The heterotrimeric G-protein subunits GNG-1 and GNB-1 form a Gbetagamma dimer required for normal female fertility, asexual development, and galpha protein levels in Neurospora crassa

Abstract

We have identified a gene encoding a heterotrimeric G protein gamma subunit, gng-1, from the filamentous fungus Neurospora crassa. gng-1 possesses a gene structure similar to that of mammalian Ggamma 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 Ggamma 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 Deltagnb-1 (Gbeta) mutants. Deltagng-1, Deltagnb-1, and Deltagng-1 Deltagnb-1 strains conidiate inappropriately in submerged cultures and are female sterile, producing aberrant female reproductive structures. Similar to previous results obtained with Deltagnb-1 mutants, loss of gng-1 negatively influences levels of Galpha 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.

FIG. 1.

Alignment of GNG-1 with other fungal Gγ protein sequences. ClustalW (http://www.embl.co.uk) was used to align Gγ protein sequences from N. crassa (Nc; GNG-1; NCU00042.1) with Gγ subunits from M. grisea (Mg; MG10193.4), G. zeae (Gz; accession no. 387411.1), A. nidulans (An; accession no. XT 4068791.1), U. maydis (Um; UM 06109.1), Botrytis cinerea (Bc; accession no. AL 114303), C. parasitica (Cp; accession no. CB 688576), T. harzianum (Th; accession no. CF875833), L. edodes Gg1 (Le; accession no. AAP 13581.1), S. pombe Git11 (Sp; accession no. NP 596681), and S. cerevisiae Ste18p (Sc; accession no. CAA 89613). BOXSHADE (www.ch.embnet.org) was used to indicate identical (black shading) and similar (gray shading) amino acid residues.

FIG. 2.

Structure of the N. crassa gng-1 genomic region and construction of Δgng-1- and Δgng-1 gng-1⁺-rescued strains. (A) gng-1 genomic clone and gene replacement vector. The grey area indicates the gng-1 ORF, and the hatched region corresponds to the gene conferring hygromycin resistance, hph, under control of the A. nidulans trpC promoter. The dashed lines illustrate the region replaced by hph that is between the second SpeI and first KpnI sites. The open triangles indicate intron positions (−511 to −197; +162 to +258). The arrows show the direction of transcription of gng-1 and hph. Abbreviations for restriction sites: N, NcoI; EV, EcoRV; Sp, SpeI; S, SalI; K, KpnI; B, BamHI; C, ClaI; St, StuI; E, EcoRI; X, XbaI; Sm, SmaI. KpnI², SpeI³, and the unique XbaI and SmaI are artifacts of cloning. pSVK3 was the probe used for Southern analysis (see the legend to panel B). pSVK7 was used as the gene replacement construct, while the portion of gng-1 in pSVK17 was present in the his-3-targeted rescue construct. (B) Expression of gng-1 and gnb-1 during the N. crassa life cycle. Samples from wild-type strain 74A tissues (20 μg of total RNA) were subjected to Northern analysis using as probes a 1,074-bp PCR product amplified from pBR2 for detection of the gnb-1 transcript and a 279-bp PCR product amplified from pSVK1 to detect the gng-1 ORF. The tissues used in the experiment were as indicated. C,conidia; S¹, 8-h submerged cultures; S², 16-h submerged cultures; M, cultures grown for 3 days at 30°C on solid VM in the dark; P, cultures grown for 6 days at 25°C on SCM under light. Amounts of the major RNA species are shown as loading controls. (C) Southern analysis. Genomic DNA was digested with NcoI, and the 1.8-kb SalI-EcoRV fragment from pSVK3 was used as a probe. Strains 5-5-3, 5-5-8, and 5-5-12 are purified homokaryotic Δgng-1 mutants. Strain 5-4 is a Δgnb-1 Δgng-1 double mutant. (D) Northern analysis of mutant and wild-type strains. Samples containing 20 μg of total RNA isolated from 16-h submerged cultures were subjected to Northern analysis using a 1,074-bp PCR product amplified from pBR2 to detect the gnb-1 transcript and a 279-bp PCR product amplified from pSVK1 to detect gng-1 mRNA. The strains used in the analysis are 74A (wild type), Δgng-1 (5-5-12), Δgnb-1 (42-8-3), and Δgng-1 + gng-1⁺ 113-1. rRNA loading controls are as in panel B. (E) GNG-1 and GNB-1 protein levels in the wild type and mutants. Samples containing 30 μg of protein from plasma membrane fractions of 16-h submerged cultures were subjected to Western analysis using the GNG-1 and GNB-1 antibodies. The strains used in the analysis were 74A (wild type), Δgng-1 (5-5-12), Δgnb-1 (42-8-3), Δgnb-1 Δgng-1 5-4, and Δgng-1 + gng-1⁺ 113-1. The asterisk indicates a nonspecific band in the GNB-1 Western blot.

