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Dimer Required for Normal Female Fertility, Asexual Development, and G
Protein Levels in Neurospora crassa
Department of Plant Pathology, University of California—Riverside, Riverside, California
Received 24 September 2004/ Accepted 18 November 2004
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ABSTRACT |
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subunit, gng-1, from the filamentous fungus Neurospora crassa. gng-1 possesses a gene structure similar to that of mammalian G 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 G 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 
gnb-1 (Gß) mutants. gng-1, gnb-1, and gng-1 gnb-1 strains conidiate inappropriately in submerged cultures and are female sterile, producing aberrant female reproductive structures. Similar to previous results obtained with gnb-1 mutants, loss of gng-1 negatively influences levels of G
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. |
INTRODUCTION |
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dimer performs numerous roles during the signal transduction process (for reviews, see references 14 and 32), including membrane targeting of the subunit (23), recognition of receptors (46), activation of downstream effectors (14), and modulation of different proteins affecting signal intensity or duration (47). Multiple isoforms, including 6 Gß and 12 G subunits, have been identified in mammals (14, 32, 50). In mammals, a major challenge for in vivo identification of Gß dimers and establishment of their roles in particular signaling pathways arises from the variety of possible combinations between ß and subtypes.
In contrast to the situation with mammals, only one Gß subunit is present in all sequenced fungal genomes (http://www.yeastgenome.org; http://www.genedb.org/genedb/pombe/index.jsp; http://www.broad.mit.edu/annotation/fungi) (27). For the budding yeast Saccharomyces cerevisiae, previous studies have indicated that the Ste4p Gß functions as a positive regulator of the pheromone response in haploid cells by activation of the downstream mitogen-activated protein kinase cascade, leading to cell cycle arrest, shmoo formation, cell fusion, and karyogamy (for reviews, see references 22 and 42). Gpa1p, the G protein that interacts with Ste4p, functions as a negative regulator of the pathway. In the fission yeast Schizosaccharomyces pombe, the Gß subunit Git5 is required for glucose sensing and mating through activation of cyclic AMP (cAMP) signaling (45). In the basidiomycete human pathogenic fungus Cryptococcus neoformans, deletion of the Gß subunit gene GPB-1 results in sterility and defective monokaryotic fruiting (72). Mutation of the Gß gene sfaD from the filamentous fungus Aspergillus nidulans leads to hyperactive conidiation (asexual sporulation) and reduced vegetative growth (56). In the chestnut blight pathogen Cryphonectria parasitica, disruption of the cpgb-1 Gß subunit gene negatively affects virulence, conidiation, pigmentation, and hyphal branching, while stimulating growth on vegetative solid medium (40). In Magnaporthe grisea, the causative agent of rice BLAST disease, mutants disrupted in the Gß subunit MGB1 exhibit reduced growth and conidiation, defective appressorium formation, and reduced intracellular cAMP levels (51). Loss of gnb-1 in the filamentous fungus Neurospora crassa leads to inappropriate conidiation in submerged culture, altered mass accumulation on solid medium, production of aberrant fertilized female reproductive structures, reduced intracellular cAMP levels, and low levels of all three G subunits (80).
G subunits belong to a large family of small proteins consisting of 68 to 75 amino acids with different primary structures in various species (6, 20, 28). All G proteins contain the CaaX box motif at the carboxy terminus that is subject to posttranslational modification, including isoprenylation and subsequent carboxyl methylation (28, 82). This posttranslational modification of G subunits determines the subcellular localization of the Gß complex, in that it targets the heterodimer to the plasma membrane (36, 48, 58). The carboxy-terminal modification of G is also necessary for effective interaction of Gß with other proteins, including G, downstream effectors, and receptors (12).
Only a single G subunit gene has been identified in the yeasts S. cerevisiae (STE18) and S. pombe (git11) (45, 76). In S. cerevisiae, previous studies have demonstrated that haploid cells of opposite mating type lacking the STE18 or STE4 gene are unable to mate (76). Genetic studies indicate that Ste4p binds to Ste18p, and various ste18 mutations have been isolated that either suppress or enhance phenotypic defects of ste4 alleles (15, 77). Furthermore, Ste18p has been shown to physically interact with Ste4p (15, 34, 64) and to tether the Gß dimer to the plasma membrane (9, 34, 64). Deletion of the git11 gene in S. pombe confers phenotypes associated with defects in the glucose-sensing (cAMP) pathway. git11 cells are defective in glucose repression of both fbp1 (encoding fructose-1,6-bisphosphatase) and sexual development, and they resemble cells lacking either gpa2 G or git5 Gß (45, 73). Moreover, a physical interaction between Git11p and Git5p has been demonstrated by coimmunoprecipitation (45).
To date, G proteins have not been characterized in any filamentous fungal species. In this study, we present the identification, isolation, and characterization of a predicted G subunit, gng-1, from the fungus N. crassa. gng-1 and gnb-1 gng-1 mutants were isolated and analyzed for phenotypes during vegetative growth as well as asexual and sexual development. Levels of the three G proteins and mRNA levels were analyzed, and intracellular amounts of cAMP were quantitated. Evidence for a physical association between GNG-1 and GNB-1 in vivo was probed using coimmunoprecipitation. Our results indicate that GNG-1 and GNB-1 form a functional Gß heterodimer that is essential for normal asexual sporulation and female fertility in N. crassa.
