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 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 Site-directed mutations altering the CAAX box of Ste18, the yeast pheromone-response pathway G gamma subunit.

Genetics ◽  
1994 ◽  
Vol 137 (4) ◽  
pp. 967-976 ◽  
Author(s):  
M S Whiteway ◽  
D Y Thomas
Keyword(s):  
G Protein ◽  
Protein Function ◽  
Dominant Negative ◽  
Minor Effect ◽  
Pheromone Response ◽  
Gamma Subunit ◽  
Pheromone Response Pathway ◽  
C Terminus ◽  
A Minor ◽  
In Cells

Abstract The STE18 gene encodes the gamma subunit of the G protein which functions in the Saccharomyces cerevisiae pheromone-response pathway. The STE18 gene product undergoes a post-translational processing at the carboxyl terminus directed by the CCAAX box motif CCTLM110. A variety of site-directed mutations of this sequence have been constructed to test the role of this motif on Ste18 function. Mutations which change or eliminate the cysteine at position 107 abolish Ste18-dependent mating, and thus the cysteine (C107) is essential for Ste18 function. However, inactivation of the prenyltransferase by disruption of DPR1 has only a minor effect on Ste18-dependent mating. Mutation of cysteine 106 to serine significantly reduces but does not eliminate Ste18 function. Deletion of the C-terminal TLM sequence or modification of the ultimate methionine to lysine, arginine or leucine, all changes which do not affect the CAAX box cysteines, have only minor effects on Ste18-dependent mating. Intriguingly, these latter mutations dramatically compromise Ste18 function in cells which are deleted for Gpa1, the alpha subunit of the G protein. In addition, overexpression of these mutant versions of STE18 causes a dominant negative phenotype and inhibits the constitutive mating response generated by GPA1 deletion in cells which contain a functional STE18 gene. These results suggest that the C terminus of Ste18 and the Gpa1 protein have overlapping roles in some aspect of yeast G protein function such as membrane targeting.

scholarly journals G-Protein β Subunit of Cochliobolus heterostrophus Involved in Virulence, Asexual and Sexual Reproductive Ability, and Morphogenesis

2004 ◽  
Vol 3 (6) ◽  
pp. 1653-1663 ◽  
Author(s):  
Sherif Ganem ◽  
Shun-Wen Lu ◽  
Bee-Na Lee ◽  
David Yu-Te Chou ◽  
Ruthi Hadar ◽  
...  
Keyword(s):  
Map Kinase ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Cochliobolus Heterostrophus ◽  
Wild Type ◽  
Heterotrimeric G Protein ◽  
Reproductive Ability ◽  
Pathway Gene ◽  
Mitogen Activated Protein ◽  
Nuclear Degradation

ABSTRACT Previous work established that mutations in mitogen-activated protein (MAP) kinase (CHK1) and heterotrimeric G-protein α (Gα) subunit (CGA1) genes affect the development of several stages of the life cycle of the maize pathogen Cochliobolus heterostrophus. The effects of mutating a third signal transduction pathway gene, CGB1, encoding the Gβ subunit, are reported here. CGB1 is the sole Gβ subunit-encoding gene in the genome of this organism. cgb1 mutants are nearly wild type in vegetative growth rate; however, Cgb1 is required for appressorium formation, female fertility, conidiation, regulation of hyphal pigmentation, and wild-type virulence on maize. Young hyphae of cgb1 mutants grow in a straight path, in contrast to those of the wild type, which grow in a wavy pattern. Some of the phenotypes conferred by mutations in CGA1 are found in cgb1 mutants, suggesting that Cgb1 functions in a heterotrimeric G protein; however, there are also differences. In contrast to the deletion of CGA1, the loss of CGB1 is not lethal for ascospores, evidence that there is a Gβ subunit-independent signaling role for Cga1 in mating. Furthermore, not all of the phenotypes conferred by mutations in the MAP kinase CHK1 gene are found in cgb1 mutants, implying that the Gβ heterodimer is not the only conduit for signals to the MAP kinase CHK1 module. The additional phenotypes of cgb1 mutants, including severe loss of virulence on maize and of the ability to produce conidia, are consistent with CGB1 being unique in the genome. Fluorescent DNA staining showed that there is often nuclear degradation in mature hyphae of cgb1 mutants, while comparable wild-type cells have intact nuclei. These data may be genetic evidence for a novel cell death-related function of the Gβ subunit in filamentous fungi.

