Dysfunctional Mitochondria Modulate cAMP-PKA Signaling and … · 2013. 2. 7. · signaling (Schefﬂer 2001; McBride et al. 2006). In yeast cells that lack the wild-type mitochondrial

INVESTIGATION

Dysfunctional Mitochondria Modulate cAMP-PKASignaling and Filamentous and Invasive Growth of

Saccharomyces cerevisiaeAnu Aun,1 Tiina Tamm,1 and Juhan Sedman2

Department of Biochemistry, Institute of Molecular and Cell Biology, University of Tartu, Tartu 51010, Estonia

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 responseto environmental changes. In this study, we analyzed defective filamentous and invasive growth of the yeast Saccharomyces cerevisiaestrains that have a dysfunctional mitochondrial genome (rho mutants). We found that the morphogenetic defect of rho mutants wascaused by specific downregulation of FLO11, the adhesin essential for invasive and filamentous growth, and did not result from generalmetabolic changes brought about by interorganellar retrograde signaling. Transcription of FLO11 is known to be regulated by severalsignaling pathways, including the filamentous-growth-specific MAPK and cAMP-activated protein kinase A (cAMP-PKA) pathways. Ouranalysis showed that the filamentous-growth-specific MAPK pathway retained functionality in respiratory-deficient yeast cells. Incontrast, the cAMP-PKA pathway was downregulated, explaining also various phenotypic traits observed in rho mutants. Thus, ourresults indicate that dysfunctional mitochondria modulate the output of the conserved cAMP-PKA signaling pathway.

MITOCHONDRIAL metabolic activities are coordinatedwith the availability of nutrients through several con-served signaling pathways. In the budding yeast Saccharo-myces cerevisiae, the cAMP-activated protein kinase A(cAMP-PKA) pathway can modulate the enzyme content ofmitochondria, reactive oxygen species generation, antiox-idant defense system, and mitochondrial protein import(Dejean et al. 2002; Hlavatá et al. 2003; Chevtzoff et al.2005; Feliciello et al. 2005; Hlavatá et al. 2008; Schmidtet al. 2011). Downregulatation of the target of rapamycin(TOR) pathway leads to an increase of mitochondrial re-spiratory complexes (Bonawitz et al. 2007; Pan and Shadel2009). The Snf1 pathway regulates the switch from glyco-lytic energy production to mitochondrial respiration in re-sponse to low-glucose and ADP levels (Ulery et al. 1994;Mayer et al. 2011).

Disturbed mitochondrial metabolism, in turn, can affecta broad range of cellular activities through aberrant fluxes ofmetabolites, formation of reactive oxygen species, or cellular

signaling (Scheffler 2001; McBride et al. 2006). In yeastcells that lack the wild-type mitochondrial genome (rhocells), the retrograde signaling pathway (RTG) is activated,leading to changes in nuclear gene expression and readjust-ments of carbohydrate and nitrogen metabolism (Liu andButow 2006). Homologs of RTG genes have not been foundin higher organisms, but the central stress regulator NF-kBhas been proposed to fulfill similar functions (Srinivasanet al. 2010).

Two recent reports indicate that dysfunctional mitochon-dria can directly interfere with signaling pathways thatmediate nutritional information to the yeast cell. In rhocells, the main target of the TOR pathway, the Sch9 kinase,is dephosphorylated, suggesting downregulation of the path-way (Kawai et al. 2011). Mitochondrial dysfunction can alsointerfere with the regulation of autophagy by modulating theactivity of the cAMP-PKA pathway (Graef and Nunnari 2011;Kawai et al. 2011).

Several of the signaling pathways that regulate mitochon-drial metabolism are also required to activate an elaboratedifferentiation program leading to pseudohyphal or filamen-tous growth (Brückner and Mösch 2012). Under specificnutrient-poor conditions, yeast cells switch to a unipolar bud-ding pattern and form physically attached elongated cellsthat can invade the growth substrate (Gimeno et al. 1992;

Copyright © 2013 by the Genetics Society of Americadoi: 10.1534/genetics.112.147389Manuscript received July 11, 2012; accepted for publication November 5, 20121These authors contributed equally to this work.2Corresponding author: University of Tartu, Riia 23B-218, Tartu 51010, Estonia. E-mail:[email protected]

Genetics, Vol. 193, 467–481 February 2013 467

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Kron et al. 1994; Roberts and Fink 1994). Pseudohyphaldifferentiation can be initiated by nitrogen starvation or inlow-glucose media (Gimeno et al. 1992; Cullen and Sprague2000). It is also induced with fusel alcohols, the end prod-ucts of amino acid catabolism in yeast, suggesting that in-termediary metabolism can modulate the interpretation ofnutritional signals received by the cells (Dickinson 1996;Lorenz et al. 2000; Jin et al. 2008).

Nutritional clues are sensed and filament formation isregulated through the activation of complex and partiallyinterconnected pathways, most notably the filamentous-growth (FG)-specific MAPK cascade and the cAMP-PKApathway (Liu et al. 1993; Roberts and Fink 1994; Robertsonand Fink 1998; Pan and Heitman 1999). The pathways con-verge on FLO11 (MUC1), encoding a cell-surface glycopro-tein that is essential for the morphogenetic switch (Liu et al.1993; Roberts and Fink 1994; Lo and Dranginis 1998;Robertson and Fink 1998; Pan and Heitman 1999; Ruppet al. 1999).

Evidence that mitochondrial genes play a role in fila-mentation has been obtained from large-scale studies andgenetic screens of filamentation-defective mutants (Lorenzet al. 2000; Kang and Jiang 2005; Jin et al. 2008). Whilerespiratory-deficient yeast mutants appear to be defective infilament formation, the underlying mechanism remains un-clear. Activation of the RTG pathway has been suggested toinhibit filamentous growth by changing nuclear gene expres-sion in rho cells (Liu and Butow 2006; Jin et al. 2008).Somewhat contradictory results, however, have demon-strated that inactivation of the RTG signaling blocks invasivegrowth of respiratory-competent cells (Chavel et al. 2010).

Here we scrutinize the filamentous and invasive growthproperties of rho mutants and show that they can undergomorphogenetic change; however, the cells do not expressthe cell-surface adhesin Flo11. Analyses of the RTG, FGMAPK, and cAMP-PKA signaling pathways indicate thatthe dysfunctional state of mitochondria modulates signalingthrough the cAMP-PKA pathway and that this results indownregulation of FLO11.