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MATERIALS AND METHODS |
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(33).
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gene was initially identified during homology searches (BLAST) (1) of the N. crassa cDNA database at the University of Oklahoma (http://www.genome.ou.edu) using the protein sequence of S. cerevisiae Ste18p. Two cDNA clones, b7a10ne and a8h02ne, encoding hypothetical proteins similar to G subunits, were identified. The 1.2-kb insert of a8h02ne was used to screen a BARGEM-7
genomic library (53). Two positive plaques were obtained and converted to double-stranded plasmids (53), and they were subsequently subjected to Southern analysis using the insert from a8h02ne as a probe. Both cDNA clone a8h02ne and one of the genomic clones (designated #31; insert size, 4.5 kb) were sequenced (Core Sequencing Facility, Department of Microbiology and Molecular Genetics, University of Texas—Houston Medical School). The entire sequence of the cDNA clone a8h02ne and a partial sequence from one of the genomic clones were analyzed. The sequence of the gng-1 open reading frame (ORF) identified in genomic clone #31 was used to search the N. crassa genome database (http://www.broad.mit.edu/annotation/fungi/neurospora) using BLAST searches and was found to correspond to predicted protein NCU000421. The gng-1 replacement mutation and complementation by gng-1+ in trans. The gng-1 ORF is located only 790 bp away from the 3' end of the insert in genomic clone #31. To make a gene replacement construct, a larger genomic clone (#2231) with an insert size of 6.5 kb was used (see Fig. 2A). The gng-1 gene was replaced with the hph gene encoding hygromycin B phosphotransferase under control of the A. nidulans trpC promoter as follows. The hph cassette was first removed from pCSN44 (66) using BamHI and SalI and was subsequently cloned into pBlueScript KS+ (Stratagene), generating pSVK5. KpnI and SpeI were used to excise the hph fragment from pSVK5; this fragment was then used to replace the portion of the gng-1 ORF between the KpnI site and the second SpeI site of the genomic clone, yielding pSVK7 (Fig. 2A). pSVK7 contains 2.5 kb of 5'-flanking DNA and 2.4 kb of 3'-flanking DNA extending from the EcoRI to EcoRV sites in the gng-1 genomic clone. Ten-day-old conidia of N. crassa wild-type strain 73a (Table 1) were electroporated with 1 µg of pSVK7 linearized with SphI, as described previously (37, 69), and transformants were selected on sorbose medium (13) containing hygromycin B. Genomic DNA was extracted from transformants by using the Puregene kit according to the manufacturer's protocol (Gentra Systems, Minneapolis, Minn.). To identify homologous and ectopic integrations, genomic DNA from transformants was subjected to Southern analysis after digestion with NcoI (37). The 1.8-kb 5' DNA flank (SalI-EcoRV) from pSVK3 was used as a probe. Heterokaryotic gene replacement strains without ectopic integrations were crossed to the wild-type strain 74A (Table 1). The progeny were selected on sorbose medium with hygromycin B. Purity of strains was verified by Southern analysis as described above.
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gng-1 matA strain (5-12) was crossed to a his-3 mata strain (his3a) and gng-1 his-3 progeny were selected (see Table 1). A gng-1 his-3 strain (113-1) was transformed by electroporation with pSVK17, and transformants were plated on histidine-free sorbose medium supplemented with hygromycin B. Heterokaryons containing the wild-type gng-1 allele integrated at the his-3 locus were identified by Southern analysis using the 1.8-kb 5' DNA flank fragment (excised using SalI-EcoRV) from pSVK3 as a probe (data not shown). Genomic DNA was digested with ApaI. Heterokaryons with homologous recombination at the his-3 locus were isolated after microconidiation (21) to obtain gng-1::hph+ gng-1+::his-3+ strains.
Isolation of gnb-1 gng-1 double mutants.
Based on phenotypic analysis, both gnb-1 and gng-1 mutants are female sterile (see Fig. 3). To isolate gnb-1 gng-1 double mutants, a forced heterokaryon was made between gnb-1 his-3 mata and the helper strain am1 ad-3B cyh-1 (FGSC 4654), and it was used as a gnb-1 female in crosses (29) (Table 1). Conidia from a gng-1 strain of opposite mating type (matA) were used as the male. The presence of the gng-1 and gnb-1 mutations in progeny was verified by Southern analysis as described above (for gng-1) or as described previously (for gnb-1 [80]).
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For Northern analysis, total RNA was extracted from tissue ground in liquid nitrogen using a previously described protocol (5). Samples containing 20 µg of total RNA were subjected to Northern analysis as described elsewhere (57). Probe templates were generated as follows. For gng-1, a 279-bp PCR product was amplified from the gng-1 cDNA clone pSVK1 by ExTaq (Takara, New York, N.Y.) using the 5GNG1 and 3GNG1 primers (Table 2). pSVK1 contains the entire gng-1 ORF (without introns) cloned in pET11a (Invitrogen, Carlsbad, Calif.). For gnb-1, a 1,074-bp PCR product was amplified from cDNA clone pBR2 using primers LEXA-GNB1-BAMH-FW and LEXA-GNB1-PST-RV (Table 2). Plasmid pBR2 corresponds to the entire ORF (no introns) of gnb-1 amplified by reverse transcriptase PCR (Access RT-PCR; Promega) and subsequently cloned into the pGEM-T vector (Promega). A 5.6-kb EcoRI-ClaI fragment from pPNO5 (37) was the source of gna-1, while a 967-bp PCR product corresponding to gna-2 was amplified from cDNA clone 13M2A5-2 (68) using GNA-2-ECORI-FW and GNA-2-BAMHI-RV as oligomers (Table 2). A template for gna-3 was generated by amplification of a 1,068-bp PCR product from pAK1 (41) using GNA3THAFW and GNA3THARV as primers (Table 2). All probe templates were labeled using the random primer method according to the manufacturer's protocol (Promega).
