(Received for publication, May 20, 1997, and in revised form, June 24, 1997)
From the Institut für Mikrobiologie,
Georg-August-Universität Göttingen, D-37077
Göttingen, Germany, the § Whitehead Institute for
Biomedical Research, Cambridge, Massachusetts 02142, and the
Albert Einstein College of Medicine, Department of Molecular
Pharmacology, Bronx, New York 10461
The small GTP-binding protein Ras and
heterotrimeric G-proteins are key regulators of growth and
development in eukaryotic cells. In mammalian cells, Ras functions to
regulate the mitogen-activated protein kinase pathway in response to
growth factors, whereas many heterotrimeric GTP-binding protein
-subunits modulate cAMP levels through adenylyl cyclase as a
consequence of hormonal action. In contrast, in the yeast
Saccharomyces cerevisiae, it is the Ras1 and Ras2 proteins
that regulate adenylyl cyclase. Of the two yeast G-protein
-subunits
(GPA1 and GPA2), only GPA1 has been
well studied and shown to negatively regulate the mitogen-activated protein kinase pathway upon pheromone stimulation.
In this report, we show that deletion of the GPA2 gene
encoding the other yeast G-protein -subunit leads to a defect in
pseudohyphal development. Also, the GPA2 gene is
indispensable for normal growth in the absence of Ras2p. Both of these
phenotypes can be rescued by deletion of the PDE2 gene
product, which inactivates cAMP by cleavage, suggesting that these
phenotypes can be attributed to low levels of intracellular cAMP. In
support of this notion, addition of exogenous cAMP to the growth media
was also sufficient to rescue the phenotype of a GPA2
deletion strain. Taken together, our results directly demonstrate that
a G-protein
-subunit can regulate the growth and pseudohyphal
development of S. cerevisiae via a
cAMP-dependent mechanism. Heterologous expression of
mammalian G-protein
-subunits in these yeast GPA2
deletion strains could provide a valuable tool for the mutational
analysis of mammalian G-protein function in an in vivo null
setting.
When shifted to nitrogen starvation conditions, diploid cells of the yeast Saccharomyces cerevisiae switch from normal vegetative growth to filamentous growth (1-3). This switch in developmental patterning is regulated by intracellular signaling pathways.
Many signaling molecules have been identified that control filamentous growth. These include the genes encoding the protein kinase Ste20p (a homologue of mammalian p65PAK protein kinases), Ste11p (a MEKK or MAPK kinase kinase),1 Ste7p (a MEK or MAPK kinase), and the transcription factor Ste12p (4). Furthermore, during nitrogen starvation the Ras2 protein, in addition to activating adenylyl cyclase (5, 6), induces filamentous growth by stimulating the MAPK pathway (7). This pathway also regulates pheromone responsiveness via the evolutionary conserved Cdc42p/Ste20p/MAPK module (7). These observations suggest that in response to a nutritional signal, the MAPK pathway is activated by a cascade of small G-proteins, similar to the activation of the Jun N-terminal kinase pathway in mammalian cells (8).
Although the pheromone response pathway and the pathway regulating
filamentous growth share a common MAPK module, the upstream regulators
of each pathway seem to be specific to a given pathway. For example,
neither the G-protein -subunit (Gpa1p) nor the G
/
-protein subunits (Ste4p/Ste18p) of the heterotrimeric G-protein that regulates the pheromone response pathway (9) are involved in pseudohyphal development. Thus, the upstream regulators in the signaling pathway that controls filamentous growth remain unknown.
In the present study, we examine whether the G-protein -subunit
encoded by GPA2 regulates filamentous growth. Using a
genetic approach, we show that Gpa2p modulates pseudohyphal development via a cAMP-dependent pathway. In addition, Gpa2p is
necessary for normal growth on rich medium in cells that lack a
functional Ras2 protein, implicating Gpa2p as an important regulator of
normal cell growth.
