using the PCR product as a probe. The full-length open reading frame spans 1,344 nucleotides including the stop codon. The deduced primary structure of the protein (447 amino acid residues) strongly resembles that of Gpa1p, the G-protein α subunit from Saccharomyces cerevisiae involved in the mating pheromone response pathway. Nevertheless, unlike disruption ofGpa1 from S. cerevisiae, disruption ofKlGpa1 rendered viable cells with a reduced capacity to mate. Expression of a plasmidic KlGpa1 copy in a ΔKlgpa1 mutant restores full mating competence; hence we conclude that KlGpa1p plays a positive role in the mating pathway. Overexpression of the constitutive subunit KlGpa1p(K364) (GTP bound) does not induce constitutive mating; instead it partially blocks wild-type mating and is unable to reverse the sterile phenotype of ΔKlgpa1 mutant cells. K. lactis expresses a second Gα subunit, KlGpa2p, which is involved in regulating cyclic AMP levels upon glucose stimulation. This subunit does not rescue ΔKlgpa1 cells from sterility; instead, overproduction of KlGpa2p slightly reduces the mating of wild-type cells, suggesting cross talk within the pheromone response pathway mediated by KlGpa1p and glucose metabolism mediated by KlGpa2p. The ΔKlgpa1 ΔKlgpa2 double mutant, although viable, showed the mating deficiency observed in the single ΔKlgpa1 mutant.

scholarly journals The Gα Subunit Signals through the Ste50 Protein during the Mating Pheromone Response in the Yeast Kluyveromyces lactis

2011 ◽  
Vol 10 (4) ◽  
pp. 540-546 ◽  
Author(s):  
Edith Sánchez-Paredes ◽  
Laura Kawasaki ◽  
Laura Ongay-Larios ◽  
Roberto Coria
Keyword(s):  
G Protein ◽  
Kluyveromyces Lactis ◽  
Adaptor Protein ◽  
Mitogen Activated Protein Kinase ◽  
Physical Interaction ◽  
Α Subunit ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Mating Pathway ◽  
Mating Signal

ABSTRACTYeast mating signal transduction pathways require a heterotrimeric G protein composed of Gα, Gβ, and Gγ subunits connected to a mitogen-activated protein kinase (MAPK) module. While inSaccharomyces cerevisiaeelimination of Gα induces constitutive activation of the mating pathway, inKluyveromyces lactisit produces partial sterility, which indicates thatK. lactisGα (KlGα) is required to positively activate mating. We use physical interaction experiments to determine that KlGα interacts with the adaptor protein KlSte50p. The Ras association (RA) domain of KlSte50p favored interaction with the GDP-bound KlGα subunit, and when the KlGα protein is constitutively activated, the interaction drops significantly. Additionally, KlSte50p strongly associates with the MAPK kinase kinase (MAPKKK) KlSte11p through its sterile alpha motif (SAM) domain. Genetic experiments placed KlSte50p downstream of the G protein α subunit, indicating that KlGα may stimulate the mating pathway via KlSte50p. Fusion of KlSte50p to the KlGβ subunit partially eliminated the requirement of KlGα for mating, indicating that one contribution of KlGα to the mating pathway is to facilitate plasma membrane anchoring of KlSte50p.

scholarly journals Characterization Of a G Protein α Subunit Encoded Gene From The Dimorphic Fungus-Tremella Fuciformis

2021 ◽  
Author(s):  
Hanyu Zhu ◽  
Dongmei Liu ◽  
Liesheng Zheng ◽  
Liguo Chen ◽  
Aimin Ma
Keyword(s):  
G Protein ◽  
Type Strain ◽  
Wild Type Strain ◽  
Pcr Analysis ◽  
Wild Type ◽  
Dimorphic Fungus ◽  
Α Subunit ◽  
Tremella Fuciformis ◽  
Group I ◽  
G Protein Α Subunit

Abstract Tremella fuciformis is a dimorphic fungus which can undertake the reversible transition between yeast and pseudohypha forms. G protein α subunit (Gα) carries different signals to regulate a variety of biological processes in eukaryotes, including fungal dimorphism. In this study, a novel Gα subunit encoded gene, TrGpa1, was firstly cloned from T. fuciformis. The TrGpa1 open reading frame has 1059 nucleotides, and encodes a protein which belongs to the group I of Gαi superfamily. Furthermore, the role of TrGpa1 in the T. fuciformis dimorphism was analysed by gene overexpression and knockdown. Stable integration of the target gene into the genome was confirmed by PCR and Southern blot hybridization. Transformants with the highest and lowest TrGpa1 expression levels were selected via quantitative real-time PCR analysis and Western blot. Each transformant was compared with the wild-type strain about the morphological change under different environmental factors, including pH values, temperature, cultural time, inoculum size, and quorum-sensing molecules (farnesol and tyrosol). Comparing with the wild-type strain, the overexpression transformant always had higher ratios of pseudohyphae, while the knockdown transformant had less proportions of pseudohyphae. Therefore, the TrGpa1 involves in the dimorphism of T. fuciformis and plays a positive role in promoting pseudohyphal growth.