FIG. 3.

Phenotypic characterization during the sexual cycle. (A) Fertilized structure (perithecium) formation. Strains were cultured on solid SCM medium at 25°C for 6 days in light prior to fertilization with wild-type conidia of opposite mating type (74a or 74A). Arrows indicate perithecia (enlarged dark bodies) formed after fertilization. Photographs were taken at ×25 magnification. (B) Trichogyne attraction. Microconidia from strain 74a or 74A were used as male cells to attract trichogynes of strains (genotypes indicated on the figure) of opposite mating type. Growth and orientation of trichogynes were monitored microscopically, and photographs were taken at ×500 magnification. Arrows indicate the direction of trichogyne growth or coiling events.

FIG. 4.

Phenotypes in submerged culture. Cultures grown for 16 h at 30°C under submerged conditions with shaking were photographed at ×400 magnification. Arrows indicate conidiophores formed in Δgng-1 and Δgnb-1 cultures.

FIG. 5.

Analysis of Gα protein and transcript levels. The strains used in the analysis were 74A (wild type), Δgng-1 (5-5-12), Δgnb-1 (42-8-3), Δgnb-1 Δgng-1 5-4, and Δgng-1 + gng-1⁺ 113-1. (A) Gα protein levels in 16-h submerged cultures. Samples containing 30 μg of protein from plasma membrane fractions were subjected to Western analysis using specific antisera (see Materials and Methods). The asterisk indicates a nonspecific band. (B) Gα protein levels in VM plate cultures. Protein samples were as indicated in panel A. (C) Gα protein levels in SCM plate cultures. Protein samples were as indicated in panel A. (D) Analysis of gna-1, gna-2, and gna-3 transcript levels. Total RNA was extracted from 16-h submerged cultures, and 20 μg was subjected to Northern analysis using a 5.6-kb EcoRI-ClaI genomic fragment from pPNO5, a 967-bp gna-2 PCR product amplified from plasmid 13M2A5-2, or a 1,068-bp gna-3 PCR product amplified from pAK1 as probes. The amounts of the two major rRNA species are indicated as a loading control.

FIG. 6.

Coimmunoprecipitation of GNB-1 with GNG-1. (A) Levels of GNB-1, FLAG-GNG-1, and GNG-1 proteins in plasma membrane fractions. Plasma membrane fractions were prepared from 16-h submerged cultures of gng-1⁺ (74A), Δgng-1⁺ FLAG-GNG-1 (5A), and Δgng-1 his-3 (113) strains. Only strain 5A expresses the FLAG-GNG-1 fusion protein (see Materials and Methods). Samples containing 50 μg of total protein were resolved on 10% (GNB-1) or 15% (GNG-1 and FLAG-GNG-1) SDS-PAGE gels. GNB-1, GNG-1, and FLAG antisera were used for Western analysis (see Materials and Methods). Nonspecific bands are indicated by asterisks. (B) Immunoblot analysis after coimmunoprecipitation. The FLAG-GNG-1 protein in extracts from the indicated strains in panel A was immunoprecipitated using anti-FLAG M2-agarose (see Materials and Methods), and the precipitated proteins were examined by immunoblot analysis using anti-FLAG, anti-GNG-1, or anti-GNB-1 antibodies.