Materials and Methods

Yeast strains and media

Yeast strains used in this study are listed in Table 1. Genedeletions were generated by one-step PCR-based gene disrup-tion, replacing the complete ORFs with kanMX6, natMX6, orhphMX6 cassettes (Janke et al. 2004; Hentges et al. 2005).Respiration-deficient rho2 and rho0 strains were generatedusing two methods. First, deletions of RPO41 or MIP1 genescaused restructuring (rho2, strain SCS-146) or complete loss(rho0, strain SCS-139) of mitochondrial DNA (mtDNA),respectively. Second, ethidium bromide treatment was usedto generate rho2 and rho0 mutants (strains SCS-160 andSCS-150) (Foury 2002). The respiratory deficiency of thestrains was tested on a nonfermentable carbon source. Loss

of mtDNA was verified by 49,6-diamidino-2-phenylindolestaining.

Cells were grown at 30� unless specified otherwise ineither YPD medium (1% Bacto yeast extract, 2% Bacto pep-tone, 2% glucose) or synthetic complete medium withouturacil and leucin (SC ura2leu2) to select for transformants(Sherman 2002). Agar (2%) was added to solid media. Fil-amentous growth was assayed on solid low-nitrogen (SLAD)medium (2% glucose, 50 mM ammonium sulfate, 0.17%yeast nitrogen base without amino acid and ammonium sul-fate) supplemented with 1% isobutanol (Lorenz et al. 2000).Strains were complemented for auxotrophic mutations withpRS-series plasmids (Sikorski and Hieter 1989) and grownfor 5–7 days to obtain equal colony size. Colony morphologywas examined with an Olympus BX61 microscope usinga ·10 Plan Olympus objective and bright-field optics. Theimages were captured with an Olympus DP70 cooled CCDcamera. For the invasive growth assay, the strains weregrown on YPD plates for 3–7 days to obtain similar patchdensity. Plates were washed under a gentle stream of waterand photographed before and after the wash (Roberts andFink 1994). The bcy1D strains were analyzed at 25� for in-vasive growth and glycogen accumulation.

Plasmids

Plasmids used in this study are listed in Table 2. PlasmidspLG669-Z FLO11 6/7 and 9/10 were constructed as de-scribed (Rupp et al. 1999). FLO11 promoter fragments(21400 to 21000 bp from the FLO11 start codon corre-sponds to fragment 6/7 and 22000 to 21600 bp to frag-ment 9/10, respectively) were amplified from genomic DNAand cloned into a XhoI-digested pLG669-Z (Guarente andPtashne 1981). Genomic loci of TPK2 (2579 to +566 bp)(Pan and Heitman 1999), BCY1 (2420 to +400 bp), SFL1(2683 to +719 bp), and FLO8 (21032 to +497 bp) (vanDyk et al. 2003) were amplified with PCR and cloned be-tween XhoI and BamHI restriction sites into the pRS426vector (Christianson et al. 1992). A minus sign (“2”) indi-cates nucleotides upstream from the start codon, and a plussign (“+”) indicates nucleotides downstream from the stopcodon of the respective gene.

Cell elongation measurement

Cell elongation was determined as described (Mösch andFink 1997). Strains were grown on SLAD medium supple-mented with 1% isobutanol for 2 (rho+) or 4 (rho mutant)days at 30� to obtain equal colony size. The length-to-width(l/w) ratio of at least 200 cells from multiple colonies wasquantified using light microscopy. Cells were divided intotwo classes: yeast form cells with a l/w ratio of 1:1 to 1:2and pseudohyphal cells (PH) with a l/w ratio .2.

Reporter gene assay

A reporter plasmid containing the URA3 gene (Table 2) wastransformed with pRS315 containing the LEU2 gene (Sikorskiand Hieter 1989) into yeast strains to compensate for

468 A. Aun, T. Tamm, and J. Sedman

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auxotrophic mutations. Cells were pregrown in SC ura2leu2

medium, inoculated into fresh medium to an OD600 of �0.3,and grown for 4–5 hr to an OD600 of �1.0–1.5 (exponentialgrowth phase). For the 24-hr time point, cells were grown for24 hr. For 5-hr growth on SLAD, cells were grown for 20 hr,washed twice with 2% glucose, transferred into SLAD me-dium, and grown for 5 hr as described (Rupp et al. 1999).Starvation conditions were induced by plating �105 cellsfrom exponentially growing cultures onto SC ura2leu2 platesand incubating for 3 days. Cells were scraped off the plate,washed twice with 2% glucose, and assayed for reporter

activity (Madhani and Fink 1997; Sabbagh et al. 2001).b-galactosidase activity was determined with the use of or-tho-nitrophenyl-b-galactoside as a substrate and expressedas Miller Units [nmol of o-nitrophenol produced/(mg of to-tal protein*ml*min)] (Rose and Botstein 1983).

Quantitative PCR analysis

Total RNA was isolated from cultures (15 ml) grownexponentially in YPD medium or from patches grown for 3or 7 days on YPD plates (Mai and Breeden 1997). Five micro-grams of total RNA were treated with DNase I, followed by