scholarly journals Mice with Deficiency of G Protein γ3 Are Lean and Have Seizures

2004 ◽  
Vol 24 (17) ◽  
pp. 7758-7768 ◽  
Author(s):  
William F. Schwindinger ◽  
Kathryn E. Giger ◽  
Kelly S. Betz ◽  
Anna M. Stauffer ◽  
Elaine M. Sunderlin ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Gene Targeting ◽  
Wild Type ◽  
Phenotypic Changes ◽  
Reduced Body ◽  
Γ Subunit ◽  
Body Weights ◽  
The Brain ◽  
Increased Susceptibility

ABSTRACT Emerging evidence suggests that the γ subunit composition of an individual G protein contributes to the specificity of the hundreds of known receptor signaling pathways. Among the twelve γ subtypes, γ3 is abundantly and widely expressed in the brain. To identify specific functions and associations for γ3, a gene-targeting approach was used to produce mice lacking the Gng3 gene (Gng3 −/−). Confirming the efficacy and specificity of gene targeting, Gng3 −/− mice show no detectable expression of the Gng3 gene, but expression of the divergently transcribed Bscl2 gene is not affected. Suggesting unique roles for γ3 in the brain, Gng3 −/− mice display increased susceptibility to seizures, reduced body weights, and decreased adiposity compared to their wild-type littermates. Predicting possible associations for γ3, these phenotypic changes are associated with significant reductions in β2 and αi3 subunit levels in certain regions of the brain. The finding that the Gng3 −/− mice and the previously reported Gng7 −/− mice display distinct phenotypes and different αβγ subunit associations supports the notion that even closely related γ subtypes, such as γ3 and γ7, perform unique functions in the context of the organism.

scholarly journals sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs.

1994 ◽  
Vol 14 (7) ◽  
pp. 4501-4508 ◽  
Author(s):  
X J Chen ◽  
G D Clark-Walker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Function ◽  
Kluyveromyces Lactis ◽  
Functional Equivalence ◽  
Striking Difference ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Dna Targeting ◽  
Mating Efficiency ◽  
Type Gene

A Kluyveromyces lactis mutant, hypersensitive to the DNA-targeting drugs ethidium bromide (EtBr), berenil, and HOE15030, can be complemented by a wild-type gene with homology to SIR2 of Saccharomyces cerevisiae (ScSIR2). The deduced amino acid sequence of the K. lactis Sir2 protein has 53% identity with ScSir2 protein but is 108 residues longer. K. lactis sir2 mutants show decreased mating efficiency, deficiency in sporulation, an increase in recombination at the ribosomal DNA locus, and EtBr-induced death. Some functional equivalence between the Sir2 proteins of K. lactis and S. cerevisiae has been demonstrated by introduction of ScSIR2 into a sir2 mutant of K. lactis. Expression of ScSIR2 on a multicopy plasmid restores resistance to EtBr and complements sporulation deficiency. Similarly, mating efficiency of a sir2 mutant of S. cerevisiae is partially restored by K. lactis SIR2 on a multicopy plasmid. Although these observations suggest that there has been some conservation of Sir2 protein function, a striking difference is that sir2 mutants of S. cerevisiae, unlike their K. lactis counterparts, are not hypersensitive to DNA-targeting drugs.

Plant heterotrimeric G protein function: insights from Arabidopsis and rice mutants

2004 ◽  
Vol 7 (6) ◽  
pp. 719-731 ◽  
Author(s):  
Laetitia Perfus-Barbeoch ◽  
Alan M Jones ◽  
Sarah M Assmann
Keyword(s):  
G Protein ◽  
Protein Function ◽  
Heterotrimeric G Protein
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