overlapping functions for two g protein a subunits in neurospora

Copyright 0 1997 by the Genetics Society of America

Overlapping Functions for Two G Protein a Subunits in Neurospora CTctSsLT

Rudeina A. Xiaohui Lu,* Patricia S. Rowley,* Gloria E. Turner*'* and Katherine A. Borkovich"

*Department of Microbiology and Molecular Genetics, University of Texas-Houston Medical School, Houston, Texas 77030, tDepartment of Plant Pathology and Microbiology, Texas A L3 M University, College Station, Texas 77843 and $Department of

Chemistv and Biochemistry, University of California at Los Angeles, Los Angeles, Calqornia 90024 Manuscript received February 18, 1997 Accepted for publication June 11, 1997

ABSTRACT Heterotrimeric G proteins, consisting of a, ,8 and y subunits, mediate a variety of signaling pathways in

eukaryotes. We have previously identified two genes, gnu-1 and gnu-2, that encode G protein a subunits in the filamentous fungus Neurospora crassa. Mutation of gna-1 results in female infertility and sensitivity to hyperosmotic media. In this study, we investigate the expression and functions of gnu-2. Results from Western analysis and measurements of gnu-2 promoter-lac2 fusion activity indicate that gnu-2 is expressed during the vegetative and sexual cycle of N. crussa in both A and a mating types. Activating mutations predicted to abolish the GTF'ase activity of GNA-2 cause subtle defects in aerial hyphae formation and conidial germination. Extensive phenotypic analysis of Agna-2 strains did not reveal abnormalities during vegetative or sexual development. In contrast, deletion of gnu-2 in a Agna-1 strain accentuates the Agna-1 phenotypes. Agna-1 Agna-2 strains have a slower rate of hyphal apical extension than Agna-1 strains on hyperosmotic media. Moreover, Agna-1 Agna-2 mutants have more pronounced defects in female fertility than Agna-l strains. We propose that gnu-1 and gnu-2 have overlapping functions and may constitute a gene family. This is the first report of G protein a subunits with overlapping functions in eukaryotic microbes.

E VERY eukaryotic cell has the ability to respond to hundreds of chemical and physical signals. Many

of these ligands bind to seven helix transmembrane receptors and trigger a flow of information that is trans- duced into the cell interior by heterotrimeric (aPy ) G proteins (BIRNBAUMER 1992). Ligand binding to the receptor promotes the exchange of GTP for GDP on the a subunit of the G protein and dissociation of the a and Py subunits. Depending on the signaling pathway, one or both subunits can activate downstream ef- fectors (HERSKOWITZ 1995; NEER 1995), which in turn generate an intracellular response. Ga genes have been isolated from both higher and lower eukaryotes (NEER 1995; BORKOVICH 1996). In mammals, Ga subunits have been grouped into four classes, G a s , G a i , Gaq and GaI2, based on amino acid sequence identity (SIMON et ul. 1991). In contrast, the limited number of cloned Ga subunits has not allowed this type of sequence classification in eukaryotic microbes.

Our laboratory has cloned two genes encoding Ga subunits (gnu-1 and gnu-2), from the filamentous fungus Neurospora crussu (TURNER and BORKOVICH 1993). GNA-1 is most homologous to members of the mammalian Gai family and was the first microbial Gcu subunit to be classified in a family found in higher organisms. Deletion of gnu-l causes female infertility, increased

Coffespondingauthw: Katherine A. Borkovich, Department of Micro- biology and Molecular Genetics, University of Texas-Houston Medical School, 6431 Fannin St., JFB 1.765, Houston, TX 77030. E-mail: [email protected]

Genetics 147: 137-145 (September, 1997)

sensitivity to hyperosmotic media and other morpho- logical abnormalities (IVEY et ul. 1996). N. crussu gnu-2 encodes an Ga subunit that is not a member of any Ga family in higher organisms. Based on amino acid sequence, GNA-2 is most related to Pneumocystis curinii PCG1, N. crussu GNA-1 and Schizosucchuromyces pombe Gpalp (54.7, 49.4 and 47.7% identical, respectively; (OBARA et ul. 1991; TURNER and BORKOVICH 1993; SMU- LIAN et ul. 1996). The function of PCGl in the opportu- nistic human fungal pathogen P. carinii is unknown (SMULIAN et ul. 1996). Gpa-1 is essential for mating and sexual sporulation in the fission yeast S. pombe (OBARA et ul. 1991). Evolutionary clustering studies performed before the cloning of P. curinii pcgl indicated that N. crussu gnu-2 is more closely related to S. pombe @a1 than to N. crussu gnu-1 (WILKIE and YOKOYAMA 1994).

In this study, we investigate the expression and functions of N. crassu gnu-2. We report the organization of this gene including transcription start sites, intron positions and putative upstream regulatory elements. We describe the effects of targeted gene replacement and constitutively activating mutations in gnu-2. In addition, we present evidence that Agnu-1 Agnu-2 strains are more impaired in female fertility and osmotic sensitivity than Agnu-1 strains. We hypothesize that gnu-I and gna- 2 have overlapping functions in N. crussu.

