Dysfunctional mitochondria modulate cAMP-PKA signaling and filamentous and invasive growth of Saccharomyces cerevisiae

Abstract

Mitochondrial metabolism is targeted by conserved signaling pathways that mediate external information to the cell. However, less is known about whether mitochondrial dysfunction interferes with signaling and thereby modulates the cellular response to environmental changes. In this study, we analyzed defective filamentous and invasive growth of the yeast Saccharomyces cerevisiae strains that have a dysfunctional mitochondrial genome (rho mutants). We found that the morphogenetic defect of rho mutants was caused by specific downregulation of FLO11, the adhesin essential for invasive and filamentous growth, and did not result from general metabolic changes brought about by interorganellar retrograde signaling. Transcription of FLO11 is known to be regulated by several signaling pathways, including the filamentous-growth-specific MAPK and cAMP-activated protein kinase A (cAMP-PKA) pathways. Our analysis showed that the filamentous-growth-specific MAPK pathway retained functionality in respiratory-deficient yeast cells. In contrast, the cAMP-PKA pathway was downregulated, explaining also various phenotypic traits observed in rho mutants. Thus, our results indicate that dysfunctional mitochondria modulate the output of the conserved cAMP-PKA signaling pathway.

Figure 1

Dysfunctional mitochondria interfere with signaling pathways regulating FLO11 transcription. (A) Filamentous growth of rho⁺ (SCΣ-48), rho⁺ tec1Δ (SCΣ-114), and respiratory-deficient rho mutants (SCΣ-139, -146, -150, -160). Yeast cells were grown for 3 days (rho⁺ strains) or 5 days (rho mutants) on a low-nitrogen (SLAD) medium (top) or SLAD supplemented with 1% isobutanol (middle). Invasion into agar was visualized after washing the plates under a stream of water (bottom). (B) Magnification of colonies marked in A. Percentage of long pseudohyphal (PH) cells with length-to-width ratio >2 is indicated below panels (n = 200). (C) Invasive growth assay. Strains were grown on YPD plates for 3 days (rho⁺ strains) or 7 days (rho mutants). Plates were washed under a stream of water and photographed before (top) and after (bottom) the wash. (D–F) P_FLO11::lacZ reporter activity in rho⁺ cells, rho mutants, and respective tec1Δ strains (SCΣ-114, -248, -250, -246, -271) carrying pYEp355-FLO11::lacZ. β-Galactosidase activity was measured from cells growing exponentially (D) or for 24 hr (E) in SC ura⁻leu⁻ medium or for 5 hr on SLAD medium (F). (G) Analysis of FLO11 transcript levels with quantitative PCR in rho⁺ (SCΣ-48) and rho⁰ mip1Δ (SCΣ-139). Strains were grown exponentially in YPD or patched onto YPD plates for 3 and 7 days before total RNA isolation. The results were normalized to the geometric average of two housekeeping genes (UBC6 and ARP6) and expressed relative to the exponentially growing rho⁺ strain. (D–G) Error bars represent standard deviations of three independent measurements. (H and I) Filamentous and invasive growth of strains ectopically expressing FLO11 (+P_TEF::FLO11). Strains were transformed with pB4126 and assayed for haploid filamentation and invasion as described for A and C. Bars, 100 μm.

Figure 2

RTG pathway is activated in both rho⁺ cells and rho mutants during starvation. (A) Signal transduction through the RTG pathway. Ovals indicate positive, rectangles negative, regulators of the pathway; shading indicates mutant used in this study. (B and C) The effect of RTG2 deletion on filamentous (B) and invasive (C) growth. Filamentation and invasion of rho⁺ (SCΣ-48), rho mutants (SCΣ-139, -146), and respective rtg2Δ strains (SCΣ-272, -276, -289) were analyzed as described for Figure 1, A and C. Bar, 100 μm. (D) P_FLO11::lacZ reporter activity in cells carrying pYEp355-FLO11::lacZ. (E and F) P_CIT2::lacZ reporter activity in cells carrying pEC261. β-Galactosidase activity was measured from cells growing exponentially in SC ura⁻leu⁻ medium (D and E) and on selective plates for 3 days (F). (G) Analysis of CIT2 transcript levels with quantitative PCR in rho⁺ (SCΣ-48) and rho⁰ mip1Δ (SCΣ-139). Strains were grown exponentially in YPD or patched onto YPD plates for 3 and 7 days. Data were normalized and expressed as described for Figure 1G. (D–G) Error bars indicate standard deviations of three independent measurements.