Table 1 Yeast strains used in this study

Strain (S1278bgenetic background) Relevant genotype Genotype Source

SCS-48 rho+ MATa [rho+] ura3-52 leu2D0 Steffen RuppSCS-139 rho0 mip1D MATa [rho0] ura3-52 leu2D0 mip1D::natMX6 This studySCS-146 rho2 rpo41D MATa [rho2] ura3-52 leu2D0 rpo41D::kanMX6 This studySCS-150a rho0 MATa [rho0] ura3-52 leu2D0 This studySCS-160a rho2 MATa [rho2] ura3-52 leu2D0 This studySCS-114 rho+ tec1D MATa [rho+] ura3-52 leu2D0 tec1D::natMX6 This studySCS-248 rho0 mip1D tec1D MATa [rho0] ura3-52 leu2D0 mip1D::natMX6 tec1D::hphMX6 This studySCS-250 rho2 rpo41D tec1D MATa [rho2] ura3-52 leu2D0 rpo41D::kanMX6 tec1D::hphMX6 This studySCS-246 rho0 tec1D MATa [rho0] ura3-52 leu2D0 tec1D::hphMX6 This studySCS-271 rho2 tec1D MATa [rho2] ura3-52 leu2D0 tec1D::hphMX6 This studySCS-272 rho+ rtg2D MATa [rho+] ura3-52 leu2D0 rtg2D::natMX6 This studySCS-276 rho0 mip1D rtg2D MATa [rho0] ura3-52 leu2D0 mip1D::natMX6 rtg2D::hphMX6 This studySCS-289 rho2 rpo41D rtg2D MATa [rho2] ura3-52 leu2D0 rpo41D::kanMX6 rtg2D::hphMX6 This studySCS-291 rho0 rtg2D MATa [rho0] ura3-52 leu2D0 rtg2D::hphMX6 This studySCS-281 rho- rtg2D MATa [rho2] ura3-52 leu2D0 rtg2D::hphMX6 This studySCS-312 rho+ tpk2D MATa [rho+] ura3-52 leu2D0 tpk2D::hphMX6 This studySCS-326 rho0 mip1D tpk2D MATa [rho0] ura3-52 leu2D0 mip1D::natMX6 tpk2D::hphMX6 This studySCS-328 rho2 rpo41D tpk2D MATa [rho2] ura3-52 leu2D0 rpo41D::kanMX6 tpk2D::hphMX6 This studySCS-324 rho0 tpk2D MATa [rho0] ura3-52 leu2D0 tpk2D::hphMX6 This studySCS-330 rho2 tpk2D MATa [rho2] ura3-52 leu2D0 tpk2D::hphMX6 This studySCS-316 rho+ bcy1D MATa [rho+] ura3-52 leu2D0 bcy1D::hphMX6 This studySCS-346 rho0 mip1D bcy1D MATa [rho0] ura3-52 leu2D0 mip1D::natMX6 bcy1D::hphMX6 This studySCS-344 rho2 rpo41D bcy1D MATa [rho2] ura3-52 leu2D0 rpo41D::kanMX6 bcy1D::hphMX6 This studySCS-348 rho0 bcy1D MATa [rho0] ura3-52 leu2D0 bcy1D::hphMX6 This studySCS-350 rho2 bcy1D MATa [rho2] ura3-52 leu2D0 bcy1D::hphMX6 This studySCS-320 rho+ sfl1D MATa [rho+] ura3-52 leu2D0 sfl1D::hphMX6 This studySCS-335 rho0 mip1D sfl1D MATa [rho0] ura3-52 leu2D0 mip1D::natMX6 sfl1D::hphMX6 This studySCS-332 rho2 rpo41D sfl1D MATa [rho2] ura3-52 leu2D0 rpo41D::kanMX6 sfl1D::hphMX6 This studySCS-334 rho0 sfl1D MATa [rho0] ura3-52 leu2D0 sfl1D::hphMX6 This studySCS-337 rho2 sfl1D MATa [rho2] ura3-52 leu2D0 sfl1D::hphMX6 This studya rho mutants isolated by ethidium bromide treatment.

Table 2 Plasmids used in this study

Plasmid Description Source

pB4126 PTEF::FLO11, URA3, CEN Gerald R. Fink (Voynov et al. 2006)pYEp355-FLO11::lacZ PFLO11::lacZ, URA3, 2m Gerald R. Fink (Rupp et al. 1999)pBHM275 PFRE(TEC1)::lacZ, URA3, 2m Gerald R. Fink (Madhani and Fink 1997)pEC261 PCIT2::lacZ, URA3, CEN Chris A. Kaiser (Chen and Kaiser 2003)pLG669-Z PCYC1::lacZ, URA3, 2m NBRP of the MEXT (Guarente and Ptashne 1981)pLG669-Z FLO11 6/7 PFLO11 6/7::lacZ, URA3, 2m This studypLG669-Z FLO11 9/10 PFLO11 9/10::lacZ, URA3, 2m This studypRS426-TPK2 TPK2, URA3, 2m This studypRS426-BCY1 BCY1, URA3, 2m This studypRS426-SFL1 SFL1, URA3, 2m This studypRS426-FLO8 FLO8, URA3, 2m This study

NBRP of the MEXT: National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology (Japan).

Mitochondria Regulate Yeast Filamentation 469

cDNA synthesis from 1 mg of RNA with RevertAid M-MuLVReverse Transcriptase (Fermentas) according to the manufac-turer’s instructions. One-twentieth of the synthesized cDNAwas used as a template for quantitative real-time PCR. Quan-titative PCR was performed in a 10-ml reaction volume withthe Maxima SYBR Green qPCR Master Mix (Fermentas) usingan ABI Prism 7900HT Fast Real-Time PCR System in standardconditions (denaturation at 95� for 15 min; 40 cycles of de-naturation at 95� for 15 sec and annealing at 60� for 1 min).Melt-curve profiles were generated to confirm specificity of am-plified fragments. The length of PCR products was �160 bp(primer sequences used are available upon request). Geneexpression was quantified by the comparative CT method(Schmittgen and Livak 2008). For normalization of mRNA lev-els, the geometric mean of two housekeeping genes (UBC6,ARP6) was used. Suitability of UBC6 and ARP6 as housekeepinggenes was determined by the geNorm program (Vandesompeleet al. 2002; Teste et al. 2009). The average and standard devi-ations were calculated from three independent experiments.

Heat-shock sensitivity

Exponentially growing cells in YPD medium were exposed to52� for 4–12 min and plated onto YPD plates. After growthat 30� for 3–5 days, the colonies were counted. Viability wasexpressed as a percentage of cells forming colonies after theheat shock relative to the number of colonies in the un-treated samples (Hlavatá et al. 2003).

Glycogen staining

Cells were streaked onto YPD plates and grown for 3–6 daysat 30� or 25�. Plates were exposed to iodine vapor until theappearance of a brown coloration, indicating the presence ofglycogen stores, and then photographed.

Trehalase assay

Precultures were diluted into fresh YPD medium and grownfor 24 hr at 25� when the wild-type cells had passed thediauxic shift. Cell extracts were prepared and a trehalaseassay was carried out as described (Carrillo et al. 1994),except 10 mM MES/KOH (pH 6.8) was used in reactionmixtures. Glucose concentration in the supernatant was de-termined with Glucose Liquidcolor (Human GmbH). Proteinconcentration was determined with the Bradford method.Trehalase activity was expressed as nanomoles of glucosereleased/(mg of total protein*min) (Carrillo et al. 1994).

The average and standard deviations for reporter con-struct, heat-shock sensitivity, and trehalase assays werecalculated from three independent cultures, and experi-ments were performed at least twice.

Results

Functional mitochondrial genome is required forfilamentous and invasive growth of budding yeast

Previous genome-wide screens of genes required for yeastfilamentous growth have indicated that respiratory-deficient

yeast cells are defective in filament formation (Kang andJiang 2005; Jin et al. 2008). However, it is unclear if respi-ratory deficiency generates defects in filamentous growthas a result of general remodeling of cellular metabolism orby interference with specific signaling pathways that regu-late the filamentation response. To investigate the role ofmitochondria in the filamentous and invasive growth in de-tail, we compared the ability of wild-type rho+ and respira-tory-deficient rho haploid cells of the S1278 background toform filaments and invade agar substrates. Mutant strainslacking the mitochondrial genome (rho0) or retaining non-functional short fragments of mitochondrial DNA (rho2)were constructed either by ethidium bromide mutagenesisor by disruption of RPO41 orMIP1 involved in mitochondrialgenome maintenance (Genga et al. 1986; Fangman et al.1990).