MATERIALS AND METHODS

Strains and growth conditions: Table 1 lists the N. crussa strains used in this study. N. crassa strains were cultured in

138 R. A. Baasiri et al.

TABLE 1

N. crassa strains ________-

Strain Genotype Comments Source

74 A Wild type A R. L. WEISS, UCLA 73 a Wild type a R. L. WEISS, UCLA FGSC #6103 his-? A J. J. LOROS, Dartmouth KB5 Amtr pdx-1 A IVEY et al. (1996) #3 his-? a 73a X 6103 This study 5x3s his-? pdx-1 Amtr a KB5 x #3 This study

3-7 Agna-1 :: mtr+ his-2 pdx-1 a IVEY et al. (1996)

PY"4 pyr-4 (a or A ) R. L. WEISS, UCLA

SGMl Agna-1 :: mtr+ al-3 A 3-7 X 2082 This study

FGSC #2082 al-3, A FGSC

T3-2 pyr-4 pyrG+ A PPY'G Tx This study

A334 Agna-2::pyrG+ pyr-4 a This study a29-1 Agna-Z::pyrG+ pyr-4 A This study

A33-2 Agna-2::pyrG+ pyr-4 A This study

3-7-6 hir-3+ :: pDE3 (lacZ+)pdx-l a pDE3 Tx This study 3-5-5 his-?+ : : pDE3 (lacZ+)pdx-l a pDE3 Tx This study 4-6-7 his-3+ : : pSR4 (gna-Z+)pdx-l a pSR4 Tx This study 5-7-7 his-?+ : : pSR5 ( p a - 2 Q205L)pdx-1 u pSR5 Tx This study

8-6-3 his-?+:: pSR8 (5'-gna-2::lacZ)pdx-l u lacZ fusion This study

21c AgnaZ::pyrG+ a 334 x SGMl This study

3c Agna2::pyrG+ Agna-l ::mtr+ A 33-4 X SGMl This study

6-2-2 hi.~-3+ : pSR6 (gnu-2 R179C)pd~-I pSR6 Tx This study

17b al-? gna-2+ gnu-l + A 33-4 X SGMl This study

16c Agna-1 ::mtr+ al-3 A 334 X SGMl This study 1 Sa Agna-1 :: mtr+ al-3 A 33-4 X SGMl This study

21d AgnaZ::pyrG+ Agna-1 :: mtr+ A 33-4 X SGMl This study

Tx, transformant; FGSC, Fungal Genetics Stock Center, Kansas City, KS.

Vogel's minimal medium (VM), synthetic crossing medium (SCM), or sorbose plating medium (SPM), and his- and pdx- auxotrophs supplemented as previously described (IVEY et al. 1996). Medium for pyr-4 mutant strains was supplemented with either 100 pg/ml (for vegetative growth) or 20 mg/ml (for sexual growth) undine. Hygromycin B was used at 200 pg/ml in media. Sexual crosses were conducted as previously described (DAVIS and DESERRES 1970). Heterokaryotic transformants were purified to homokaryons by repeated plating on SPM with selection unless stated otherwise. Plasmids were maintained in Escherichia coli strain DH5a (HANAHAN 1983).

Genomic clone analysis and primer extension: A gna-2+ gene clone was isolated from a XJ1 genomic library (ORBACH et al. 1986) using the gna-2 cDNA clone (13M2A5; TURNER and BORKOVICH 1993) as a probe. A 4.1-kb BamHI fragment was subcloned into pGEM4Z (Promega), yielding plasmid pXHL3 (see Figure 1A). However, the insert in pXHL3 contains only 500 bp 3' from the translational stop codon, and we had determined in preliminary experiments that this amount of flanking DNA was not sufficient to obtain a reason- able homologous recombination frequency during gene replacement experiments. Therefore, to obtain a larger carboxy terminal fragment of the gna-2+ gene, a cosmid library of N. crassa DNA (VOLLMER andYANOFSKY 1986) was screened using the polymerase chain reaction (PCR) with gnu-2specific primers. Positive cosmids were obtained from pools X23:E2 and X24:E12. A 9.&kb Hind111 fragment containing gna-p (the amino acid coding region is in the center) was subcloned from a cosmid into pBluescript I1 K S + (Stratagene; pXCOh- 111). The entire BamHI-XbaI fragment depicted in Figure 1A was obtained by ligating the insert from pXHL3 and a 0.78- kb BamHI-XbaI fragment from pXCOhIII into pBluescript I1

SK' (Stratagene; pXHL29). Sequence analysis of the BamHI- XbaI region was as previously described (IVEY et al. 1996). The GenBank/EMBL accession number for the gnu-2 genomic clone is AF004846.

Total RNA from &hr germlings of strain 74A was isolated as previously described (YARDEN et al. 1992), hut using sand and liquid N2 to break open cells. RNA was treated with RQ1 DNase (Promega) according to the manufacturer's recommendations. Primers (10 pmol) were end-labeled (SAMBROOK et al. 1989) and annealed to 10 pg of total RNA at 65" for 5 min, and 42" for 1 hr. Primer extension was carried out at 42" for 2 hr, and products analyzed as described (SAMBROOK et al. 1989).

DNA constructs: A 2.4kb BamHI-StuI fragment from pXHL3 containing the gnu-2 5' region and the first 50 bases of the gna-2+ coding sequence was subcloned into BamHI- SmaI digested pGEM4Z (pRAB10). A BamHI-EcoRI (EcoRI site from vector polylinker) fragment from pRAB10, an EcoRI-NdeI (NdeI filled-in using the klenow fragment of DNA polymerase; SAMBROOK et al. 1989) fragment from ppyrG containing the Aspergillus nidulanspyrGgene (OAKLEY et al. 1987), and a XbaI- NcoI (NcoI filled-in using Klenow) fragment of the gnu-2 3' region from pXCohIII were inserted into vector pGEM7Zf (Promega) digested with BamHI and XbaI to yield pRABl1, the gene replacement construct (Figure 1A).

A 7.4kb HzndIII-EcoRI fragment from pXCohIII containing the gnu-2 gene and a HindIII-KpnI fragment from pCSN44 (STABEN et al. 1989) containing the Hygromycin B resistance gene (hph) were inserted into Kpd-EcoRI digested pGEM-3Z to yield pRAB7, the rescue construct.