All
strains used are congenic to the 1278b genetic background (4, 10). A
haploid gpa2 deletion strain was constructed by transforming
a disruption cassette in which the GPA2 open reading frame
has been replaced by the LEU2 gene. The ras1 and
ras2 mutations were constructed by using the disruption
cassettes pras1::HIS3 and
pras2::URA3, respectively (11). The
pde2 deletion strain was constructed by transforming a
polymerase chain reaction-based disruption cassette with a marker
conferring G418 resistance. For the filamentous growth assay, the
strains were made nutritionally prototrophic by transforming the
respective vectors of the pRS series of Centromer-based plasmids (12).
Standard yeast culture medium was prepared essentially as described
(13). Low ammonium medium plates for scoring pseudohyphal growth were
prepared as described (1). Standard procedures were used for yeast
transformation and genetic manipulations. Addition of cAMP was
performed by streaking a 100 mg/ml solution of the sodium salt of cAMP
(Sigma) in sodium phosphate buffer (pH 6.5) onto the assay plates to
obtain a final concentration of 5 mM. Sodium phosphate
buffer was added to the control plates.
The growth assay for filament formation and light microscopy of microcolonies were performed as described (1, 14).
Iodine StainingStrains were grown on rich medium for 2 days at 30 °C. The plates were then exposed to the vapor of elementous iodine for 30 s and photographed immediately.
Previous studies using a yeast strain deleted for GPA2 have been conducted by other investigators (15). However, no discernible phenotype was described, and pseudohyphal development was not examined (15).
To evaluate if the yeast G-protein -subunit, Gpa2p, regulates the
signaling cascade that controls pseudohyphal development, we
constructed a diploid strain homozygous for a deletion of the GPA2 open reading frame. In our strain background,
pseudohyphal development of the cells deleted for GPA2 was
strongly inhibited as compared with a corresponding wild-type strain
(Fig. 1, upper left and
right panels). Only occasionally could we detect filamentous outgrowth.
Assuming that Gpa2p acts linearly via Ras2p on the MAPK pathway to
regulate pseudohyphal development, a ras2 deletion mutant should exhibit the same phenotype as a gpa2,ras2
double mutant. Fig. 1, lower left panel shows that a ras2
deletion strain is still capable of forming filaments; however, the
cells constituting the filaments are round rather than being elongated.
A gpa2,ras2 double deletion strain exhibited the additive
phenotype of the respective single mutants, i.e. very few
filaments with round cells. This result suggests that Gpa2p and Ras2p
do not act on the same pathway leading to pseudohyphal development, but
rather that Gpa2p activates pseudohyphal development through a pathway parallel to the Ras2p pathway. These experiments were confirmed by
testing the transcriptional activation of the Ste12p-dependent FG::LacZ reporter gene (7) (that depends on a
functional MAPK pathway), which in a diploid gpa2
deletion strain under starvation condition was not significantly
altered (data not shown).
On rich medium, we also observed a very slow growth phenotype for the
gpa2,ras2 double deletion strain, while each of the single
mutants grew as the wild-type (Fig. 2,
upper panel). Interestingly, such a synthetic growth
phenotype was not observed when a gpa2 deletion was combined
with a ras1 deletion (Fig. 2, lower panel). This
observation provides further genetic evidence for a differential function of the RAS1 and RAS2 genes.
During normal haploid growth, Ras2p regulates intracellular cAMP levels
(5). Therefore, we wondered if Gpa2p may influence cAMP levels in
parallel to Ras2p. A combinatorial absence of the two proteins could
lower the cAMP levels so drastically that cells would grow only very
slowly. This hypothesis was tested by iodine staining of intracellular
glycogen, a well established inverse-proportional in vivo
measurement of intracellular cAMP levels (5). Compared with the
wild-type strain, both of the single mutants, gpa2 and ras2
, respectively, showed only slightly darker staining
(i.e. reduced intracellular cAMP levels; Fig.
3). For the ras2
strain this result was expected and is consistent with previous studies. The
double mutant gpa2
,ras2
, however, showed very dark
staining indicating very low intracellular cAMP levels; this
observation demonstrates a strong synthetic effect of both
mutations.