Filamentous growth of rho mutants and rho+ cells wasassayed on low-nitrogen (SLAD) medium supplementedwith 1% isobutanol (Figure 1A, middle panels). Invasivegrowth was monitored on the same medium or on YPD afterwashing non-adherent cells off the plates (Figure 1, A and C,bottom panels). All tested respiratory-deficient strains weredefective in both filament formation and agar invasion,and no variation could be observed between different rho0

and rho2 mutants. The rho cells formed fewer short and lessbranched filaments compared to the rho+ strain (Figure 1B).However, we also noted that, despite the obvious defect infilamentation, the rho mutant cells appeared to be elon-gated. Seventy-eight percent of rho cells had pseudohyphalgeometry, and this was only slightly less than the corre-sponding fraction of elongated cells of rho+ strain (84%)under similar growth conditions (Figure 1B). Compared tothe rho+ tec1D strain, the rho mutants retained some resid-ual filament formation and invasion. TEC1 encodes a tran-scription factor activated by the FG MAPK cascade (Figure3A) and is required for both isobutanol-induced filamenta-tion and invasive growth (Mösch et al. 1996; Lorenz et al.2000). Our results demonstrate that rho cells exhibit defec-tive invasive growth. We also confirm that a functional mi-tochondrial genome is required for a wild-type level offilamentation, as reported earlier (Kang and Jiang 2005;Jin et al. 2008).

Dysfunctional mitochondria interfere with signalingpathways regulating FLO11 transcription

The weak invasive phenotype and defective filament forma-tion led us to ask if the adhesin FLO11, required for bothfilament formation and agar invasion (Lambrechts et al.1996; Lo and Dranginis 1998; Robertson and Fink 1998),is expressed in rho cells during starvation.

To analyze FLO11 promoter activity, we first exploiteda PFLO11::lacZ reporter carrying the whole 3-kb promoterof FLO11 (Rupp et al. 1999). b-Galactosidase activity wasmeasured in cells grown either exponentially or under star-vation conditions in SC ura2leu2 or SLAD medium (Figure 1,D–F). The activity of the PFLO11::lacZ reporter was severely


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Figure 1 Dysfunctional mitochondria interfere with signaling pathways regulating FLO11 transcription. (A) Filamentous growth of rho+ (SCS-48), rho+

tec1D (SCS-114), and respiratory-deficient rho mutants (SCS-139, -146, -150, -160). Yeast cells were grown for 3 days (rho+ strains) or 5 days (rhomutants) on a low-nitrogen (SLAD) medium (top) or SLAD supplemented with 1% isobutanol (middle). Invasion into agar was visualized after washingthe plates under a stream of water (bottom). (B) Magnification of colonies marked in A. Percentage of long pseudohyphal (PH) cells with length-to-widthratio .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 (rhomutants). Plates were washed under a stream of water and photographed before (top) and after (bottom) the wash. (D–F) PFLO11::lacZ reporter activity inrho+ cells, rho mutants, and respective tec1D strains (SCS-114, -248, -250, -246, -271) carrying pYEp355-FLO11::lacZ. b-Galactosidase activity wasmeasured from cells growing exponentially (D) or for 24 hr (E) in SC ura2leu2 medium or for 5 hr on SLAD medium (F). (G) Analysis of FLO11 transcriptlevels with quantitative PCR in rho+ (SCS-48) and rho0 mip1D (SCS-139). Strains were grown exponentially in YPD or patched onto YPD plates for 3 and7 days before total RNA isolation. The results were normalized to the geometric average of two housekeeping genes (UBC6 and ARP6) and expressedrelative 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 (+PTEF::FLO11). Strains were transformed with pB4126 and assayed for haploidfilamentation and invasion as described for A and C. Bars, 100 mm.


reduced in rho mutants compared to the rho+ strain. Underall conditions tested, the FLO11 reporter activity in rhomutants was similar to the rho+ tec1D strain.

To confirm these findings, the mRNA level of FLO11 wasanalyzed with quantitative PCR in rho+ and rho0 mip1Dcells grown exponentially or patched onto YPD plates toinduce invasive growth. We found that, in the rho0 mip1Dmutant, the FLO11 mRNA level was reduced �4-fold in ex-ponential cultures and 10-fold under invasive growth con-ditions (Figure 1G).

We next tested if respiratory-deficient mutants are able toshift to pseudohyphal growth by ectopically expressingFLO11. The expression of FLO11 under the control of theconstitutive TEF promoter resulted in a filamentation andinvasion phenotype of rho mutants that was indistinguish-able from the rho+ strain (Figure 1, H and I). In contrast,FLO11 expression in the rho+ tec1D strain only partially re-stored filamentation, suggesting that other Tec1 targets areessential for filament formation at a wild-type level.

Our results show that the expression of FLO11 is down-regulated in respiratory-deficient yeast cells and that ectopicexpression of FLO11 can restore filamentous and invasivegrowth of the mutants. Since extensive rearrangements ofnuclear gene expression patterns take place in rho cells(Epstein et al. 2001; Traven et al. 2001), we next focusedon the interorganellar (RTG) signaling pathway that canmediate these changes.

RTG pathway is activated in both rho+ strains and rhomutants during starvation

Activation of the RTG-signaling pathway in rho mutantsleads to compensatory readjustments in cellular metabolismand nuclear gene expression (Liu and Butow 2006). Thesensor of mitochondrial dysfunction is the cytoplasmic pro-tein Rtg2, which regulates the subcellular localization of thetranscriptional activators Rtg1 and Rtg3 (Figure 2A) (Sekitoet al. 2000). Two recent reports have reached conflictingconclusions as to whether the RTG-signaling pathway is re-quired for yeast invasive growth and filament formation ornot. First, the RTG pathway regulators Rtg1 and Rtg2 havebeen shown to be required for invasive growth of rho+ cells(Chavel et al. 2010). In contrast, it was also suggested that,in respiratory-deficient yeast strains, the activation of RTGsignaling is at least partially responsible for defective fila-ment formation (Jin et al. 2008). Therefore, we next aimedto clarify the role of RTG signaling in filamentous growth inboth the rho+ strain and respiratory-deficient mutants.

We inactivated retrograde signaling by disrupting thepathway positive regulator RTG2 and then monitored fila-mentous and invasive growth (Figure 2, B and C). Inacti-vation of the RTG pathway did not change the defectivephenotype of respiratory-deficient mutants (Figure 2, Band C). The invasion phenotype of the rho+ rtg2D strainwas also not affected, but a reduction in filament forma-tion was observed (Figure 2, B and C). Different othermutants have been characterized where filament formation

is affected without reduction in invasive growth, includingthe bud-site selection and cell polarity mutants (Mösch andFink 1997; Lorenz et al. 2000). Similarly, FLO11 overex-pression in the rho+ tec1D background restored invasivegrowth to a wild-type level, but filament formation wasrescued only partially (Figure 1, H and I).