Two constitutively activating mutations (R179C and Q205L; JOHNSON et al. 1994) were made in gnu-2 using site-directed

N . crnssn p n - 2 139

A

\

Hhdm \Ndd 9 =CnTGorCAAAG f i o A ~ h d m \

W = van~cription s ~ a n site

= i n m -1 lkb

B

P-gal S.A. NA 124 241 183 ND ND

74A 73a EN,, GNAP

I.B. 0 4 16 VM W

FIGURE 1.-(A) Gene structure of pa-2. The striped area is the coding region, with the A of the translational initiation ATG designated as + l . The four intron positions are indicated (V) . Transcription start sites from primer extension analysis are at -902, -695 and -582. A pyrimidine rich region (GUM et al. 1987) is at -603 to -621, a putative TATA box at -657, and C m G or c.&%AG elements (BAxE\~~\NIS and LANDSWN 1995) are found at -289, -547, -639, -1836 and -1968. The dotted lines mark the position of the firG gene replacement. The UamHI site followed by an asterisk is an artifact of cloning; it is a Snu3AI site in the chromosome. (B) &galactosidase assays and Western analysis using GNA-2 antiserum. 74A and 7% are wild-type strains of opposite mating type; 0 ,4 and 16 indicate the hour of germination in liquid V M . I.B., GNA-2 inclusion bodies; W, solid VM cultures; W , solid SCM cultures. The migration position of GNA-2 protein is indicated on the right side of the figure. P-galactosidase specific activities (P-gal S.A, as Miller units/mg protein) were measured using strain 8-63; standard deviations for 0, 4 and 16 hr samples are 9, 43 and 33, respectively. Wild-type strain 3-7-6 transformed with control plasmid pDE3 has a 0-galactosidase S.A. = 0. ND, not determined; NA, not applicable.

mutagenesis essentially as previously described (KUNKEI. ~t al. 1987). Plasmid pSR3 contains the 3' 1.25-kb NcoI-XhnI fragment, with the X6nI site converted to EcoRI using linkers. Plasmid pSR4 contains the wild-type allele of gnn-2. It was constructed by insertion of a 3.6-kb RamHI-NcoI fragment from pXHL3 (5' region) and a 1.25-kb Ncol-EcoRI fragment from pSR3 (3' portion) into BgfiI-EcoRI digested pDE3 (a vector that allows targetting to the his-? locus of N . mussn; EBBOLE 1990).

The template for mutagenesis (plasmid pSR2) contains the 1.42-kb BnmHI-At1 fragment from pXHL3 in vector m13mp19. To clone the mutations, a 1.3Rkh BstEII-PstI fragment from pSR4, a 1.42-kb AtI-BnmHI fragment of the pSR2 mutagenized replicative form DNA and a 8&kb BnmHI-BstEII fragment from pSR4 were ligated. The plasmids containing the Q205L and R179C mutations are pSR5 and pSR6, respectively.

The pa -2 promoter lacZfusion construct (pSR8) was made by insertion of a 2.42-kb BamHI-StuI fragment from pXHL3- 18 into pDEl (ERDOIX 1990) digested with BgZlI and SmnI. N. m s u transformation and strain construction: Transfor-

mation of N. crmsn was hy electroporation of conidia, as de-

scribed (VASN 1995; IWY et n/. 1996). Strain / ~ , - - 4 w a s elcctro- porated with 2 pg linearized pWBI 1 or 1 pg ppyrG to obtain A,pm-2 deletion mutants or control strains. I-Ictrrokaryotic transformants were crossed to jy4strains to isolate homokaryotic strains.

To obtain strains containing p o - 2 activated alleles or thc p a - 2 IcrcZ fusion, strain 5x3s (Tahle 1 ) was clcctroporated with supercoiled pSR5, pSR6 or pSR8. .5X3S was also elcctro- porated with pSR4 to ohtain strains with two wild-type copies of gnn-2+, or with pDE3 t o obtain isogenic It lS-Ji- controls.

To obtain A p n - I Apcr-2strains. an nl-?strain (F<;SC 2082) was crossed to Agnn-l strain 3-7 ( I \w pt crl. I!W6). DNA from 18 white (a/-3) ascospore progeny was analyzed hy PCR using primers OMP19 and 20 (f-hXATIiMIASS et crl. 1989) to dcter- mine which were Apn-I . One strain (SGMI) was crossed to Apn-2 strain A33-4. Ascospore octads were collcctcd on a previously dcscrihetl medium (MIETLESRERG 1988), hilt with 4% agar. LJnordered ascospores wcre picked and heat- shocked in \W slants (DA\-IS and DIXRRIS 1970). Progeny were screened for segregation of the nl-? gcnc (orangr vs. white color) and for the allele of gvn-1 and/or ,prr-2hy South- ern analysis (see helow). Strains that were Apn-I . A ~ N - 2 , or wild type were also isolated from the progcny. To complemcnt the Apm2mutation in the douhle mutants, pRZB7 was clec- troporated into strain 3c (Tahle I ) . Transformants containing a single ectopic copy of fi.nn-2' were identified h y Southern analysis and purified.

Southern analysis and phenotypic characterization: A! crnsso genomic DNA was isolated and subjected to Southern analysis as previously dcscribcd (I\w et nl. 1996). To identify A,pn-2 strains, genomic DSA was digested with I-lintlIlI antl prohed with a 1.3-kh BnmHI-B,g/II fragment from pXHI,i which contains the 5' end of pa-2. To identify strains with p a - 2 activated alleles or the p n - 2 promoter-/ncZ construct, genomic DNA was digested with XbrrI, antl pH301 (contains a 1 .%kb fragment from the 5' end of the N. mnssn hi.r-3 gene: supplied by DASIIX ERROI.E) was used as a probe. A,pn-l strains were identified as previously descrihed (I\TY et nl. 1996).

Phenotypic analysis of N. rrnxw strains, including apical extension rates, sexual fertility assays and photomicroscopy were performed as previously described (IWY et n/. 1996).