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To produce a specific antiserum for GNG-1, the amino acid sequence corresponding to the extreme amino terminus (plus a cysteine for coupling to the resin: CQYASRDVGDPSQIKKN) was synthesized (United States Biological, Swampscott, Mass.) and used as an antigen to produce a rabbit polyclonal antibody (Cocalico Biologicals, Reamstown, Pa.). The plasma membrane fraction was isolated from strains as described above. Samples containing 30 µg of total protein were separated on a SDS-15% PAGE gel and transferred to a PVDF membrane (Millipore Corp., Bedford, Mass.). The primary antibody was used at a dilution of 1:3,000. The secondary antibody treatment and chemiluminescence system were the same as those described above.
Coimmunoprecipitation studies.
A construct containing the gng-1 ORF with the FLAG epitope tag at the amino terminus was targeted to the his-3 locus in a gng-1 his-3 strain to facilitate coimmunoprecipitation experiments. To generate a FLAG fusion construct, the GNG1-FLAG-XBA-FW primer was engineered to contain a 24-bp sequence encoding the FLAG epitope (DYKDDDDK) (7). The gng-1 ORF was amplified by PCR (LA Taq; Takara) from pSVK1 using GNG1-FLAG-XBA-FW and GNG1-FLAG-ECOR-RV as oligomers (Table 2) with designed XbaI (5' end) and EcoRI (3' end) restriction sites. The resulting 323-bp PCR product was cloned into pGEM-T (Promega, Madison, Wis.), yielding pBR5. A 319-bp insert containing the FLAG-gng-1 fusion construct was subsequently released from pBR5 with XbaI and EcoRI and was cloned in the his-3-targeting vector pMF272 (26), generating pBR6. pMF272 was originally constructed for overexpression of green fluorescent protein (GFP) fusion proteins under control of the N. crassa ccg-1 promoter (26). In pBR6, the GFP gene has been replaced with the XbaI-EcoRI fragment from pBR5. Ten-day-old conidia from gng-1 his-3 strain #113 were transformed with pBR6, and transformants were plated on FIGS plates. Strains with homologous recombination events were identified by Southern analysis using the 8.8-kb HindIII fragment from pRAUW122 as a probe, and homokaryons were purified using the microconidiation technique (21).
For coimmunoprecipitation experiments, conidia were inoculated in 500 ml of liquid VM at a final concentration of 106 cells/ml. Cultures were incubated in the dark at 30°C with shaking at 200 rpm for 16 h, harvested by filtration, and ground in liquid nitrogen. The plasma membrane fraction was isolated, and protein concentrations were determined as described above. To solubilize membrane-associated proteins, samples containing 2 mg of total protein were adjusted to 360 µl with the extraction buffer (see above). Subsequently, 40 µl of 5% Triton X-100 was added, and the solution was incubated on ice for 15 min. The mixtures were then centrifuged (21,000 x g for 15 min at 4°C) to remove insoluble material. The supernatant was diluted with an equal volume of 2x coimmunoprecipitation buffer (20 mM Tris-Cl [pH 7.5], 300 mM NaCl), and 80 µl of anti-FLAG M2-agarose slurry (Sigma, St. Louis, Mo.) was added. The suspension was incubated at 4°C on a rotating shaker for 3 h. Afterwards, the agarose beads were collected by centrifugation (1,000 x g for 1 min at 4°C) and washed twice with ice-cold 1x Tris-buffered saline. An aliquot (50 µl) of 2x sample buffer (25 mM Tris-HCl [pH 6.8], 4% SDS, 20% [vol/vol] glycerol, 0.004% bromphenol blue) was added to the agarose beads, and the mixture was incubated at 95°C for 3 min. The samples were then centrifuged (21,000 x g for 30 s at room temperature). Aliquots of supernatant (40 µl) were then resolved using a 10 (GNB-1 detection) or 15% (GNG-1 detection) SDS-PAGE gel, and the proteins were subsequently transferred to PVDF membranes (Millipore Corp.). Western analysis was performed as described above, using anti-FLAG M2 monoclonal (1:1,000; Sigma), anti-GNG-1 (1:3,000), and anti-GNB-1 (1:5,000) as primary antibodies.
Phenotypic analysis. To determine apical extension rates, 1 µl of a conidial suspension was inoculated in the center of VM plates and the plates were incubated at 30°C in the dark. The colony diameter was measured at 2-h intervals. To analyze phenotypes in submerged cultures, liquid VM was inoculated with conidia at a final concentration 106 cells/ml and incubated with shaking at 200 rpm for 16 h at 30°C. Cultures were then viewed and photographed using a BX41 fluorescent microscope and a C-4040 digital camera (Olympus, Lake Success, N.Y.). Unfertilized (6-day-old protoperithecia) and fertilized (3-day-old perithecia) female tissues were grown on SCM plates in light and were observed using an SZX9 stereomicroscope with an ACH 1x objective lens outfitted with the C-4040 digital camera (Olympus).
