scholarly journals The Heterotrimeric G-Protein Subunits GNG-1 and GNB-1 Form a Gβγ Dimer Required for Normal Female Fertility, Asexual Development, and Gα Protein Levels in Neurospora crassa

2005 ◽  
Vol 4 (2) ◽  
pp. 365-378 ◽  
Author(s):  
Svetlana Krystofova ◽  
Katherine A. Borkovich
Keyword(s):  
Neurospora Crassa ◽  
G Protein ◽  
Magnaporthe Grisea ◽  
Cryphonectria Parasitica ◽  
Normal Female ◽  
Submerged Cultures ◽  
Heterotrimeric G Protein ◽  
Developmental Defects ◽  
Protein Levels

ABSTRACT We have identified a gene encoding a heterotrimeric G protein γ subunit, gng-1, from the filamentous fungus Neurospora crassa. gng-1 possesses a gene structure similar to that of mammalian Gγ genes, consisting of three exons and two introns, with introns present in both the open reading frame and 5′-untranslated region. The GNG-1 amino acid sequence displays high identity to predicted Gγ subunits from other filamentous fungi, including Giberella zeae, Cryphonectria parasitica, Trichoderma harzianum, and Magnaporthe grisea. Deletion of gng-1 leads to developmental defects similar to those previously characterized for Δgnb-1 (Gβ) mutants. Δgng-1, Δgnb-1, and Δgng-1 Δgnb-1 strains conidiate inappropriately in submerged cultures and are female sterile, producing aberrant female reproductive structures. Similar to previous results obtained with Δgnb-1 mutants, loss of gng-1 negatively influences levels of Gα proteins (GNA-1, GNA-2, and GNA-3) in plasma membrane fractions isolated from various tissues of N. crassa and leads to a significant reduction in the amount of intracellular cyclic AMP. In addition, we show that GNB-1 is essential for maintenance of normal steady-state levels of GNG-1, suggesting a functional interaction between GNB-1 and GNG-1. Direct evidence for a physical association between GNB-1 and GNG-1 in vivo was provided by coimmunoprecipitation.

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 G Protein α Subunit Genes Control Growth, Development, and Pathogenicity of Magnaporthe grisea

1997 ◽  
Vol 10 (9) ◽  
pp. 1075-1086 ◽  
Author(s):  
Shaohua Liu ◽  
Ralph A. Dean
Keyword(s):  
Neurospora Crassa ◽  
G Protein ◽  
Vegetative Growth ◽  
Magnaporthe Grisea ◽  
Cryphonectria Parasitica ◽  
Appressorium Formation ◽  
Α Subunit ◽  
Targeted Deletion ◽  
Control Growth ◽  
G Protein Α Subunit

Three G protein α subunit genes have been cloned and characterized from Magnaporthe grisea: magA is very similar to CPG-2 of Cryphonectria parasitica; magB is virtually identical to CPG-1 of Cryphonectria parasitica, to gna1 of Neurospora crassa, and to fadA of Emericella nidulans; and magC is most similar to gna2 of Neurospora crassa. Homologous recombination resulting in targeted deletion of magA had no effect on vegetative growth, conidiation, or appressorium formation. Deletion of magC reduced conidiation, but did not affect vegetative growth or appressorium formation. However, disruption of magB significantly reduced vegetative growth, conidiation, and appressorium formation. magB¯ transformants, unlike magA¯ and magC¯ transformants, exhibited a reduced ability to infect and colonize susceptible rice leaves. G protein α subunit genes are required for M. grisea mating. magB¯ transformants failed to form perithecia, whereas magA¯ and magC¯ transformants did not produce mature asci. These results suggest that G protein α subunit genes are involved in signal transduction pathways in M. grisea that control vegetative growth, conidiation, conidium attachment, appressorium formation, mating, and pathogenicity.

scholarly journals Large G protein α-subunit XLαs limits clathrin-mediated endocytosis and regulates tissue iron levels in vivo

2017 ◽  
Vol 114 (45) ◽  
pp. E9559-E9568 ◽  
Author(s):  
Qing He ◽  
Richard Bouley ◽  
Zun Liu ◽  
Marc N. Wein ◽  
Yan Zhu ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
G Protein ◽  
Critical Role ◽  
Hek293 Cells ◽  
Activity Levels ◽  
Α Subunit ◽  
Proteomic Screen ◽  
Protein Levels ◽  
G Protein Α Subunit