Overexpression of GNA-2 in E. coli and generation of a polyclonal antiserum: Primers that w o r ~ l t l amplify a 240-hp PCR fragment with a Me1 site at the translational initiation codon of pa-2, and maintain the Ps/l sitc in the CDS, wcrc used in reactions with 13M2A5 as template. The 240-hp prod- uct was suhcloned into pCEM47, (Promega) to yicld pKB29. A 1.2-kh fragment containing the carhoxy terminus of gnn-2 from 13M2A.5 and the N d ~ 1 - A t 1 insert from pKB29 were then inserted into pETlla (Novagen) to yield pKR3O.

E. coli strain BL21 DE3 (SrrDlER et nl. 1 9 9 0 ) containing plasmid pKB30 was cultured to allow overproduction of GNA- 2 protein according to the manufacturer's recommendations (Novagen). Inclusion bodies (I.B.) containing insoluhle GNA- 2 were purified, electrophoresed, and GXA-2 protcin excised in gel slices essentially as described (1~13 et nl. 1996). GNA-2 from diced gel slices was eluted hy overnight incuhation at 70" in a solution containing 10 mu TrisCI, 100 mlr NaCI, 10 m u EDTA, and 0.05% SDS, pH 8.0. The mixture was centri- ftlged and the supernatant dialyzed at room temperature against 50 mv TrisCI, 1 mu EDTA, 2 >I NaCI, pH 7.5 to precipitate GNA-2. After centrifugation at 40,000 rpm in a Beckman type 70Ti rotor at 4" for 1 hr, the pellet was used as an antigen in rabbits (Cocalico Biologicals, Inc.).

Western blot analysis and P-galactosidase assays: For West- ern hlot analysis, 7-8-day-old conidia l'rom >Y. onssn strains were either used to inoculate liquid I'M to a final conccntra-


tion of lo' conidia/ml or the center of VM and SCM plates with cellophane overlays. Liquid cultures were shaken at 250 rpm for 0-16 hr at 30". VM plate cultures were grown in the dark at 30" until the colony reached the edge, while SCM plate cultures were grown for 7 days in constant light at room temperature. Conidia or mycelia were collected and subcellu- lar protein fractions were isolated as previously described (TURNER and BORKOVICH 1993). Western blot analysis was performed (IVEY et al. 1996), using Anti-GNA-2 or Anti-GNA- 1 polyclonal rabbit antiserum as the primary antibody at a 1:300 or 1:lOOO dilution, respectively.

For &galactosidase assays, total protein from 8day-old conidia, 4 and IGhr germlings was extracted and P-galactosidase specific activities determined as previously described (MILLER 1972; CORROCHANO et al. 1995).

RESULTS

The entire gnu-2 gene resides on a 9.4kb Hind111 fragment isolated from a cosmid library of N. crmsu genomic DNA (VOLLMER and YANOFSKY 1986). A 4.1- kb Suu3AI-XbuI fragment of this region was sequenced (Figure 1A). The three start sites found for the gnu-2 transcript during primer extension (data not shown) are in the vicinity of several putative transcriptional regulatory elements in the 5' region of the gene (Figure 1A). Sequence comparison of the genomic and cDNA clones demonstrates that gnu-2 contains four introns. Two of these introns are in positions conserved within the Ga, and two within the Gai/Ga, subunit gene fami- lies (reviewed in BORKOVICH 1996). Furthermore, three of the intron positions in N. crussu gnu-2 are also conserved in the pcgl Ga gene from P. curinii. Such conser- vation of intron positions has also been observed for the N. crassu gnu-1 gene and its mammalian and fungal Gai homologues (IVEY et ul. 1996).

Western analysis demonstrates that the GNA-2 protein is present in conidia, germinated liquid cultures and cultures grown on high and low nitrogen solid medium from both mating types (Figure 1B). To fur- ther explore regulation of gnu-2 during conidial germination, we constructed a gnu-2 promoter fusion with the E. coli lac2 gene. Using this promoter, P-galactosidase levels peak at 4 hr into germination (Figure lB), consistent with the results of Western analysis. Pre- viously, Northern analysis demonstrated that the gnu-2 transcript is more abundant in 4 h r germlings than in conidia (TURNER and BORKOVICH 1993). Our data com- bined with these previous results suggest that the regulation of p a - 2 expression is at the transcriptional level in N. crussu.

We made Agnu-2 strains by targeted integration of a construct in which most of the gnu-2 amino-acid coding sequence was replaced with the BrG gene. p y r G is an A. nidulans gene that can complement N. crmsu Pyr-4 gene mutations (G. E. TURNER, unpublished observations). Southern analysis was used to identify heterokaryotic transformants and Agnu-2 homokaryons after purifica- tion (Figure 2A). Western blot analysis demonstrated that a 40.6-kD immunoreactive protein is present in wild-

type but not Agnu-2 strains (Figure 2B). The size of the reactive species is similar to the 41.4kD molecular mass previously predicted from the amino acid sequence of GNA-2 (TURNER and BORKOVICH 1993).

Extensive phenotypic analysis of Agnu-2 strains re- vealed no obvious defects. Conidia from mutants were of the same size, had the same germination rate, and cultures accumulated the same mass as wild-type strains at both normal and suboptimal temperatures (data not shown). Agnu-2 strains had wild-type tolerance to hyperosmotic media (Figure 4A; data not shown). When Agnu-2 strains were used as either female or male par- ents in homozygous or heterozygous crosses, protoperithecia were of wild-type size and amount, normal perithecia were developed, and viable ascospores were produced (Figure 5; data not shown).