For trichogyne pheromone attraction assays (7, 8, 44), cultures were grown for 6 days on 2% water agar. Chemoattraction between trichogynes and microconidia was observed using a BX41 fluorescent microscope with UM Plan Fluorite objective lenses (Olympus) as described above.
Measurement of intracellular steady-state cAMP levels. For measuring in vivo cAMP levels, 16-h submerged cultures and tissues grown on VM plates for 3 days at 30°C in the dark and SCM plates incubated at 25°C in constant light were ground in liquid nitrogen and extracted as previously described (38). cAMP levels were quantified using a protein binding assay following the manufacturer's instructions (Amersham Pharmacia Biotech, Piscataway, N.J.). The protein concentration was determined using the bichinonic acid method (Pierce) as described elsewhere (38).
Nucleotide sequence accession number. The GenBank accession number for the gng-1 cDNA clone a8h02ne is AY823297.
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RESULTS |
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subunit Ste18p were identified using BLAST (1) in the Neurospora database at the University of Oklahoma (http://www.genome.ou.edu). The gng-1 ORF is 279 bp, and the predicted GNG-1 protein consists of 93 amino acid residues with a molecular mass of 10 kDa (Fig. 1). GNG-1 shows relatively high identity to G proteins from other filamentous fungi: 90% to G. zeae, 92% to C. parasitica, 86% to Trichoderma harzianum, 86% to M. grisea, 65% to A. nidulans, and 55% to Ustilago maydis. Interestingly, N. crassa GNG-1 shares only 40% identity with S. cerevisiae Ste18p (76) and even less identity with S. pombe Git11 (9%) (45), indicating evolutionary divergence between filamentous fungi and yeasts. In addition, as a group, fungal G proteins display very little identity (less than 20%) to mammalian G proteins (data not shown). Predicted G proteins from U. maydis (UM 06109.1) and M. grisea (MG10193.4) were identified in genome databases at http://www.broad.mit.edu/annotation/fungi. However, the positions of introns and exons in the two genes were predicted incorrectly by the automatic gene caller. Therefore, both genes were annotated manually, and the resulting protein sequences were used in the alignment (Fig. 1).
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genomic library (53). The screen resulted in isolation of two genomic clones designated #31 (4.5-kb insert) and #2231 (6.5-kb insert). The entire nucleotide sequence of the a8h02ne cDNA and partial nucleotide sequence of genomic clone #31 were used to determine the gene structure of gng-1 (Fig. 2A). The gng-1 gene contains one 96-bp intron in the ORF, from +162 to +257. Another 315-bp intron is present in the 5'-untranslated region (UTR) of the mRNA (–510 to –196). All of the exon-intron boundaries conform to the GT-AG rule for intron splice sites. The sequence of the 5' region upstream of the gng-1 ORF was obtained (http://www.broad.mit.edu/annotation/fungi/neurospora) and analyzed for potential transcriptional regulatory motifs (Fig. 2A). No identifiable pyrimidine-rich regions (31) or TATA box consensus sequences (75) were present. Nevertheless, two putative transcriptional regulatory motifs were observed: one CTTTG at –320 (4) and one CCAAT box at –453 (31).
In order to elucidate the expression of gng-1 throughout development, Northern analysis was used to examine gng-1 transcript levels in conidia, 8- and 16-h submerged cultures, and VM and SCM plates. gnb-1 message levels were also measured during the experiment. A 1.2-kb gng-1 transcript was detected in all tissues (Fig. 2B and data not shown). This size is similar to the insert sizes (1,198 bp) of the two independent cDNA clones (b7a10ne and a8h02ne). The results show that gng-1 is differentially expressed during the life cycle of N. crassa and that the highest expression levels of gng-1 are in 8-h submerged cultures and protoperithecial tissue from SCM plates (Fig. 2B). The lowest levels of gng-1 were detected in conidia and in tissues grown on VM plates. Comparison of gnb-1 and gng-1 message levels shows that these two genes share a similar expression pattern (Fig. 2B) (80). A possible exception is on SCM plates, where gng-1 may have higher relative expression levels than gnb-1. Observation of similar expression profiles has also been reported for the single Gß and G in Dictyostelium discoideum (83).
Deletion of gng-1 by targeted gene replacement and isolation of a gng-1 gng-1+-complemented strain.
