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 and Katherine A. Borkovich"
E. Turner*'*
*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 activityindicate 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 formationand conidial germination. Extensive phenotypic analysis ofAgna-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 functionsand 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 ofinformation thatis transduced into the cell interior by heterotrimeric ( a P y ) G proteins (BIRNBAUMER 1992). Ligand bindingtothe receptor promotes the exchange of GTP for GDP on the a subunit of the G protein and dissociation of the a and P y subunits. Depending on the signaling pathway, one or both subunits can activate downstream effectors (HERSKOWITZ1995; NEER 1995),which in turn generate anintracellular response. Ga genes have been isolated from both higher and lower eukaryotes (NEER 1995; BORKOVICH 1996). In mammals, G a subunits have been grouped into four classes, G a s , G a i , Gaq and GaI2, based on amino acid sequence identity (SIMONet ul. 1991). In contrast, the limited number of cloned G a 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 Microbiology and Molecular Genetics, Universityof 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 morphological abnormalities (IVEYet ul. 1996). N. crussu gnu-2 encodes an G a subunit that is not a member of any G a 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; SMULIAN et ul. 1996). The function of PCGl in the opportunistic 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 (WILKIEand YOKOYAMA1994). In this study, we investigate the expression and functions of N. crassu gnu-2. We report the organization of this gene includingtranscription start sites, intron positions and putative upstream regulatory elements. We describe the effects of targeted gene replacement and constitutively activatingmutations in gnu-2. In addition, we present evidence that Agnu-1Agnu-2 strains are more impaired in female fertility and osmotic sensitivity than Agnu-1 strains. We hypothesize that gnu-I and gna2 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
TABLE 1 N. crassa strains ________-
Comments Strain
yrG+
4
R.A. Baasiri et al.
pyr-4
Agna-Z::pyrG+
74 A 73 a FGSC #6103 KB5 #3 5x3s SGMl 3-7 FGSC #2082 PY"4 T3-2 A33-2 A334 a29-1 3-7-6
3-5-5 4-6-7 5-7-7 6-2-2
8-6-3 17b AgnaZ::pyrG+ 21c 16c 1Sa 3c AgnaZ::pyrG+ 21d
Genotype Wild type A Wild type a his-? A Amtr pdx-1 A his-? a his-? pdx-1 Amtr a Agna-1 ::mtr+ al-3 A Agna-1 ::mtr+ his-2 pdx-1 a al-3, A pyr-4 (a or A ) A Agna-2::pyrG+ pyr-4 A Agna-2::pyrG+ pyr-4 a A hir-3+:: pDE3 (lacZ+)pdx-l a his-?+ : :pDE3 (lacZ+)pdx-l a his-3+: :pSR4 (gna-Z+)pdx-l a his-?+ : :pSR5 ( p a - 2 Q205L)pdx-1 u hi.~-3+ : pSR6 (gnu-2R179C)pd~-I his-?+::pSR8 (5'-gna-2::lacZ)pdx-l u al-? gna-2+ gnu-l + A a Agna-1 ::mtr+ al-3 A Agna-1 ::mtr+ al-3 A Agna2::pyrG+ Agna-l ::mtr+ A Agna-1 ::mtr+ A
73a X 6103 KB5 x #3 3-7 X 2082
PPY'G Tx
pDE3 This Tx pDE3 Tx pSR4 Tx pSR5 Tx pSR6 This Tx lacZ fusion 33-4 X SGMl 334 x SGMl 334 X SGMl 33-4 X SGMl 33-4 X SGMl 33-4 X SGMl
R. L. WEISS,UCLA R.L. WEISS,UCLA J. J. LOROS,Dartmouth IVEY et al. (1996) This study This study This study IVEYet al. (1996) FGSC R. L. WEISS, UCLA This study This study This study This study study This study This study This study study This study This study This study This study This study This study 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 pdxauxotrophs 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 (DAVISand 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 haddetermined in preliminaryexperiments that this amount of flanking DNA was not sufficient to obtain areasonable homologous recombination frequency during gene replacement experiments. Therefore, toobtain a largercarboxy 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 (Stratagene; pXCOh111). The entire BamHI-XbaI fragment depicted in Figure 1A was obtained by ligating the insert from pXHL3 and a 0.78kb BamHI-XbaI fragment from pXCOhIII into pBluescript I1 K S +
SK' (Stratagene; pXHL29). Sequence analysis of the BamHIXbaI region was as previously described (IVEYet 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 (YARDENet al. 1992), hut using sand and liquid N2to break open cells. RNA was treated with RQ1 DNase (Promega) according to the manufacturer'srecommendations. 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 BamHISmaI 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; et al. 1989) fragment from ppyrG containing the SAMBROOK et al. 1987), anda XbaIAspergillus nidulanspyrGgene (OAKLEY 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 (STABENet 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
A
\
9
W
=CnTGorCAAAG = van~criptions ~ a site n
=inm
k lb
fioA~hdm
1-
\
Hhdm
\Ndd
B
P-gal S.A. NA 124 241 183 ND ND 74A 73a
139
s scribed (VASN1995; IWY et n/. 1996). Strain / ~ , - - 4 w aelcctroporated 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. .5X3Swas also elcctroporated with pSR4 to ohtain strains with two wild-type copies of gnn-2+, or with pDE3 t o obtain isogenic ItlS-Ji- controls. T o obtain A p n - I Apcr-2strains. an nl-?strain (F