We introduced two mutations in gnu-2 (R179C and Q205L; gnu-2) that in other systems are dominant in trans and activate Ga proteins by reducing their GTPase activity UOHNSON et ul. 1994). The mutation constructs were targeted to the his-? locus of a gnu-2+ strain (EB BOLE 1990), yielding gnu-2+ gnu-2 strains. Strains har- boring an extra copy of gnu-2' (gnu-2+ gnu-2') or the his-?+ gene alone (gnu-2+ pDE3) were made by trans- forming the appropriate constructs into the gnu-2+ strain. The gnu2 promoter fragment used to drive expression of the introduced constructs is the same one that successfully directed the expression of the l a d gene when inserted at the his-3 locus (Figure 1B).

gnu-2' gnu-2' strains are similar to gnu-2' pDE3 strains (data not shown). However, gnu-2+ gnu-2 strains have two subtle phenotypes. The first is a faster germination rate in shaking liquid cultures; strains with the gnu-2 allele have longer germ tubes at 4 hr (Figure 3), but still accumulate the same amount of mass as wild type (data not shown). These gnu-2' gnu-2 strains also show increased production of aerial hyphae on solid media (data not shown). However, the gnu-p mutations do not cause any other discernible defects during the vegetative or sexual cycle (data not shown).

To determine if there is a relationship between gnu- l and gnu-2, we investigated the outcome of deleting both genes. N. crussu Agnu-1 Agnu-2 strains were recov- ered as progeny from crossing an ul-? Agna-1 strain to an ul-7 Agnu-2 strain. The genotype of strains was confirmed by both Southern (data not shown) and Western analysis (Figure 2B). Western analysis also indi- cates that deletion of either Ga gene does not alter the level of the remaining Ga protein in single mutants (Figure 2B).

The Agnu-1 Agnu-2 strains and various controls were tested for sensitivity to elevated levels of sorbitol, KC1 and NaCl (Figure 4, A and B). The Agnu-2 strains are indistinguishable from wild type on hyperosmotic media, whereas Agnu-1 strains are more sensitive, as previously described ( IVEY et uZ. 1996). Correspondingly,

4.0 - kb

1 -F;i ,I

-G NA-2 -G N A- 1

FIGL.RI;. 2.--lsolation o f A p o - 2 strains. ( A ) Southern analysis of I-li~~dIlI-digested gcnomic DNA. Lanes: MT, wild type jgr-4 strain TJ-3: H, hrtcrokanotic Apw-2 mutant a29; 1-3, homokaryotic Agno-2 strains a29-I, A33-2, ancl A33-4. The wild-type p7o- 2 locus resides on a 9.4-kh I-lindlII fragment; replacement of pn-2' w i t h jtyrc' produces one 3.9-kh fragment. (R) Western hlot analysis. The lane marked I.B. contains GNA-I or GNA-2 inclusion hodies; all other lanes contain 20 pg plasma memhrane protein. The first t w o Ianrs arc strains w i t h the j1~r.l genetic hxkground; all other lanes are from cross progeny (see Tahle I ) . Antisera were t o GNA-2 and G N h l (top ancl hottom panels, rrspcctively). The migration positions of GNtI-2 and GNA-I protrin are intlicatrd on the right side of the figure. MT, w i l d type; AI, Apo-I; A2, Agno-2. Lanes are wild-typr T3-3; Ag71o-2 mutant A J M ; inclusion hodies; wild-type 1 ih; A,q~cr-I 18a; A,q?n-2 21 c; Apo-I A,po-2 3c, and A-po-I A,po-2 + pRABi 3c-7-1.

the Ap(r-1 A p n - 2 strains are several-fold more sensitive than A p n - 1 strains (Figure 4, A and B).

The Agnu-I Agnn-2 strains were also examined for sexual cycle defects. On SCM, both wild-type and Agnn- 2strains produce protoperithecia that develop into normal perithecia after fertilimtion, and viable ascospores are produced (Figure 5, A and B; data not shown). In accordance with previous results (IIW pt nl. 1996), Apcr-I strains produce protoperithecia; however, most of these protoperithecia do not mature into perithecia. The perithecia that do develop arc abnormal with abundant emanating hyphae (Figure .X). In contrast, although double mutant strains produce protoperithecia (data not shown), perithecia are exceedingly rare and abnormal (Figure 5D). Thus, mutation of gna-2 intensi- fies the sexual cycle defects of a A p n - 1 mutant. The estimated differences in perithecial formation are 85% of wild-type for Agm-2, 35% for A p o - I and 0.6% for

To confirm that the accentuated phenotypes exhibited by the double mutants wcre due to the absence of the p n - 2 gene, A < p n - I A p u - 2 strains containing an ectopic copy o f the g71~-2+ gene (pRAB7 strains) were obtained by electroporation. pRAR7 strains exhibited a rate of apical extension that was identical to that of A p n - I strains on hyperosmotic media (Figure 4B; data not shown). The rescued pRAR7 strains are similar to Ape-1 controls in the amount (54% of wild type) and appearance of abnormal perithecia after fertilization (Figure 5E).

April A<p0-2.

DISCUSSION

Deletion o f p n - 2 does not result in major abnormalities during vegetative or sexual development in N. unssn. One interpretation for a lack of phenotvpes is that pcr-2 is required for the growth of A'. rrnssu in its natural environment and not in a laboratory setting. Alternatively, p n t r - I and p n - 2 may he functionally re- dundant. This hypothesis is substantiated by the obser- vation that A p n - I A,qns.-2 strains are more impaired

in the processes affected by the Agnsnrr-1 mutation (I\w P/ nl. 1996), including osmotic sensitivity and female infertility. In addition, the levels of GNA-1 and GNA-2 are not altered in Apn-201- Agnn-I strains, respectively. Therefore, the lack of phenotypes observed in A p n - 2 strains may result from the activity of GNA-I, which is still present. Furthermore, because GNA-2 can not cover for GNA-I in Agnn-I mutants, the activity or level of GNA-I predominates over that of GNA-2 in N. crnssn.