A gng-1 mutant was isolated after electroporation of a wild-type strain with a construct in which the gng-1 ORF was replaced by the hygromycin B cassette (Fig. 2A) (66). Genomic DNA from transformants was digested with NcoI and subjected to Southern analysis using the 1.8-kb DNA fragment (SalI-EcoRV) from pSVK3 as a probe (Fig. 2C). Under these conditions, the wild-type strain produces a 5.7-kb hybridizing fragment, while a 2.8-kb fragment is detected in gng-1 nuclei (Fig. 2C). Transformants exhibiting homologous recombination at the gng-1 locus were crossed to a wild-type strain of opposite mating type to produce homokaryotic gng-1 mutant progeny. The genotype of homokaryons was verified by Southern analysis (data not shown). gng-1 gnb-1 double mutants were constructed by crossing the gnb-1 as a female, with sheltering in a heterokaryon (see Materials and Methods). Northern analysis showed that gng-1 and gnb-1 gng-1 strains lack gng-1 mRNA (Fig. 2D). Western analysis using a rabbit polyclonal antibody raised against a GNG-1 peptide sequence (see Materials and Methods) demonstrated that gng-1 and gnb-1 gng-1 mutants do not produce the corresponding GNG-1 protein (Fig. 2E).
The gng-1 mutation was complemented in trans using the 6.5-kb gng-1 genomic fragment in the his-3 targeting vector pRAUW123 (2). Transformants were screened for conferral of histidine prototrophy. Homokaryons were obtained by using microconidial isolation (21). Both gng-1 mRNA and GNG-1 protein were detected at appreciable levels in gng-1 gng-1+-complemented strains (Fig. 2D and E).
gng-1 strains are female sterile and male fertile.
In N. crassa, sexual development is induced by nitrogen starvation, with formation of female reproductive structures (protoperithecia) containing specialized hyphae, termed trichogynes (55). Trichogynes exhibit chemotropic growth towards male gametes (conidia or other vegetative cells) of opposite mating type (9), followed by fusion and recruitment of a male nucleus to the base of the protoperithecium. The nuclei from the male and female parents recognize one another and migrate to croziers (ascogenous hyphae), where they undergo mitosis. Subsequent fusion of male and female nuclei is followed by two meiotic divisions and one episode of postmeiotic mitosis. Each resulting ascus contains eight homokaryotic, haploid ascospores. About 200 to 400 asci are enclosed in each mature fruiting body (perithecium).
Previous studies have shown that gnb-1 mutants are female sterile but are fertile as males during sexual crosses (80). gnb-1 mutants are able to form protoperithecia but fail to develop fruiting bodies after fertilization (80) (Fig. 3A). gng-1 strains and gng-1 gnb-1 double mutants exhibit a phenotypic pattern identical to that of gnb-1 strains (Fig. 3A). Although they produce reproductive structures, development of normal perithecia after fertilization is blocked (Fig. 3A), and no ascospores are produced (data not shown). In contrast, gng-1 gng-1+-rescued strains are phenotypically identical to the wild type (Fig. 3A).
Our laboratory has demonstrated that gnb-1 mutants are deficient in both trichogyne attraction and perithecial development (44, 80). In order to determine whether a similar defect is present in gng-1 strains or gng-1 gnb-1 double mutants, microconidia of opposite mating type were applied at a distance from wild-type, gng-1, gnb-1, or gng-1 gnb-1 double mutant protoperithecia. Growth of trichogyne tips towards male cells was then followed microscopically (8, 44). In a previous study (44), gna-1 and gnb-1 mutants did not display directional migration but instead grew in random directions and failed to undergo fusion with male cells, even when in direct contact. Similarly, trichogynes of gng-1 and gnb-1 gng-1 strains did not respond to microconidia and exhibited random orientation on the agar surface during this analysis (Fig. 3B). gng-1 gng-1+-complemented strains resembled the wild type, with normal trichogyne migration and fusion with microconidia (Fig. 3B). These data support the hypothesis that GNA-1 and Gß (GNB-1/GNG-1) are essential for trichogyne chemotropism during the pheromone response and for subsequent fusion with male gametes. The observations from previous work suggested that GNA-1 is coupled to PRE-1 (the matA pheromone receptor), because pre-1 strains exhibit the same defects in trichogyne chemoattraction as gna-1 mutants (44).
gng-1 mutants conidiate inappropriately in submerged culture.
During vegetative growth, N. crassa produces tubular filaments (hyphae) characterized by tip-based polarized growth. We analyzed the rate at which strains extended vegetative hyphae on VM medium. Apical extension rates of gnb-1 and gng-1 single and double mutants are similar to those of the wild type and gna-3 mutants (41, 80, and data not shown) but differ from those of gna-1 strains that display reduced apical extension rates (37).
Asexual spore formation (conidiation) is induced in wild-type strains of N. crassa cultured on solid medium. In contrast, submerged cultures form vegetative nonconidiating hyphae unless starved for carbon or nitrogen or exposed to stress conditions, such as high temperature (54, 67). Our laboratory previously showed that gna-1, gna-3, and gnb-1 strains conidiate inappropriately in submerged culture; in the case of gna-1 strains, submerged conidiation is cell density dependent (39, 41, 80).
The conidiation patterns of gnb-1, gng-1, and gnb-1 gng-1 mutants cultured on solid medium are similar (80 and data not shown), with the mutants exhibiting shorter aerial hyphae and increased conidiation relative to the wild type. Like gna-1, gna-3, and gnb-1 strains, gng-1 single and gnb-1 gng-1 double mutants also form conidia in 16-h submerged cultures (Fig. 4). Rescued gng-1 gng-1+ strains are phenotypically identical to the wild type (Fig. 4).
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gng-1 and gnb-1 mutants have decreased levels of intracellular cAMP.