Alterations in the activity/levels of the extralarge G protein α-subunit (XLαs) are implicated in various human disorders, such as perinatal growth retardation. Encoded by GNAS, XLαs is partly identical to the α-subunit of the stimulatory G protein (Gsα), but the cellular actions of XLαs remain poorly defined. Following an initial proteomic screen, we identified sorting nexin-9 (SNX9) and dynamins, key components of clathrin-mediated endocytosis, as binding partners of XLαs. Overexpression of XLαs in HEK293 cells inhibited internalization of transferrin, a process that depends on clathrin-mediated endocytosis, while its ablation by CRISPR/Cas9 in an osteocyte-like cell line (Ocy454) enhanced it. Similarly, primary cardiomyocytes derived from XLαs knockout (XLKO) pups showed enhanced transferrin internalization. Early postnatal XLKO mice showed a significantly higher degree of cardiac iron uptake than wild-type littermates following iron dextran injection. In XLKO neonates, iron and ferritin levels were elevated in heart and skeletal muscle, where XLαs is normally expressed abundantly. XLKO heart and skeletal muscle, as well as XLKO Ocy454 cells, showed elevated SNX9 protein levels, and siRNA-mediated knockdown of SNX9 in XLKO Ocy454 cells prevented enhanced transferrin internalization. In transfected cells, XLαs also inhibited internalization of the parathyroid hormone and type 2 vasopressin receptors. Internalization of transferrin and these G protein-coupled receptors was also inhibited in cells expressing an XLαs mutant missing the Gα portion, but not Gsα or an N-terminally truncated XLαs mutant unable to interact with SNX9 or dynamin. Thus, XLαs restricts clathrin-mediated endocytosis and plays a critical role in iron/transferrin uptake in vivo.

scholarly journals Selection of Gβ Subunits with Point Mutations That Fail to Activate Specific Signaling Pathways In Vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G Protein in Dictyostelium discoideum

1998 ◽  
Vol 9 (10) ◽  
pp. 2949-2961 ◽  
Author(s):  
Tian Jin ◽  
Mario Amzel ◽  
Peter N. Devreotes ◽  
Lijun Wu
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Dictyostelium Discoideum ◽  
Actin Polymerization ◽  
Point Mutations ◽  
Cellular Responses ◽  
Receptor System ◽  
Heterotrimeric G Protein ◽  
Downstream Effectors

In Dictyostelium discoideum, a unique Gβ subunit is required for a G protein–coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Gα2, and Gβ genes completely impair all these responses. To dissect specificity in Gβγ signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gβ genes and isolated Gβ alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gβ subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gβγ by making point mutations on Gβ. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gβs and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtβγ interacts with its regulators, Gα and phosducin.

scholarly journals Genetic relationships between the RACK1 homolog cpc-2 and heterotrimeric G protein subunit genes in Neurospora crassa

PLoS ONE ◽  
2019 ◽  
Vol 14 (10) ◽  
pp. e0223334 ◽  
Author(s):  
Amruta Garud ◽  
Alexander J. Carrillo ◽  
Logan A. Collier ◽  
Arit Ghosh ◽  
James D. Kim ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
G Protein ◽  
Genetic Relationships ◽  
Protein Subunit ◽  
Heterotrimeric G Protein

scholarly journals Heterotrimeric G proteins promote FLS2 protein accumulation through inhibition of FLS2 autophagic degradation

2018 ◽  
Author(s):  
Jimi C. Miller ◽  
Stacey A. Lawrence ◽  
Nicole K. Clay
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
G Proteins ◽  
Protein Complex ◽  
Heterotrimeric G Proteins ◽  
Immune Signaling ◽  
Heterotrimeric G Protein ◽  
Protein Levels ◽  
Autophagic Degradation ◽  
Protein Components

ABSTRACTFLAGELLIN-SENSITIVE 2 (FLS2) is a plant immune receptor that binds bacterial flagellin to activate immune signaling. This immune signal is transduced by a heterotrimeric G protein complex at the plasma membrane and activates downstream signaling. However, it is unknown whether the heterotrimeric G proteins have functions at other subcellular locations away from the plasma membrane. Here, we show that components of the heterotrimeric G protein complex stabilize FLS2 protein levels by inhibiting the autophagic degradation of FLS2. Using genetic analysis, we determined that mutations of G protein components resulted in reduced immune signaling in part due to decreased FLS2 protein levels. Furthermore, reduction of FLS2 protein levels was caused by elevated proteasomal and autophagic degradation of FLS2. Genetic inhibition of autophagy in G protein mutants rescued FLS2 levels and immunity. Our findings suggest that the heterotrimeric G protein components, in addition to being part of the heterotrimeric G protein complex that transduces signals at the plasma membrane, also function away from the plasma membrane to control FLS2 protein levels. These results expand the functional capacity of the heterotrimeric G protein complexes in plant immunity.

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