Strains containing activating mutations in the ~ n n - 2 gene ( p . n - P ) elaborate longer germ tubes in liquid culture and produce more abundant aerial hyphae when cultured on solid medium. Preliminary results from our laboratory indicate that strains that contain an activated allele of p n - I ( , pn - l * ) as the only source of the g.no-1 gene produce abundant, long aerial hyphae on plates (Q. YANG and K. BORKOWCH, unpublished observations). The common effect on aerial hyphal development by activating mutations in either p n - 2 or pncr-1 provides more evidence that these two genes have overlapping functions in N. rmssn.

By analogy to systems such as Dir/yxtdium. discoidmm and Cnpnor/mbdi/is rlqnns, in which multiple Ga proteins are hypothesized to bind the same p subunit (LILLY PI d . 1993; ZWML P/ nl 1996), GNA-I and GNA- 2 might share the same py dimer in N. rrnssn. In such a scenario, loss or activation of GNA-I or GNA-2 would result in a higher level of free p-y. Furthermore, if 0-y is the major signaling unit, A p n - 1 and ,pn- l* , and A p n - 2 a n d p n - P strains should exhibit similar phenotypes. However, as mentioned above, strains with deletion or activating mutations in <qnn-I or p n - 2 exhibit different phenotypes. For example, Agnn-I strains produce short aerial hyphae ( ~ T Y Pt nl. 1996) while gnn-I* strains elaborate abundant long aerial hyphae and A p n - 2 strains produce normal aerial hyphae while pn- P strains exhibit dense aerial hyphae. T~ILIS, the data do not support sequestration of p-y as the only signaling function for , pn - I and p n - 2 in IV. crnssn.

Other models to explain the data are that GNA-I and GNA-2 regulate parallel signaling pathways that con-

142 R. A. Baasiri d nl.

i

verge, modulate the same pathway at different steps or control the same pathway with differing efficiency. If GNA-1 and GNA-2 regulate the same effector system, then p a - I and pn-2 would represent a gene family. Although they are prevalent in higher organisms (SI- MON pt nl. 1991), this would be the first example of a Ga subunit gene family in eukaryotic microbes. A precedent for a gene family in which deletion of one of the members has no phenotypes exists in the SSA subfamily of hsp70 genes of Snrchnrnmyws rpyp[lisinp (LINDQUIST and CRAIG 1988). ssnl ssn2 mutants do not grow at elevated temperatures, while ssn4 mutants are phenotypically wild type. However, ssnl ssn2 ssn4 mutants (analogous to the A p n - I Apn-2double mutant) are more impaired in the ssal ssn2 phenotype; they are inviable at all temperatures.

The results indicate that p n - I and pa-2 are essential for female fertility in N. mnssn. A role for Ga proteins in the sexual cycle has been demonstrated in the yeasts S. cprpoisinp and 5'. Pomhp (BORKOWCH 1996), the worm C. t4yyzn.s (MENDEL pt 01. 1995; SEGAIAT pt nl. 1995), and

A

OS I

Wild Type

B

L

Agm-2 I

I Additions:

None Sorbitol KC1 IiaC1

n

Relevant Genotype

FIGURE 4.-Deletion of p n - 2 accentuates the phenotypes of A p e 1 strains. (A) Apical extension rates on hyperosmotic media. Strains are w i l d type ( I 7h), Apn-1 (18a). Apa-2 (21c) and Apn-I Apo-2 (2113). (B) Colonvgrowth for A p o - I 18a, A p o - I Apo-2 3c and p W 7 rescued 3c-7-1 strains on VM + 0.75 LI NaCI.

the insect Drosojlhiln mdanognslm (PARK!! and WIESCHAUS 1991). In C. PIPgnns, mutation of the Gai superfamily protein gon-I leads to defects in both male and female reproduction. The common finding that mutation of a Gai superfamily gene in C. Phgnns and N. CT(ISS~ causes defects in sexual reproduction suggests a similar role for Gai superfamily members in mammals.

Another major phenotype of Apn-I Apn-2 mutants is sensitivity to hyperosmotic media. Resistance to osmotic stress, which includes desiccation and exposure to high concentration solutions, is important to the viability of numerous cell types, from bacteria to mammals (reviewed in BURG PI nl. 1996). In bacteria, there

FIGURE 5.--l'hrnotypcs tlrlring rhr S ~ X I I ; ~ cycle. Pcrithccia o n S(:SI ~vcre photographcd i clays I"'stli.rtilization. (A) Wild type (17h): (€5) A p n - 2 (21c); ( C ) Ag-nn-l (Ific); (D) A.pwl-1 Agxo-2 (Jc: a rare aberrant pcrithecium is shown); and (E) Apo- I Agm-2 + pRAR7 strain ?IC-7-1.

are at least three systems that mediate the response to hyperosmotic conditions; hvo of these are known to involve histidine kinase signaling pathways (RC'RG PI nl. 1996). In the yeast S. cmtwi.$icrP, there are hvo known pathways for responding to hyperosmotic conditions which feed into a common mitogen-activated protein kinase (MAPK) pathway. One branch of this system includes the SHOl SH8 domain protein (MAEDA rt n/. 1995). The other is a multistep phosphorelav histidine kinase-aspartate receiver module (POSAS P/ crl. 1996). In mammals, there are three MAPK pathways that are activated by exposure to osmotic stress. However, no histidine kinases have been identified in mammals, and the upstream osmosensors are unknown (BURG P/ d . 1996).