Study of fungal G subunits has revealed functions for these proteins in regulation of cAMP levels. In N. crassa, GNA-1 is required for GTP-dependent adenylyl cyclase activity, while GNA-3 regulates the levels of the adenylyl cyclase protein (CR-1) (38, 41). Levels of cAMP are greatly reduced in both submerged and plate cultures of gna-3 mutants, and many defects of gna-3 strains can be reversed by supplementation with cAMP (41). On the other hand, gna-1 mutants have normal intracellular cAMP levels during submerged growth but low levels in cultures grown on solid media. The normal concentration of cAMP in submerged cultures may result from a compensatory mechanism involving reduced cAMP-phosphodiesterase activity (38). gna-2 mutants have normal cAMP amounts in submerged cultures and on VM plates but smaller amounts on SCM solid medium (38). gna-1 gna-2 strains have normal cAMP levels in submerged cultures but greatly reduced concentrations on VM and SCM plates (38). Similar to gna-1 and gna-2 strains, gnb-1 mutants have normal levels of cAMP in submerged cultures but low cAMP levels on VM (Table 3) (80) and SCM plates (Table 3). Furthermore, like gna-1 and gna-2 mutants, gnb-1 strains have normal levels of CR-1 protein but reduced GTP-dependent adenylyl cyclase activity (80).
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gng-1 strains. As expected, gng-1 strains have concentrations of cAMP very similar to those of gnb-1 mutants (Table 3). Wild-type amounts of cAMP are produced in submerged cultures, while reduced levels are obtained when gng-1 mutants are cultured on VM (55% of wild type) or SCM (21% of wild type) plates.
gng-1 strains have reduced Gß and G protein levels.
Gß and G subunits form a tight complex and are not known to dissociate from one another in vivo. Coexpression of the Gß and G subunit and the presence of an intact CaaX domain in the G protein are required for plasma membrane targeting (58, 60). Mutation of G genes has been shown to suppress the level of Gß protein(s) in various organisms (34, 62, 71). To determine whether a similar mechanism exists in N. crassa, the plasma membrane fraction of gng-1 and gnb-1 mutants was subjected to Western analysis using GNG-1- and GNB-1-specific antisera (Fig. 2E). The results demonstrate that the amount of GNB-1 was reduced 
60% in gng-1 mutants (Fig. 2E) and that GNG-1 is almost completely absent from the plasma membrane of gnb-1 mutants (Fig. 2E). We were not able to detect GNG-1 in nonmembrane fractions of wild-type or mutant strains (data not shown), presumably due to low concentrations of GNG-1 in the cytosol. Interestingly, the levels of GNB-1 protein in cytosolic fractions from the gng-1 mutant and wild-type are comparable (data not shown), demonstrating that the major reduction in GNB-1 levels occurs in plasma membrane fractions of the gng-1 strain. The effect of the mutations appears to be largely posttranscriptional, as either normal (gng-1 in gnb-1) or 50% reduced (gnb-1 in gng-1) levels of the corresponding mRNAs are present in those cases where the partner protein is absent (Fig. 2D). In addition, the reduced amount of gnb-1 in gng-1 mutants is similar to that of rescued gng-1 gng-1+ strains that have normal levels of GNB-1 (Fig. 2E) and are phenotypically comparable to the wild type.
Tethering of the Gß protein by isoprenylated G also facilitates interactions between Gß and its other partner protein, G, at the plasma membrane. Deletion of the G subunit can not only affect the levels of Gß but also affect the levels of G proteins. For example, it has been shown in mice that G7 is required for the stability of a G-protein heterotrimer (olfß7), in that loss of G7 results in an 82% reduction in Golf protein levels in Gng7–/– mutant mice (61). Deletion of the mouse G3 gene, which results in a phenotype distinct from that of Gng7–/– mice, leads to reduced levels of Gß2 and Gi3 proteins. And, as mentioned above, deletion of the Gß gene gnb-1 suppresses the level of G subunits in N. crassa (80).
Because GNG-1 is the only G subunit in N. crassa and, by extension, is the only G subunit capable of interacting with GNB-1, it was reasonable to test whether loss of gng-1 would affect expression of the three G proteins. Western analysis was used to measure levels of G proteins in wild-type, gnb-1, gng-1, and gnb-1 gng-1 strains in three different tissues: 16-h submerged cultures and VM and SCM plate cultures (Fig. 5A, B, and C). The amounts of GNA-1, GNA-2, and GNA-3 were significantly diminished in all mutants analyzed, and the magnitude of the reduction was almost identical. There were significant differences observed in the levels of single G proteins in 16-h submerged cultures. GNA-1 and GNA-2 levels were greatly reduced in all mutants, while changes in GNA-3 were much more subtle (30 to 50%). The amount of all G proteins was dramatically lowered in VM and SCM plate cultures. To determine whether the effects on G protein levels were pre- or posttranscriptional, we examined levels of mRNA for the gna-1, gna-2, and gna-3 genes in 16-h submerged cultures of gng-1 and gnb-1 mutants (Fig. 5D). Similar to previous results from our laboratory (80), G message amounts were either normal (gna-1 and gna-3) or reduced only 50% (gna-2), consistent with mainly posttranscriptional regulation of G subunit levels in both gng-1 and gnb-1 mutants.
