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. 1999 Sep;19(9):6110-9.
doi: 10.1128/MCB.19.9.6110.

The yeast trimeric guanine nucleotide-binding protein alpha subunit, Gpa2p, controls the meiosis-specific kinase Ime2p activity in response to nutrients

M Donzeau  1 , W Bandlow
Affiliations

The yeast trimeric guanine nucleotide-binding protein alpha subunit, Gpa2p, controls the meiosis-specific kinase Ime2p activity in response to nutrients

M Donzeau et al. Mol Cell Biol. 1999 Sep.

Abstract

Saccharomyces cerevisiae Gpa2p, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein (G protein), is involved in the regulation of vegetative growth and pseudohyphal development. Here we report that Gpa2p also controls sporulation by interacting with the regulatory domain of Ime2p (Sme1p), a protein kinase essential for entrance of meiosis and sporulation. Protein-protein interactions between Gpa2p and Ime2p depend on the GTP-bound state of Gpa2p and correlate with down-regulation of Ime2p kinase activity in vitro. Overexpression of Ime2p inhibits pseudohyphal development and enables diploid cells to sporulate even in the presence of glucose or nitrogen. In contrast, overexpression of Gpa2p in cells simultaneously overproducing Ime2p results in a drastic reduction of sporulation efficiency, demonstrating an inhibitory effect of Gpa2p on Ime2p function. Furthermore, deletion of GPA2 accelerates sporulation on low-nitrogen medium. These observations are consistent with the following model. In glucose-containing medium, diploid cells do not sporulate because Ime2p is inactive or expressed at low levels. Upon starvation, expression of Gpa2p and Ime2p is induced but sporulation is prevented as long as nitrogen is present in the medium. The negative control of Ime2p kinase activity is exerted at least in part through the activated form of Gpa2p and is released as soon as nutrients are exhausted. This model attributes a switch function to Gpa2p in the meiosis-pseudohyphal growth decision.

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Figures

FIG. 1
FIG. 1
Ime2CT specifically binds to Gpa2p. Strain MD211 was transformed with pCUP1-HAIME2CT together with an empty plasmid, pRS426 (lane 1), or with pCUP1-HisGPA2 (lane 2). Extracts from cells grown on lactate medium containing CuSO4 were purified on Ni-NTA columns. Bound proteins were analyzed by Western blotting using anti-Gpa2p and anti-HA antibodies.
FIG. 2
FIG. 2
Gpa2p bound to GTP physically interacts with Ime2p in vitro. Purified E. coli Gpa2p-His6 was preincubated with either GDP (lanes 2 and 4) or GTPγS (lanes 3 and 5) and loaded onto glutathione resin carrying GST (lanes 2 and 3) or GST-Ime2p (lanes 4 and 5). Resin-bound proteins were analyzed by Western blotting with anti-Gpa2p antibodies and by Coomassie blue staining for GST and GST-Ime2p. Lane 1, Gpa2p-His6 purified from E. coli; lane M, molecular weight standards.
FIG. 3
FIG. 3
Gpa2p inhibits sporulation in the presence of nitrogen or glucose. (A) Wild-type (wt) and Δgpa2/Δgpa2 mutant strains were grown on glucose-rich medium, shifted to SPO medium containing 4 mM NH4Cl, and incubated for 10 days. Sporulation was measured by counting the asci. (B) The strain wild-type (◊), a Δgpa2/Δgpa2 strain (■) transformed with empty vectors, and a Δime2/Δime2 Δgpa2/Δgpa2 strain cotransformed either with pP584-IME2 and pG0304 (○) or with pP584-IME2 and an empty vector (▴) were cultured on SD medium and shifted to SPO medium containing increasing concentrations of glucose as indicated. (C) Experiment performed as for panel B except that cells were shifted to SPO medium containing increasing concentrations of NH4Cl as indicated. Values represent averages of at least three independent experiments.
FIG. 4
FIG. 4
The constitutively active Gpa2p acts in a cAMP-independent pathway to regulate meiosis. A bcy1-tpk1w1 diploid strain transformed with either pVT-GPA2G132V (G132V), pVT-GPA2/G299A (G299A), or an empty plasmid (vector) was incubated in SPO medium for 2 days. Sporulation was scored by counting the asci. Values represent averages of at least three independent experiments.
FIG. 5
FIG. 5
Ime2p and the presence of acetate inhibit pseudohyphal development. Filamentous growth of the diploid strain CEN.PK2 transformed with an empty plasmid (A) or with pP584-IME2 overexpressing Ime2p (B) and of the isogenic Δgpa2/Δgpa2 mutant diploid strain (C) was inspected after 4 days of growth on SLAD medium. Strain CEN.PK2 strain was also tested for filamentous growth on SLADA medium (D).
FIG. 6
FIG. 6
Gpa2p does not promote proteolytic degradation of Ime2p. (A) Strain MD211 was transformed with pP584-IME2-His together with pRS426 as a control (−Gpa2p) or with pG0304 (+Gpa2p). As a negative control, MD211 was transformed with plasmids pRS423 and pRS426 (Control). Transformants were grown in SPO medium containing 10 mM NH4Cl (+N), in SPO medium (−N), or in YPD medium (G). After purification on an Ni-NTA column using the same protein amounts, bound proteins were analyzed by Western blotting using anti-His6 antibodies. (B) MD211 was transformed with either pRS426 as a control (−Gpa2p) or pG0304 (+Gpa2p). Cells were grown in YPD medium (G), in SPO medium (−N), or in SPO medium containing 10 mM NH4Cl (+N). Total extracts were analyzed by Western blotting using anti-Gpa2p antibodies. Control, Gpa2-His6 recombinant protein from E. coli.
FIG. 7
FIG. 7
Nitrogen influences interaction between Gpa2p and Ime2p in vivo. Copurification of Gpa2p and Ime2p-His6 was performed as follows. Strain MD211 was cotransformed with pG0304 and either pRS423 as a control (lanes 2 and 3) or pP584-IME2His6 (lanes 4 and 5). Cells were grown in SPO medium (−N; lanes 2 and 4) or in SPO medium containing nitrogen (+N; lanes 3 and 5). Total cell extracts were loaded onto an Ni-NTA column, and bound proteins were analyzed by Western blotting with purified anti-Gpa2p and anti-His6 antibodies (a). Aliquots of total cell extracts were also analyzed by Western blotting using purified Gpa2 antibodies (b). Lane 1, Gpa2p-His6 recombinant protein, purified from E. coli; lane M, molecular weight standards.
FIG. 8
FIG. 8
Autoradiograms showing that Gpa2p-His6 recombinant protein bound to GTPγS inhibits Ime2p kinase activity. (A) Histone H1 phosphorylation was assayed with GST-Ime2p purified from E. coli and [γ-32P]ATP in the presence of BSA (lane 2), Gpa2p bound to GDP (lane 3), or Gpa2p bound to GTPγS (lane 4), washed free of unbound guanine nucleotides. GST was incubated under the same conditions in the presence of BSA (lane 1). As a control, histone H1 phosphorylation by PKA was assayed in the presence of either BSA (lane 5), Gpa2-GDP (lane 6), or Gpa2-GTPγS (lane 7). (B) Ime2p-His6 purified from MD211 cells transformed with pP583-IME2His6 and grown in SPO medium was assayed for histone H1 phosphorylation in the presence of either BSA (lane 2), Gpa2p bound to GDP (lane 3), or Gpa2p bound to GTPγS (lane 4). As a control, extracts from yeast cells transformed with an empty plasmid and grown in SPO medium were used (lane 1).
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