Next, we analyzed the effect of RTG pathway inactivationon FLO11 promoter activity by using the PFLO11::lacZ re-porter. The deletion of RTG2 did not increase the activityof the PFLO11::lacZ reporter in respiratory-deficient strains(Figure 2D). Furthermore, the deletion led to a threefolddecrease in the activity of the FLO11 reporter in the rho+

strain, supporting the observation that the RTG pathway isrequired for a complete filamentous response in wild-typecells (Figure 2B) and for invasive growth as proposed earlier(Chavel et al. 2010).

RTG pathway activation in rho0 strains has been studiedmostly under nonrepressing conditions on raffinose mediumusing the non-invasive PSY142 strain (Liao et al. 1991; Liaoand Butow 1993). Since most filamentation and invasionassays are performed in the S1278b background and onthe repressive carbon source glucose, we next analyzed ifthe pathway is activated in the S1278b strain under condi-tions that stimulate filamentation. The retrograde responsewas measured with the PCIT2::lacZ reporter that reflects theexpression level of the prototypic RTG pathway target CIT2(Liao et al. 1991). In exponentially growing cultures, onlya modest twofold increase of b-galactosidase activity wasobserved in rho mutants compared to the rho+ strain, sug-gesting a weak retrograde response under these conditions(Figure 2E). In contrast, an �10-fold increase has beenreported for the PSY142 strain (Liao et al. 1991). The acti-vation of the pathway in the S1278b background was en-tirely dependent on RTG2, similarly to what has beenreported for the PSY142 strain (Figure 2, E and F) (Liaoand Butow 1993). Under starvation conditions on SCura2leu2 plates, the activity of the PCIT2::lacZ reporter wasactivated in rho mutants and the rho+ strain to comparablelevels. Compared to exponentially growing cultures, theCIT2 reporter activity increased more than 3-fold in therho+ strain and �1.3-fold in rho mutants (Figure 2F).

To confirm these results, we compared the levels ofCIT2mRNAwith quantitative PCR in the rho0 mip1Dmutantand the rho+ strain. During exponential growth, no differ-ence in CIT2 mRNA levels was detected between the rho0

mip1D mutant and the rho+ strain (Figure 2G). Under star-vation conditions, the level of CIT2 mRNA was increased inboth the rho+ strain and rho0 mip1D mutant compared toexponentially growing cells, indicating that the RTG path-way was activated upon glucose depletion in both strains(Figure 2G). Similar changes of CIT2 promoter activitywere observed with the PCIT2::lacZ reporter assay. However,we noted that the CIT2 mRNA level in the rho0 mip1D mu-tant was 3.3-fold elevated compared to the rho+ strain atthe third day of growth on YPD plates (Figure 2G, 3 dayson plate). No such difference was detected with the CIT2


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reporter on SC ura2leu2 media (Figure 2F). This could stemfrom intrinsic variations in the levels of RTG pathway activ-ity at the beginning of starvation or may reflect the differ-ent rate of nutrient depletion during the growth of the rho+

and rho mutant strains. The difference disappeared aftera prolonged incubation for 7 days under starvation condi-tions, when the level of CIT2 mRNA was �10-fold highercompared to exponentially growing cells in both the rho0

mip1D mutant and the rho+ strain (Figure 2G, 7 days onplate).

In conclusion, we found that under starvation conditionsthe activity of the RTG pathway in the rho+ strain was com-parable to that of rho mutants and that inactivation of the

pathway did not restore filamentous and invasive growth ofthe respiratory-deficient mutants. Furthermore, intact RTGsignaling was required for FLO11 expression and extensivefilament formation in the rho+ strain. Therefore, the fila-mentous and invasive growth defects of rho mutants arenot caused by active RTG signaling.

FG MAPK pathway is active in rho mutants

Since inactivation of the RTG pathway does not restoreFLO11 expression in respiratory-deficient mutants, we nextexamined the activities of two signaling pathways that areessential for FLO11 expression: the FG MAPK pathway andthe cAMP-PKA pathway.

Figure 2 RTG pathway is activated in both rho+ cells and rho mutants during starvation. (A) Signal transduction through the RTG pathway. Ovalsindicate positive, rectangles negative, regulators of the pathway; shading indicates mutant used in this study. (B and C) The effect of RTG2 deletion onfilamentous (B) and invasive (C) growth. Filamentation and invasion of rho+ (SCS-48), rho mutants (SCS-139, -146), and respective rtg2D strains (SCS-272, -276, -289) were analyzed as described for Figure 1, A and C. Bar, 100 mm. (D) PFLO11::lacZ reporter activity in cells carrying pYEp355-FLO11::lacZ.(E and F) PCIT2::lacZ reporter activity in cells carrying pEC261. b-Galactosidase activity was measured from cells growing exponentially in SC ura2leu2

medium (D and E) and on selective plates for 3 days (F). (G) Analysis of CIT2 transcript levels with quantitative PCR in rho+ (SCS-48) and rho0 mip1D(SCS-139). Strains were grown exponentially in YPD or patched onto YPD plates for 3 and 7 days. Data were normalized and expressed as described forFigure 1G. (D–G) Error bars indicate standard deviations of three independent measurements.


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The core FG MAPK cascade is composed of Ste11, Ste7,and the pathway-specific MAPK Kss1 (Liu et al. 1993;Roberts and Fink 1994). The pathway is regulated throughtransmembrane osmosensor Sho1 and mucin family mem-ber Msb2 (Figure 3A) (Cullen et al. 2000; Cullen et al.2004). Signaling through the kinase cascade leads toactivation of the transcription factors Tec1 and Ste12, fol-lowed by their cooperative binding to filamentous responseelements (FREs) in target gene promoters (Madhani andFink 1997). FREs are found in the promoters of FLO11 andof TEC1 itself (Madhani and Fink 1997; Lo and Dranginis1998).

Differences in filamentous and invasive growth of rhomutants and the rho+ tec1D strain (Figure 1, A and F) im-plied that some Tec1 target(s) were expressed in rhomutants. Therefore, we hypothesized that the FG MAPKpathway is functional in rho mutants. This was verified bymeasuring FG MAPK signaling with the FRE-dependent re-porter PTEC1::lacZ (Madhani and Fink 1997). Compared tothe rho+ strain, the activity of the PTEC1::lacZ reporter in rhomutants decreased 1.4-fold in exponentially growing cul-tures (Figure 3B) and increased 2.4-fold under starvationconditions (Figure 3C). In the tec1D strains, reporter activityalways remained at background level.