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gng-1 his-3 recipient strain #113, and his-3+ transformants were selected on minimal medium. Homologous recombination at the his-3 locus was verified by Southern analysis (see Materials and Methods); strains with such events were purified, and one of the strains (#5A) was used for coimmunoprecipitation studies. Phenotypic analysis of strain 5A showed that the FLAG-GNG-1 construct complemented some, but not all, of the gng-1 defects (data not shown). Although strain 5A conidiates abundantly during incubation on VM plates, conidiation is partially suppressed in 16-h submerged cultures; hyphal tips of strain 5A are swollen, but mature conidiophores similar to those of gng-1 or gnb-1 mutants were not observed. Strain 5A is also female fertile, producing perithecia and ascospores after fertilization.
Plasma membrane fractions were extracted from wild-type (#74A), #5A (gng-1 his-3::FLAG-GNG-1), and #113 (gng-1 his-3) strains, and proteins were solubilized with 1% Triton X-100 (see Materials and Methods). We first analyzed the amount of tagged and untagged GNG-1 proteins present in the input membrane extracts by using Western analysis (Fig. 6A). Untagged GNG-1 and FLAG-GNG-1 were detected using two different antibodies: the GNG-1-specific peptide antibody described above and anti-FLAG antiserum. The GNG-1-specific antiserum was used to determine levels of FLAG-GNG-1 or GNG-1 protein associated with the plasma membrane (Fig. 6A, top panel). Addition of the FLAG epitope results in a protein that migrates at a larger apparent molecular weight and that can be distinguished from the untagged GNG-1 protein by using the GNG-1-specific antiserum during Western analysis (see the shift in Fig. 6A, top panel). In contrast, the FLAG antibody is specific for the tagged FLAG-GNG-1 protein present in the corresponding transformants (Fig. 6A, middle panel). The level of FLAG-GNG-1 protein in strain 5A was significantly lower than the corresponding level of untagged GNG-1 in the wild type (Fig. 6A, top panel). This result may be explained by the difference in promoters, in that expression of the FLAG-GNG-1 construct is driven by the ccg-1 promoter (26). The lower level of FLAG-GNG-1 versus native GNG-1 presumably leads to the observed reduction in GNB-1 amount in the FLAG-GNG-1 strain relative to that of the wild type (Fig. 6A, bottom panel) and may explain why only partial phenotypic complementation of the gng-1 mutation was observed by using the FLAG-GNG-1 construct (data not shown).
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gng-1 his-3 recipient strains, although the former contains appreciable amounts of GNB-1 protein (Fig. 6A, bottom panel). |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein, GNG-1. Although we cannot rule out the possibility of another G with a very different sequence, previous studies of mammals and plants have shown that G proteins from the same species usually share a relatively high level of similarity (28, 49). The predicted GNG-1 protein possesses a typical G secondary protein structure (2.5 helices) (63, 83) and the conserved CaaX box motif at the carboxy terminus. As shown in other species, the CaaX motif is subjected to isoprenylation (farnesylation or geranylgeranylation) at the cysteine residue, followed by proteolytic removal of the last three amino acids and methylation of the carboxy terminus (28). If the last amino acid residue (X) of the CaaX box is M, S, Q, or A, the cysteine is a substrate for farnesylation, whereas leucine (X = L) results in geranylgeranylation (59). Amino acids at the X position of the CaaX box in characterized fungal G proteins are M (GNG-1 and Ste18p), S (Git11), or Q (Gg1 from Lentinula edodes), indicating that the CaaX motif is likely to be farnesylated.
Like Ste18p from S. cerevisiae and Git11 from S. pombe, GNG-1 contains two cysteine residues near its carboxy terminus (Fig. 1). In Ste18p, one cysteine, at position 107, is contained in the farnesyl-directing CaaX box (CTLM) (24), while the other cysteine (106) is a potential site for palmitoylation (35). In S. cerevisiae, substitution of serine for cysteine at position 106 or 107 resulted in failure of Gß to bind to the plasma membrane (35). The Cys 107 substitution also resulted in reduced steady-state levels of Ste18p, suggesting that Cys 107 farnesylation is required for Ste18p stability (35). Furthermore, previous genetic studies (30, 78) have demonstrated that yeast mutants with substitutions at either cysteine residue are unresponsive to pheromone. Further experimentation is needed to determine the importance of these two conserved cysteine residues to GNG-1 function in N. crassa.
The intron-exon boundaries and mRNA splicing patterns for several mammalian G-subunit genes have already been characterized (19, 20, 25, 28, 52). In all cases, the 5'-untranslated region of the mRNA contains one intron. A second intron is located in the ORF, and its position relative to the amino acid sequence is conserved between the G-subunit genes (20). The S. cerevisiae STE18 ORF does not contain an intron (http://www.yeastgenome.org). In contrast, both S. pombe git11 (http://www.genedb.org/genedb/pombe/index.jsp) and N. crassa gng-1 have introns in their ORFs. However, there are no reports of introns in the 5' UTRs of STE18 and git11. In this study, we have identified two introns in N. crassa gng-1 at positions that correspond to those found in mammalian G-subunit genes. We previously reported a similar phenomenon with respect to conserved intron positions in mammalian and N. crassa G genes (68). The remarkable conservation of intron positions between mammalian and N. crassa G (and G) genes suggests that these sequences play a regulatory role in mRNA synthesis or stability. Future studies will investigate these possibilities.