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 The β Subunit of the Heterotrimeric G Protein Triggers the Kluyveromyces lactis Pheromone Response Pathway in the Absence of the γ Subunit

2010 ◽  
Vol 21 (3) ◽  
pp. 489-498 ◽  
Author(s):  
Rocío Navarro-Olmos ◽  
Laura Kawasaki ◽  
Lenin Domínguez-Ramírez ◽  
Laura Ongay-Larios ◽  
Rosario Pérez-Molina ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Function ◽  
Mating Disruption ◽  
Kluyveromyces Lactis ◽  
Dominant Negative ◽  
Generic Model ◽  
Wild Type ◽  
Heterotrimeric G Protein ◽  
Pheromone Response Pathway ◽  
Γ Subunit

The Kluyveromyces lactis heterotrimeric G protein is a canonical Gαβγ complex; however, in contrast to Saccharomyces cerevisiae, where the Gγ subunit is essential for mating, disruption of the KlGγ gene yielded cells with almost intact mating capacity. Expression of a nonfarnesylated Gγ, which behaves as a dominant-negative in S. cerevisiae, did not affect mating in wild-type and ΔGγ cells of K. lactis. In contrast to the moderate sterility shown by the single ΔKlGα, the double ΔKlGα ΔKlGγ mutant displayed full sterility. A partial sterile phenotype of the ΔKlGγ mutant was obtained in conditions where the KlGβ subunit interacted defectively with the Gα subunit. The addition of a CCAAX motif to the C-end of KlGβ, partially suppressed the lack of both KlGα and KlGγ subunits. In cells lacking KlGγ, the KlGβ subunit cofractionated with KlGα in the plasma membrane, but in the ΔKlGα ΔKlGγ strain was located in the cytosol. When the KlGβ-KlGα interaction was affected in the ΔKlGγ mutant, most KlGβ fractionated to the cytosol. In contrast to the generic model of G-protein function, the Gβ subunit of K. lactis has the capacity to attach to the membrane and to activate mating effectors in absence of the Gγ subunit.

scholarly journals Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (1) ◽  
pp. 168-178 ◽  
Author(s):  
L R Kao ◽  
J Peterson ◽  
R Ji ◽  
L Bender ◽  
A Bender
Keyword(s):  
G Protein ◽  
Synthetic Lethality ◽  
Wild Type ◽  
Pheromone Response ◽  
Multicopy Suppressor ◽  
Pheromone Response Pathway ◽  
Bud Emergence ◽  
Pheromone Signaling ◽  
Two Hybrid ◽  
Inducible Gene

Akr1p, which contains six ankyrin repeats, was identified during a screen for mutations that displayed synthetic lethality with a mutant allele of the bud emergence gene BEM1. Cells from which AKR1 had been deleted were alive but misshapen at 30 degrees C and inviable at 37 degrees C. During a screen for mutants that required one or more copies of wild-type AKR1 for survival at 30 degrees C, we isolated mutations in GPA1, which encodes the G alpha subunit of the pheromone receptor-coupled G protein. (The active subunit of this G protein is G beta gamma, and G alpha plays an inhibitory role in G beta gamma-mediated signal transduction.) AKR1 could serve as a multicopy suppressor of the lethality caused by either loss of GPA1 or overexpression of STE4, which encodes the G beta subunit of this G protein, suggesting that pheromone signaling is inhibited by overexpression of Akr1p. Mutations in AKR1 displayed synthetic lethality with a weak allele of GPA1 and led to increased expression of the pheromone-inducible gene FUS1, suggesting that Akr1p normally (and not just when overexpressed) inhibits signaling. In contrast, deletion of BEM1 resulted in decreased expression of FUS1, suggesting that Bem1p normally facilitates pheromone signaling. During a screen for proteins that displayed two-hybrid interactions with Akr1p, we identified Ste4p, raising the possibility that an interaction between Akr1p and Ste4p contributes to proper regulation of the pheromone response pathway.

scholarly journals The G-Protein α-Subunit GasC Plays a Major Role in Germination in the Dimorphic FungusPenicillium marneffei

Genetics ◽  
2003 ◽  
Vol 164 (2) ◽  
pp. 487-499 ◽  
Author(s):  
Sophie Zuber ◽  
Michael J Hynes ◽  
Alex Andrianopoulos
Keyword(s):  
G Protein ◽  
Germination Rate ◽  
Yeast Cells ◽  
Class Iii ◽  
Temperature Dependent ◽  
Gene Encoding ◽  
Α Subunit ◽  
G Protein Α Subunit ◽  
Opportunistic Human Pathogen

AbstractThe opportunistic human pathogen Penicillium marneffei exhibits a temperature-dependent dimorphic switch. At 25°, multinucleate, septate hyphae that can undergo differentiation to produce asexual spores (conidia) are produced. At 37° hyphae undergo arthroconidiation to produce uninucleate yeast cells that divide by fission. This work describes the cloning of the P. marneffei gasC gene encoding a G-protein α-subunit that shows high homology to members of the class III fungal Gα-subunits. Characterization of a ΔgasC mutant and strains carrying a dominant-activating gasCG45R or a dominant-interfering gasCG207R allele show that GasC is a crucial regulator of germination. A ΔgasC mutant is severely delayed in germination, whereas strains carrying a dominant-activating gasCG45R allele show a significantly accelerated germination rate. Additionally, GasC signaling positively affects the production of the red pigment by P. marneffei at 25° and negatively affects the onset of conidiation and the conidial yield, showing that GasC function overlaps with functions of the previously described Gα-subunit GasA. In contrast to the S. cerevisiae ortholog Gpa2, our data indicate that GasC is not involved in carbon or nitrogen source sensing and plays no major role in either hyphal or yeast growth or in the switch between these two forms.

scholarly journals N-terminal fatty acylation of the α-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation

1994 ◽  
Vol 303 (3) ◽  
pp. 697-700 ◽  
Author(s):  
F Galbiati ◽  
F Guzzi ◽  
A I Magee ◽  
G Milligan ◽  
M Parenti
Keyword(s):  
Palmitic Acid ◽  
G Protein ◽  
Fatty Acyl ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Α Subunit ◽  
Metabolic Labelling ◽  
Fatty Acylation ◽  
Absolute Requirement ◽  
Cdna Construct

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

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