We next checked if, in rho mutants, the FG MAPK signalreaches the FLO11 promoter where specific regions respondto different input signals of filamentous and invasive growth(Rupp et al. 1999; Brückner and Mösch 2012). A promoterfragment known to be Tec1/Ste12-responsive [21.6 to22.0 kb upstream of initiator ATG (Rupp et al. 1999)] wasfused to the lacZ gene, and reporter activity was measured inexponentially growing cells. The FG MAPK-responsive frag-ment was activated in rho mutants and the rho+ strain tocomparable levels, with only a slight 1.2-fold difference,confirming that the FG MAPK pathway was functional inrho mutants (Figure 4B).

Furthermore, inactivation of the FG MAPK pathway bydisruption of TEC1 led to a complete loss of residual fila-mentation and invasion observed in rho mutants (Figure 3,D and E; compare to Figure 1A). Therefore, we concludethat the FG MAPK pathway is active and is responsible forthe residual filamentous and invasive growth of respiratory-deficient strains.

cAMP-PKA pathway activity is modulated inrespiratory-deficient strains

We next analyzed the functionality of the cAMP-PKA path-way in the regulation of the filamentous response. Intra- and

Figure 3 FG MAPK pathway is active and responsible for residual filamentation in rhomutants. (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) PTEC1::lacZ reporter activity in rho+ (SCS-48), rhomutants (SCS-139, -146), and respective tec1D strains (SCS-114, -248, -250) carrying pBHM275. b-Galactosidase activity was measured from cellsgrowing exponentially in SC ura2leu2 medium (B) and on selective plates for 3 days (C). Error bars indicate standard deviations of three independentmeasurements. (D and E) Filamentous (D) and invasive (E) growth of rho+ cells, rhomutants, and respective tec1D strains were analyzed as described forFigure 1, A and C. Bar, 100 mm.


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Figure 4 Activation of the cAMP-PKA pathway restores filamentous and invasive growth of respiratory-deficient strains. (A) Signal transduction throughthe 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 PFLO11 fragment::lacZ reporters in rho+ (SCS-48), rho0 mip1D (SCS-139), and rho2 rpo41D (SCS-146).Scheme of the FLO11 locus indicates positions of cAMP-PKA pathway-responsive (21.0 to 21.4 kb from FLO11 start codon, named 6/7) and FG MAPK-responsive (21.6 to 22.0 kb from FLO11 start codon, named 9/10) promoter fragments. Numbers mark nucleotides in kilobases. b-Galactosidaseactivity was measured from cells growing exponentially in SC ura2leu2 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


extracellular stimuli activate the adenylate cyclase Cyr1through the GTP-binding protein Ras2, the Gpr1-Gpa2 re-ceptor system, or Mep2 ammonium permease (Toda et al.1985; Kübler et al. 1997; Lorenz and Heitman 1997, 1998;Kraakman et al. 1999; Pan and Heitman 1999). The result-ing increased level of cAMP activates protein kinase A bydissociating a complex that consists of the inhibitory subunitBcy1 and the catalytic subunits Tpk1/2/3 (Figure 4A) (Todaet al. 1987a,b; Thevelein and de Winde 1999). Althoughredundant for viability (Toda et al. 1987b), the catalyticsubunits differentially regulate filamentous response. WhileTpk2 is required for pseudohyphal differentiation, the Tpk1and Tpk3 inhibit it (Robertson and Fink 1998; Pan andHeitman 1999).

First, the cAMP-PKA-responsive FLO11 promoter frag-ment containing the Flo8- and Sfl1-binding sites [1.0 to21.4 kb from the initiator ATG (Rupp et al. 1999; Pan andHeitman 2002)] was analyzed as a fusion to the lacZ re-porter gene. The reporter showed an approximately eight-fold decrease in activity in rho mutants compared to thewild-type strain (Figure 4B), suggesting strong downregula-tion of the cAMP-PKA pathway in rho mutants. In compari-son, the Tec1/Ste12-specific fragment showed only a 1.2-folddecrease in exponential growth conditions in rho mutants(Figure 4B).

Subsequently, we deleted or overexpressed several cAMP-PKA pathway components in rho+ and rho strains and ana-lyzed changes in filament formation and substrate invasion.We first inactivated the signaling pathway by deleting TPK2,known to block the morphogenetic switch (Robertson andFink 1998; Pan and Heitman 1999; Jin et al. 2008). TPK2disruption in rhomutants exerted modest effects on filamen-tous growth since residual filament formation was not lostas in the case of the TEC1 deletion (compare to Figure 3D),whereas residual invasion was completely abolished (Figure4C). As PKA signaling at a basal level is required for viability,some Tpk2-catalyzed phosphorylation is expected in rhomutants. This activity is apparently required for residual in-vasion but not for filament formation.

Next, we overexpressed SFL1 or BCY1, leading to thedownregulation of filamentation-specific signaling throughthe cAMP-PKA pathway in rho+ yeast cells. Sfl1 is a down-stream repressor that reduces FLO11 expression (Pan andHeitman 1999), and Bcy1 is the inhibitory subunit of PKA(Toda et al. 1987a). As expected, the overexpression of SFL1or BCY1 reduced invasion and filamentation of the rho+

strain. In rho mutants, however, residual filament formationand invasive growth was not affected (Figure 4, C and D).Notably, the residual filamentous and invasive growth of rho

mutants closely resembles rho+ strains with attenuated cAMP-PKA signaling, attained by overexpression of SFL1 or BCY1(Figure 4, C and D).

We next bypassed the requirement for cAMP-PKA activa-tion by deleting the pathway downstream inhibitor SFL1 orby overexpressing the downstream activator FLO8, bothresulting in hyperfilamentation in rho+ strains (Liu et al.1996; Robertson and Fink 1998). Both genes encode tran-scription factors that bind to the cAMP-PKA-responsive regionin the FLO11 promoter (Rupp et al. 1999; Pan and Heitman2002). Both the rho mutants and the rho+ cells displayedstrong filamentous and invasive growth phenotypes whenSFL1 was deleted or FLO8 overexpressed (Figure 4, E and F).

Finally, activation of the pathway by the deletion of BCY1or the overexpression of TPK2, known to lead to hyperfila-mentation of the rho+ strain (Pan and Heitman 1999), re-stored invasive growth of rho mutants in our assays (Figure4, E and F). However, TPK2 overexpression did not enhancefilament formation of rho mutants, indicating that invasivegrowth and formation of filaments could be regulated dif-ferentially as observed earlier (Figure 1, H and I; Figure 2, Band C) (Lorenz et al. 2000). Interestingly, the deletion ofBCY1 led to an inability of rho strains to grow on SLADmedium supplemented with 1% isobutanol (Figure 4E).