Figure 3

FG MAPK pathway is active and responsible for residual filamentation in rho mutants. (A) Signal transduction through the FG MAPK pathway. Ovals indicate positive regulators of the pathway; shading indicates mutant used in this study. (B and C) P_TEC1::lacZ reporter activity in rho⁺ (SCΣ-48), rho mutants (SCΣ-139, -146), and respective tec1Δ strains (SCΣ-114, -248, -250) carrying pBHM275. β-Galactosidase activity was measured from cells growing exponentially in SC ura⁻leu⁻ medium (B) and on selective plates for 3 days (C). Error bars indicate standard deviations of three independent measurements. (D and E) Filamentous (D) and invasive (E) growth of rho⁺ cells, rho mutants, and respective tec1Δ strains were analyzed as described for Figure 1, A and C. Bar, 100 μm.

Figure 4

Activation of the cAMP-PKA pathway restores filamentous and invasive growth of respiratory-deficient strains. (A) Signal transduction through the cAMP-PKA pathway. Ovals indicate positive, and rectangles negative, regulators of the pathway; shading indicates mutants used in this study. (B) Activity of cAMP-PKA and FG MAPK-responsive P_{FLO11 fragment}::lacZ reporters in rho⁺ (SCΣ-48), rho⁰ mip1Δ (SCΣ-139), and rho⁻ rpo41Δ (SCΣ-146). Scheme of the FLO11 locus indicates positions of cAMP-PKA pathway-responsive (−1.0 to −1.4 kb from FLO11 start codon, named 6/7) and FG MAPK-responsive (−1.6 to −2.0 kb from FLO11 start codon, named 9/10) promoter fragments. Numbers mark nucleotides in kilobases. β-Galactosidase activity was measured from cells growing exponentially in SC ura⁻leu⁻ medium and carrying pLG669-Z FLO11 6/7, pLG669-Z FLO11 9/10, or pLG669-Z (no insert). Error bars indicate standard deviations of three independent measurements. (C and D) The effect of cAMP-PKA pathway downregulation on filamentous and invasive growth. Colony morphology and invasive growth were analyzed in rho⁺ cells and rho mutants overexpressing SFL1 (pRS426-SFL1) or BCY1 (pRS426-BCY1) or containing the tpk2Δ mutation (SCΣ-312, -326, -328). (E and F) The effect of cAMP-PKA pathway activation on filamentous and invasive growth. Colony morphology and invasive growth were analyzed in rho⁺ cells and rho mutants overexpressing FLO8 (pRS426-FLO8) or TPK2 (pRS426-TPK2) or containing sfl1Δ (SCΣ-320, -335, -332) or bcy1Δ (SCΣ-316, -346, -344) mutations. Filamentation (C and E) and invasion (D and F) were analyzed as described for Figure 1, A and C. Bars, 100 μm.

Figure 5

cAMP-PKA pathway activity is downregulated in rho mutants. (A) Cellular processes controlled by PKA-mediated phosphorylation. (B) Heat-shock sensitivity of rho⁺ cells (SCΣ-48) and rho mutants (SCΣ-139, -146). Yeast cells were grown exponentially in YPD and exposed to heat shock at 52° for indicated times. Viability was expressed as the percentage of cells forming colonies after heat shock relative to untreated cells. (C) Serial dilution spot test of rho⁺ cells, rho mutants, and respective bcy1Δ strains (SCΣ-316, -346, -344) grown on YPD medium at the indicated temperatures. (D) Glycogen staining of rho⁺ cells, rho mutants, and respective bcy1Δ strains. Yeast cells were patched onto YPD and incubated for 3 and 6 days at 30° or 25°. Plates were exposed to iodine vapor. The dark color indicates the presence of glycogen stores. (E) Trehalase activity of rho⁺ cells and rho mutants and respective bcy1Δ strains. Yeast cells were grown in YPD at 25°. Error bars indicate standard deviations of three independent measurements.

Figure 6

Model showing the role of mitochondrial function in the regulation of cAMP-PKA signaling. Mitochondrial dysfunction triggers downregulation of the cAMP-PKA pathway in Σ1278b cells. This leads to a defective filamentous response despite the wild-type level activity of the RTG and FG MAPK pathways. Downregulation of the cAMP-PKA pathway results in modulation of several cellular processes in addition to filamentation in respiratory-deficient mutants. Activation is shown by arrows and inhibition by T-bars.