In N. crnssn, mutation of the histidine kinase nilc-1 causes sensitivity to hyperosmotic media (ALEX rt nl. 1996). Similarly, Ape-I and, more so, A p n - I Agnn-2 mutants are sensitive to hyperosmotic media. It is also

well documented that G proteins commonly operate upstream of MAPK modules in various organisms (HER- SKOWITZ 1995; BORKOVICH 1996; BURG p t nl. 1996; LOPEZ-~I.IJ\I\SACA PI nl. 199'7). Therefore, it is plausible that N. crmsn uses both a histidine kinase and G protein(s) to regulate MAPK pathwav(s) responsible for resistance to hyperosmotic conditions. Since mammals apparently lack histidine kinases, this may point to a role for G proteins in resistance to hyperosmotic conditions in higher organisms.

I t is intri<guing that p n - 2 has overlapping functions with a Gai (gnn-I) and an evolutionary relationship with a yeast Ga subunit (S. p o m h Gpal; M 7 1 1 . ~ ~ and YOKO- YAMA 1994). It has been proposed that the Gai family first appeared in the filamentous fungi (TURNER and BORKO\KH 1993). This hypothesis has been supported by reports of Gai family members in other filamentous fungi such as Coprinvs ron.,qrqntu.s (KOZAK P/ crl. 1995), Gyplronrctrin pnrnsitictr (CHOI rt 01. 1995) and A. nidulnns


(Yu et ul. 1996). Moreover, the completion of the S. cerevzszue genome sequence has allowed the determina- tion that this yeast does not contain sequences for a G a , homologue (our unpublished observations). Therefore, it is conceivable that gnu-2 descended from a progenitor gene homologous to those in yeasts. It has already been suggested that gnu-2 is an ancient Gai that gave rise to gnu-1 by a gene duplication event (BORKO- VICH 1996). However, this suggestion was based on the sequence characteristics of gnu-2. The work presented here supports this idea by showing that in addition to sequence homology, gnu-1 and gnu-2 also possess overlapping functions.

N. crussu presents a unique system for studying signal transduction mechanisms because it possesses Ga proteins with overlapping functions and well-defined molecular and classical genetics. Our next step is to identify the effector(s) and receptor(s) of the signaling pathways and determine if they are shared by GNA-1 and GNA-2.

We thank J. CHANDRA for technical assistance, R. WEISS, J. LOROS and D. EBBOLE for N. crassa strains and plasmids; T. VIDA and W. MARCOLIN for help with photomicroscopy and preparation of figures; R. DAVIS for assistance with tetrad dissections; G. FENG for densitome- try, and M. WINKLER and M. PLAMANN for use of equipment. We acknowledge L. ALEX, D. IVEY and Q. YANG for reviewing the manuscript. This work was supported by Texas Higher Education ARF' 011618-046 (K.A.B.), National Institutes of Health GM-48626 (KA.B.) and American Cancer Society Junior Faculty Research Award JFRA- 495 (K.A.B.).

LJTERATURE CITED ALEX, L. A., K. A. BORKOVICH and M. I. SIMON, 1996 Hyphal devel-

opment in Neurospora crmsa involvement of a twocomponent histidine kinase. Proc. Natl. Acad. Sci. USA 93: 3416-3421.

BAXEVANIS, A. D., and D. LANDSMAN, 1995 The HMGl box protein family: classification and functional relationships. Nucleic Acids Res. 23: 1604-1613.

BIRNBAUMER, L., 1992 Receptor-to-effector signalling through G proteins: roles for Pr dimers as well as a subunits. Cell 71: 1069- 1072.

BORKOVICH, K. A,, 1996 Signal transduction pathways and heterotrimeric G proteins, pp. 211-233 in The Mycota III: Biochemistly and Molecular Biology, edited by R. BRAMBL and G. A. MARZLUF. Springer-Verlag, Berlin, Heidelberg.

BURG, M. B., E. D. KWON and D. KULTZ, 1996 Osmotic regulation of gene expression. FASEB J. 10: 1598-1606.

CHOI, G. H., B. CHEN and D. L. Nuss, 1995 Virus-mediated or transgenic suppression of a Gprotein a subunit and attenuation of fungal virulence. Proc. Natl. Acad. Sci. USA 92: 305-309.

CORROCHANO, L. M., F.-R. LAUTER, D. J. EBBOLE and C. YANOFSKY, 1995 Light and developmental regulation of the gene con-10 of Neurospora crmsa. Dev. Biol. 167: 190-200.

DAVIS, R. H., and F. J. DESERRES, 1970 Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol. 71A: 79-143.

EBBOLE, D., 1990 Vectors for construction of translational fusions to P-galactosidase. Fungal Genet. Newsl. 37: 15-16.

GURR, S. J., S. E. UNKLES and J. R. KINGHORN, 1987 The structure and organization of nuclear genes of filamentous fungi, pp. 93- 139 in Gene Structure in Eukalyotic Microbes, edited by J. R. KING HORN. IRL Press Limited, Oxford, UK.

HANAHAN, D., 1983 Studies on transformation of Escherichia colzwith plasmids. J. Mol. Biol. 166: 557.

HERSKOWITZ, I., 1995 MAP kinase pathways in yeast: for mating and more. Cell 80: 187-197.

IVEY, F. D., P. N. HODGE, G. E. TURNER and K. A. BORKOVICH, 1996 The Gai homologue p a - l controls multiple differentiation pathways in Neurospora crassa. Mol. Biol. Cell 7: 1283-1297.

JOHNSON, G. L., A. M. GARDNER, C. LANCE-CARTER and N. Qw, 1994 How does the G protein, GiP, transduce mitogenic signals? J. Cell. Biochem. 54: 415-422.

KOZAK, K. R., L. M. FOSTER and I. K. ROSS, 1995 Cloning and characterization of a G protein a-subunitencoding gene from the basid- iomycete, Cqpnnus congregatus. Gene 163: 133-137.

KUNKEL, T. A,, J. D. ROBERTS and R. A. -OUR, 1987 Rapid and efficient sitespecific mutagenesis without phenotypic selection. Methods Enzymol. 154: 367-382.