Analysis of the cAMP-PKA-responsive FLO11 promoterfragment and the effects of mutants that modulate the ac-tivity of cAMP-PKA signaling in filamentous and invasivegrowth assays suggests that signaling through the cAMP-PKA pathway is downregulated in rho mutants. It has beenpreviously reported that S. cerevisiae does not tolerate loss ofmtDNA combined with activation of the cAMP-PKA pathwayby IRA2 or PDE2 deletion (Dunn et al. 2006). Our analysisdemonstrates that activation of the pathway by BCY1 dele-tion is tolerated in rich growth media, but not under starva-tion conditions with additional stress. This further supportsthe notion that rho strains do not tolerate strong activationof the cAMP-PKA pathway.

Our data indicated that downregulation of cAMP-PKAsignaling pathway could explain the defective filamentformation of rho strains. Therefore, we next analyzed phys-iological reporters of the cAMP-PKA pathway, such as heatresistance, glycogen accumulation, and trehalase activity, inthe rho+ strain and in rho mutants (Figure 5A).

The cAMP-PKA pathway suppresses stress tolerance;therefore, the mutants with a reduced pathway activity aremore resistant to heat shock (Shin et al. 1987; Theveleinand de Winde 1999; Hlavatá et al. 2003). To analyze theheat-shock sensitivity of the respiratory-deficient strains, weexposed exponentially growing cultures to a severe heat

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 tpk2D mutation (SCS-312, -326, -328). (E and F) The effect of cAMP-PKA pathway activation onfilamentous 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 sfl1D (SCS-320, -335, -332) or bcy1D (SCS-316, -346, -344) mutations. Filamentation (C and E) andinvasion (D and F) were analyzed as described for Figure 1, A and C. Bars, 100 mm.


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shock at 52�. The heat-shock tolerance of rho mutants wassubstantially increased compared to the respective rho+

strain (Figure 5B) that corresponds to the downregulatedcAMP-PKA pathway. Activation of the pathway by BCY1 de-letion led to increased heat-shock sensitivity of both rho+

and respiratory-deficient strains. Only 0.02% of cells formedcolonies on YPD plates after 4 min of incubation at 52�. Wealso tested temperature sensitivity of rho mutants and therho+ strain carrying bcy1D. The double mutants mip1Dbcy1D and rpo41D bcy1D both failed to grow above 34�(Figure 5C).

The cAMP-PKA pathway inhibits accumulation of reservecarbohydrate glycogen in actively dividing cells. Glycogencan be detected upon prolonged incubation of rho+ strainson YPD plates when the pathway becomes downregulated(François and Parrou 2001). Previously, it has been reportedthat the rho0 mutants of a non-invasive strain backgroundmobilize glycogen, and by the third day of growth on YPDplates glycogen is not detectable (Enjalbert et al. 2000). Ouranalysis demonstrates that glycogen is readily accumulatedand stored in respiratory-deficient S1278b mutants (Figure5D, top, days 3 and 6, respectively). In the rho+ S1278bstrain, the accumulation of glycogen was slower and becamedetectable by day 6. This difference points to earlier down-regulation of the cAMP-PKA pathway in rho mutants com-pared to the wild-type strain. Activation of the pathway withBCY1 deletion completely abolished the glycogen staining of

rho mutants, confirming that the glycogen content in rhomutants is dependent on cAMP-PKA pathway activity (Fig-ure 5D, bottom).

PKA activates the neutral trehalase NTH1 by direct phos-phorylation (Uno et al. 1983; Shin et al. 1987; François andParrou 2001). Enzymatic activity of neutral trehalase wasmeasured in cultures grown for 24 hr at the time pointwhere the rho+ strain had passed the diauxic shift andhad presumably downregulated the cAMP-PKA pathway.The trehalase activity in rho mutants was decreased com-pared to the respective rho+ strain (Figure 5E), indicatingthat PKA activity is downregulated in rho mutants of theS1278b background. The disruption of BCY1 increases tre-halase activity in rho mutants to the levels observed in rho+

strains. As expected, trehalase activity also increases in therho+ bcy1D strain.

Discussion

Adaptation to nitrogen starvation or a poor carbon sourcerequires the remodeling of cellular metabolism, and notsurprisingly, the signaling pathways that regulate filamenta-tion (e.g., cAMP-PKA and Snf1) also target mitochondria(Ulery et al. 1994; Kuchin et al. 2003; Feliciello et al.2005). Mitochondrial mass is increased in yeast filaments,and genetic screens of mutants showing defects in filamen-tation have identified mitochondrial proteins, indicating

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 (SCS-48) and rhomutants (SCS-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 spottest of rho+ cells, rho mutants, and respective bcy1D strains (SCS-316, -346, -344) grown on YPD medium at the indicated temperatures. (D) Glycogenstaining of rho+ cells, rhomutants, and respective bcy1D strains. Yeast cells were patched onto YPD and incubated for 3 and 6 days at 30� or 25�. Plateswere exposed to iodine vapor. The dark color indicates the presence of glycogen stores. (E) Trehalase activity of rho+ cells and rho mutants andrespective bcy1D strains. Yeast cells were grown in YPD at 25�. Error bars indicate standard deviations of three independent measurements.


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that mitochondrial metabolism and the filamentous responseare linked (Lorenz et al. 2000; Kern et al. 2004; Kang andJiang 2005; Jin et al. 2008). Our detailed analysis of S1278rho mutant yeast strains revealed a defect in isobutanol-induced filament formation and agar invasion, demonstrat-ing that mitochondrial function is critical for both processes(Figure 1).

Several explanations for the observed filamentation de-fect in rho strains can be considered. First, an insufficientsupply of metabolic energy or the shortage of some meta-bolic intermediates could block the morphogenetic switch.Alternatively, dysfunctional mitochondria could trigger orinterfere with specific signaling cascades that target thegenes required for filamentous growth. The expressionlevel of FLO11 in rho mutants was reduced, and extensivefilament formation and substrate invasion was restored byectopic expression of FLO11 (Figure 1, D–I). Therefore, weconclude that, in rho cells, a specific signal is either missingor inhibiting filament formation through the regulation oftarget genes and that there is no fundamental deficiencyin metabolic building blocks required for pseudohyphalgrowth.