The gng-1 mutant displays phenotypes identical to those observed in gnb-1 strains (44, 80), and the gnb-1 gng-1 double mutant is indistinguishable from either single mutant. Our results also demonstrate that loss of gng-1 or gnb-1 results in a significant reduction in GNB-1 or GNG-1 protein levels, respectively, from plasma membrane fractions (Fig. 2E), suggesting interdependence between GNB-1 and GNG-1 for their stability in vivo. This is similar to the situation of S. cerevisiae, in which Ste18p is barely detectable in ste4 mutants while Ste4p is reduced only 50% in ste18 cells (34). Taken together, our data support the hypothesis that GNB-1 and GNG-1 regulate identical events in N. crassa and form an active Gß complex in vivo. The finding that GNB-1 is coprecipitated with GNG-1 using an antibody directed against an epitope on GNG-1 provides strong evidence for a direct, physical association between these two proteins in vivo.
Like gnb-1 mutants (80), gng-1 strains have lower levels of G proteins than the wild type. This is in contrast to results reported for S. cerevisiae, where Gpa1p is present at normal levels and is localized to the plasma membrane in the absence of Gß (64). The major effect caused by loss of the Gß dimer in N. crassa appears posttranscriptional, because normal or appreciable levels of gna-1, gna-2, and gna-3 transcripts are produced in gng-1 and gnb-1 strains. In contrast, deletion of a single G does not greatly influence GNB-1 levels (38, 41, 43); a significant reduction in GNB-1 amount is only observed in a mutant lacking both GNA-1 and GNA-3 or all three G proteins (43). This finding suggests that the absence of multiple G proteins can influence the amount of Gß dimer anchored to the plasma membrane of N. crassa.
Many of the defects shared by gnb-1 and gng-1 strains can be explained by reduced amounts of G proteins. The female sterility of these mutants is similar to that of gna-1 and gna-1 gna-2 mutants (37, 44). These strains are defective in trichogyne attraction toward the male cell and form small aberrant perithecia with no ascospores after fertilization (37, 80). In contrast to GNA-1 and GNA-2, GNA-3 levels in submerged cultures were not greatly reduced (30 to 50%), suggesting that the Gß subunit is not crucial for GNA-3 stability in vegetative hyphal tissue. However, GNA-3 levels are significantly reduced in VM and SCM plate cultures. Based just on protein amount, it is not easy to predict the phenotypic outcome of lower GNA-3 levels in the various tissues. It is possible that GNA-3 is coupled to different receptors, and thus its turnover might be regulated differently in various cell types. On the other hand, GNB-1 may act as a direct regulator of downstream effectors, while GNA-3 is only required to regulate GNB-1 function. Such a scenario has been described for S. cerevisiae, where Gpa1p negatively regulates Ste4p function during pheromone signal transduction (18, 65, 81).
It was demonstrated previously that gnb-1 strains have low levels of intracellular cAMP when cultured on solid medium but normal amounts of cAMP in submerged culture (80). We have obtained similar results with gng-1 mutants (Table 3). The gng-1 mutant conidiates abundantly on solid medium and in submerged cultures, and phenotypically it resembles gna-1, gna-1 gna-2, and gna-3 mutants. It was hypothesized that the smaller amount of GNA-1 and GNA-2 in gnb-1 mutants is responsible for the reduction in cAMP levels (80). This hypothesis is supported by results from previous studies with both gna-1 deletion and gna-1 constitutively activated alleles (38, 39, 79). The observation of normal cAMP levels in submerged cultures of gnb-1 and gng-1 strains is similar to results determined for gna-1 and gna-1 gna-2 mutants (38). In contrast, submerged liquid cultures of gna-3 mutants produce low levels of intracellular cAMP, presumably due to reduced amounts of adenylyl cyclase protein (41). Tissue-specific effects on cAMP metabolism due to loss of a G-subunit gene have also been observed in mice, where the G7 protein regulates adenylyl cyclase activity in specific regions of the brain (61).
Some phenotypes observed in gnb-1 mutants cannot be explained by low levels of G proteins. For example, gnb-1 mutants have essentially normal apical extension rates on various media (80), while a mutant lacking all three G proteins exhibits severely restricted growth (43). A possible explanation is that although G protein amounts are reduced in gnb-1 (and gng-1) mutants, free G proteins, untethered by GNB-1, can regulate downstream effectors. A similar model for G-protein functional interactions has been suggested for S. pombe, where Gpa2 remains partially active during cAMP signaling in git5 (Gß) mutants (45).
In this study, we provide evidence that GNG-1 is the sole G subunit in N. crassa and that this protein forms a physical association with the only Gß protein, GNB-1. Levels of GNG-1 and GNB-1 are decreased in the absence of the other subunit, consistent with decreased protein stability. The GNB-1/GNG-1 Gß heterodimer acts as a unit during signaling, with loss of either protein leading to similar defects, including a severe reduction in G protein levels. Future studies will focus on elucidation of the mechanism whereby loss of GNG-1 leads to smaller amounts of GNB-1 and the three G proteins and on understanding the contribution of individual G protein subunits to regulation of downstream effectors in N. crassa.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant GM48626 from the National Institutes of Health (to K.A.B.).
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FOOTNOTES |
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