The correlation between dark iodine staining of the double mutant and
low intracellular cAMP levels was independently validated by further
deleting the PDE2 gene. This gene encodes a
phosphodiesterase that inactivates cAMP by cleavage. The absence of
Pde2p leads to an increase in intracellular cAMP levels (16, 17).
Indeed, after iodine staining, the triple mutant
gpa2,ras2
,pde2
appeared as the wild-type strain
(Fig. 3). The pde2 deletion within the gpa2
,ras2
strain also augmented cell growth to
wild-type levels (Fig. 4a).
These results clearly illustrate the necessary involvement of Gpa2p in
positively regulating cAMP levels in the absence of Ras2p.
To evaluate whether the absence of Pde2p would also facilitate the
diploid gpa2,ras2
double mutant to form filaments
under nitrogen starvation conditions, we next constructed a diploid homozygous triple mutant, gpa2
,ras2
,pde2
.
Fig. 4b shows the dramatic effect of a pde2
deletion within the gpa2,ras2 double deletion strain. As
predicted, pseudohyphal development was completely restored, resulting
in filaments similar to wild-type filaments. Importantly, a
pde2 deletion also reversed the effect of the
gpa2 and ras2
single mutants with respect to pseudohyphal development (data not shown).
Taken together, the above observations indicate that both phenotypes of Gpa2p-deficient yeast strains, (i) a defect in pseudohyphal development in diploid cells and (ii) a strong growth defect in ras2 deleted cells, are due to alterations in the regulation of cAMP levels.
Several genetic and biochemical studies in S. cerevisiae
have demonstrated that externally added cAMP is taken up by yeast cells
and can induce cAMP-dependent signals (19, 20). Fig. 5 shows that upon addition of exogenous
cAMP to the growth medium the gpa2 mutant strain behaved
as the wild-type strain, i.e. dramatically rescuing the
defect in pseudohyphal development. Note that both the
gpa2
mutant and the wild-type strain formed filaments in
the presence of cAMP (Fig. 5, lower panels), while only the
wild-type strain showed filamentous growth without added cAMP (Fig. 5,
upper panels). These results provide additional evidence
that Gpa2p normally modulates pseudohyphal development by positively
regulating cAMP levels within the yeast cell.
The present genetic evidence suggests that Gpa2p plays a role in the normal regulation of cAMP levels in yeast. We demonstrate that this regulation is important for the induction of pseudohyphae during nitrogen starvation conditions. However, on rich medium (YPD), Gpa2p is only necessary for normal growth in the absence of Ras2p.
Previously, Ras1p and Ras2p were thought to be the main regulators of cAMP in S. cerevisiae, demonstrating a role for Ras1p only in the early exponential growth phase (18). However, we have shown here that deletion of both RAS2 and GPA2 reveals that the remaining activity of Ras1p is not sufficient to induce normal cAMP levels and, thus, not sufficient to ensure wild-type-like growth. These observations argue that Gpa2p is responsible for inducing cAMP levels in a ras2 deletion strain to a considerable amount, and not only Ras1p. In addition, under low nitrogen conditions, cAMP levels regulated by Ras2p are not sufficient to induce filament formation. The presence of Gpa2p is necessary, in addition to Ras2p, to induce this dimorphic switch.
These observations suggest a new pathway in yeast by which
intracellular levels of cAMP are positively regulated by a G-protein -subunit. This pathway may be analogous to the known
Gs
-mediated pathway in mammalian cells. Whether Gpa2p
regulates cAMP levels through a direct or indirect interaction with
adenylyl cyclase remains to be elucidated. In this regard, heterologous
expression of mammalian G-protein
-subunits in these yeast
GPA2 deletion strains will provide an invaluable tool for
the mutational analysis of mammalian G-protein function in a unique
in vivo null setting.
We thank Dr. Gerald R. Fink and members of the Lisanti and Fink laboratories for helpful discussions and Dr. Steve Kron and Cora Styles for donating the pde2 deletion strain.