It is likely that an important role in the filamentousresponse is played by the interorganellar RTG cascade,known to reconfigure nuclear gene expression in yeast cellswith dysfunctional mitochondria. The RTG pathway regu-lates the supply of glutamate used as a nitrogen source, andnitrogen starvation is one of the triggers of the filamentousresponse (Gimeno et al. 1992; Liu and Butow 1999, 2006).Conflicting interpretations about the role of the RTG path-way in the filamentous response have been proposed.Downregulation of the RTG pathway by deletion of thepositive regulators RTG1 or RTG2 has been reported to in-hibit substrate invasion of respiratory-competent yeast cells(Chavel et al. 2010), indicating that the RTG pathway isrequired for filamentous growth. Consistent with these ob-servations, the expression of the pathway target gene DLD3is upregulated during isoamyl alcohol-induced filamentousresponse (Chelstowska et al. 1999; Hauser et al. 2007). Ourexperiments demonstrate that the downregulation of RTGsignaling does not restore the filamentous and invasivegrowth of respiratory-deficient strains. Moreover, we foundthat the RTG pathway is activated under starvation conditions

not only in rho mutants but also in wild-type cells and thatthe pathway stimulates the expression of FLO11 in wild-type cells (Figure 2). Therefore, our data support the con-clusion that the RTG pathway is needed for the filamentousresponse (Figure 6) (Chavel et al. 2010). It has been pre-viously reported that rtg2D strains display genomic insta-bility (Bhattacharyya et al. 2002). It is therefore possiblethat the reported mild filamentation rescue phenotype ofrtg2D strains (Jin et al. 2008) could be due to compensa-tory secondary mutations frequently observed in the rtg2Dbackground.

The key regulators of FLO11 transcription are the FGMAPK and the cAMP-PKA pathways. Our analysis indicatesthat the FG MAPK pathway is functional in rho cells and thatthe activity of the pathway is required for residual filamen-tation (Figures 3 and 6). In contrast, the cAMP-PKA pathwayis downregulated in S1278 rho strains. This conclusion issupported by the finding that the rho cells demonstrate sev-eral physiological characteristics typical for downregulatedcAMP-PKA signaling, such as increased tolerance of stress,reduced trehalase activity, and extensive accumulation ofglycogen (Figure 5). Importantly, the activation of cAMP-PKA signaling by the overexpression of TPK2 or FLO8 andby the deletion of SFL1 or BCY1 rescued the morphogeneticdifferentiation program in rho cells, directly linking the de-fect of morphogenesis and the low level of cAMP-PKA sig-naling (Figure 4, E and F; Figure 6). Our data clearlyindicate that the filamentation defect of rho mutants ofthe S1278b background can be explained by the downregu-lation of the cAMP-PKA pathway. This is supported by theanalysis of the physiological readouts of the cAMP-PKA path-way in rho mutants. However, our findings contrast witha recent report that the same pathway is upregulated inW303 rho cells during autophagy (Graef and Nunnari2011). It is possible that the different effects of dysfunc-tional mitochondria on cAMP-PKA signaling result frommultigenic variations between the two strains.

Our data demonstrate that mitochondrial metabolismactively modulates the output of the cAMP-PKA signaling,leading to the downregulation of the pathway in S1278background (Figure 6). The RTG and FGMAPK pathways arefunctional in rho cells under nitrogen starvation conditions,whereas the decreased activity of cAMP-dependent signaling

Figure 6 Model showing the role of mi-tochondrial function in the regulation ofcAMP-PKA signaling. Mitochondrial dys-function triggers downregulation of thecAMP-PKA pathway in S1278b cells.This leads to a defective filamentous re-sponse despite the wild-type level activ-ity of the RTG and FG MAPK pathways.Downregulation of the cAMP-PKA path-way results in modulation of several cellu-lar processes in addition to filamentationin respiratory-deficient mutants. Activa-tion is shown by arrows and inhibitionby T-bars.


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leads to the downregulation of FLO11 transcription andthereby to the inhibition of filamentous growth (Figure 6).

Several pathways can modulate the expression of FLO11(Brückner and Mösch 2012) and therefore we do not ruleout the possibility that other factors contribute to the re-duced filamentation phenotype of rho mutants. However,we would like to speculate that two important regulators,Snf1 and Mss1, are probably functional in rho mutants. TheSnf1 protein kinase pathway is required for glycogen accu-mulation that readily occurs in rho mutants (Figure 5D)(Thompson-Jaeger et al. 1991; Cullen and Sprague 2000;François and Parrou 2001). Mss11 is required for the inva-sion response of rho+ cells when FLO8 is overexpressed orSFL1 deleted (Gagiano et al. 1999; van Dyk et al. 2005). Sinceinvasive growth is restored in sfl1D cells or by the overexpres-sion of FLO8 in the rho background (Figure 4, E and F), it islikely that Mss11 is functional in rho strains.

An interesting question that remains unanswered is, atwhich level is the cAMP-PKA pathway downregulated in rhocells? It has been reported that ira2D and pde2D mutants donot tolerate the loss of mtDNA (Dunn et al. 2006). Thissuggests that the hyperactivation of Ras2 or the elevatedlevels of cAMP are not tolerated in respiratory-deficientstrains. Our analysis indicates that the deletion of BCY1 inrho mutants causes the temperature-sensitive growth defectat 34� (Figure 5C) and that this deletion is lethal undernitrogen starvation conditions in the presence of isobutanol(Figure 4E). Therefore, we propose that the regulatory in-teraction between the cAMP-PKA pathway and mitochon-drial function takes place upstream or at the level of theBcy1 regulatory subunit.

Our results demonstrate that mitochondrial function canactively modulate cAMP-PKA signaling and that this modelhas potential implications for higher eukaryotes. In mam-malian cells, A-kinase-anchoring proteins (AKAPs) localizePKA near the sites of cAMP generation or to PKA targets(Carlucci et al. 2008). Different AKAP1 RNA splice variantshave been shown to localize to the outer mitochondrialmembrane (OMM) (Lin et al. 1995; Chen et al. 1997).AKAPs potentially contribute to enhanced localization andtranslation of mRNAs targeted to mitochondria. In the con-text of our data, it is tempting to speculate that the tetheringof PKA signaling to OMM could also be important for sens-ing the functional state of mitochondria and for the modu-lation of PKA activity.

Acknowledgments

We thank Gerald R. Fink (Massachusetts Institute of Tech-nology), Chris A. Kaiser (Massachusetts Institute of Technol-ogy), Steffen Rupp (Fraunhofer Institute for InterfacialEngineering and Biotechnology, Germany), and the NationalBioResource Project of the Ministry of Education, Culture,Sports, Science and Technology (Japan) for plasmids andstrains; Maie Loorits for technical assistance; Arnold Kristjuhanfor critical reading of the manuscript; Laura Sedman for

language corrections; and members of the Sedman lab forfruitful discussions. This work was supported by grants 7013and 8845 (J.S.) and 9210 (T.T.) from the Estonian ScienceFoundation.

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