LILLY, P., L. WU, D. L. WELKER and P. N. DEVREOTES, 1993 A G protein P subunit is essential for Dictyostelium development. Genes Dev. 7: 986-995.

LINDQUIST, S., and E.A. CRAIG, 1988 The heat-shock proteins. Annu. Rev. Genet. 22 631-677.

LOPEZ-ILLASACA, M., P. CRESPO, P. G. PELLICI, J. S. GUTKIND and R. WETZKER, 1997 Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase y . Science 275: 394-397.

MAEDA, T., M. TAKEKAWA and H. SAITO, 1995 Activation of yeast PBS2 MAPKK by MAF'KKKs or hy binding of an SHkontaining osmosensor. Science 269: 554-558.

MENDEL, J. E., H. C. KORSWAGEN, K. S. LIU, Y. M. HAJDU-CRONIN, M. I. SIMON et al., 1995 Participation of the protein Go in multiple aspects of behavior in C. elegans. Science 267: 1652-1655.

METZENBERG, R. L., 1988 An alternate way for collecting, storing and dissecting Neurospora asci. Fungal Genet. Newsl. 35: 28.

MILLER, J. H., 1972 Assay of &galactosidase, pp. 352-355 in Experi- ments in Molecular Genetics, edited hy J. H. MILLER. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

NEER, E.J., 1995 Heterotrimeric G proteins: organizers of transmembrane signals. Cell 8 0 249-257.

OAKLEY, B. R., J. E. RINEHART, B. L. MITCHELL, C. E. OAKLEY, C. CAR- MONA et al., 1987 Cloning, mapping and molecular analysis of the prrG (orotidine8'-phosphate decarboxylase) gene of Asperg& lus nidulans. Gene 61: 385-399.

OBARA, T., M. NAKAEUKU, M. YAMAMOTO andY. KAZIRO, 1991 Isola- tion and characterization of a gene encoding a Gprotein a subunit from Schizosaccharomyces pornbe: involvement in mating and sporulation pathways. Proc. Natl. Acad. Sci. USA 88: 5877-5881.

ORBACH, M. J., E. B. PORRO and C. YANOFSKY, 1986 Cloning and characterization of the gene for P-tubulin from a benomyl-resis- tant mutant of Neurospora crassa and its use as a dominant marker. Mol. Cell. Biol. 6: 2452-2461.

PARKS, S., and E. WIESCHAUS, 1991 The Drosophila gastrulation gene concertina encodes a Ga-like protein. Cell 64: 447-458.

POSAS, F., S. M. WURGLER-MURPHY, T. MAEDA, E. A. WITTEN, T. C. THAI et al., 1996 Yeast HOG1 M A P kinase cascade is regulated by a multistep phosphorelay mechanism in the SLNl-YPD1-SSK1 "two-component'' osmosensor. Cell 86: 865-875.

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Clon- ing, Ed. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SEGALAT, L., D. A. ELKES and J. M. KAPLAN, 1995 Modulation of serotonincontrolled behaviors by Go in Caenorhabditis elegans. Science 267: 1648-1651.

SIMON, M. I., M. P STRATHMANN and N. GAUTAM, 1991 Diversity of G proteins in signal transduction. Science 252 802-808.

SMULIAN, G., M. RYAN, C. STABEN and M. CUSHION, 1996 Signal transduction in Pneumocystzs carinii: characterization of the genes (pcgl) encoding the alpha subunit of the G protein (PCG1) of Pneumocystis carinii carinii and Pneumocystis carinii ratti. Infect. Immunol. 64: 691-701.

STABEN, C., B. JENSEN, M. SINGER, J. POLLOCK, M. SCHECHTMAN et al,, 1989 Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crmsa transformation. Fungal Genet. Newsl. 36: 79-81.

STRATHMANN, M., T. M. WILKIE and M. I SIMON, 1989 Diversity of the Gprotein family: sequences from five additional subunits in the mouse. Proc. Natl. Acad. Sci. USA 86 7407-7409.

STUDIER, F., W., A. H. ROSENBERG, J. J. DUNN and J. W. DUBENDORFF, 1990 Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185: 60-89.

TURNER, G. E., and K A. BORKOVICH, 1993 Identification of a G

N. masa

protein a subunit from Neurospora crassa that is a member of the Gi family. J. Biol. Chem. 268: 14805-14811.

VANN, D. C., 1995 Electroporation-based transformation of freshly hawested conidia of Neurospora crassa. Fungal Genet. Newsl. 4% 53.

VOLLMER, S. J., and C. YANOFSKY, 1986 Efficient cloning of genes of Neurospora crassa. Proc. Natl. Acad. Sci. USA 83: 4869-4873.

WILKIE, T. M., and S. YOKOYAMA, 1994 Evolution of the G protein alpha subunit multigene family, pp. 249-270 in MolecuZurEvolu- t ion of Physiological Processes, edited by D. M. FAMBROUGH. Rocke- feller University Press, New York.

p a - 2 145

YARDEN, O., M. PIAMANN, D. J. EBBOLE and C. YANOFSKY, 1992 cot- 1, a gene required for hyphal elongation in Neurospora crmsa, encodes a protein kinase. EMBO J. 11: 2159-2166.

Yu, J. H., J. WIESER, J. and T. H. ADAMS, 1996 The Aspergillus FlbA RGS domain protein antagonizes G protein signaling to block proliferation and allow development. EMBO J. 15 5184-5190.

ZWAAL, R. R., J. AHRINGER, H. G. A. M. VAN LUENEN, A. RUSHFORTH, P. ANDERSON et al., 1996 G proteins are required for spatial orientation of early cell cleavages in C. elegans embryos. Cell 86: 619-629.

Communicating editor: R. H. DAVIS

overlapping functions for two g protein a subunits in neurospora

Documents