PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011858 Sep embe 9, 2025 1 / 28
OPEN ACCESS
Ci a ion: Schwei ze F, Bischo L, Wal e S,
Mo is S, Schmi z H-P, Heinisch JJ (2025)
The small GTPase Rho5—Ye ano he playe
in yeas glucose signaling. PLoS Gene 21(9):
e1011858. h ps://doi.o g/10.1371/jou nal.
pgen.1011858
Edi o : Ani a K. Hoppe , Ohio S a e Uni e si y,
UNITED STATES OF AMERICA
Recei ed: Feb ua y 12, 2025
Accep ed: Sep embe 1, 2025
Published: Sep embe 9, 2025
Copy igh : © 2025 Schwei ze e al
.
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RESEARCH ARTICLE
The small GTPase Rho5—Ye ano he playe
inyeas glucose signaling
F anziska Schwei ze 1, Linne Bischo 1, S e an Wal e 2, Silke Mo is3,
Hans-Pe e Schmi z1, Jü gen J. Heinisch 1*
1 Depa men o Biology/Chemis y, Di ision o Gene ics, Uni e si y o Osnab ück, Ba ba as asse,
Osnab ück, Ge many, 2 Depa men o Biology/Chemis y, Facili y o Mass Spec ome y, Uni e si y o
Osnab ück, Ba ba as asse, Osnab ück, Ge many, 3 Facul y o Biology, Ins i u e o In eg a i e Cell Biology
and Physiology, Uni e si y o Müns e , Schlosspla z, Ge many
* [email p o ec ed]
Abs ac
The small GTPase Rho5 has been shown o be in ol ed in egula ing he Bake ’s
yeas esponse o s ess on he cell wall, high medium osmola i y, and eac i e
oxygen species. These s ess condi ions igge a apid ansloca ion o Rho5 and i s
dime ic GDP/GTP exchange ac o (GEF) o he mi ochond ial su ace, which was
also obse ed upon glucose s a a ion. We he e show ha ho5 dele ions a ec ca -
bohyd a e me abolism bo h a he ansc ip omic and he p o eomic le el, in addi ion
o cell wall and mi ochond ial composi ion. Epis asis analyses wi h dele ion mu an s
in componen s o he h ee majo yeas glucose signaling pa hways indica e a p i-
ma y ole o Rho5 ups eam o he Ras2 GTPase in cAMP-media ed p o ein kinase
A signaling. Toge he wi h de e mina ions o p o ein kinase A ac i i ies, glycogen and
ehalose measu emen s hey indica e a s imula ion o Ras/cAMP signaling by Rho5.
Au ho summa y
GTPases a e molecula swi ches go e ning a a ie y o physiological p ocess-
es and signal ansduc ion cascades in all euka yo es. Rho5 is a membe o
he small g oup o i e Rho- ype GTPases in he model yeas Saccha omyces
ce e isiae which modula es se e al s ess esponse pa hways. In his wo k,
he unc ion o Rho5 in yeas glucose signaling has been add essed igge ed
by he esul s o p o eome and RNA sequence analyses. Di e en dele ion and
hype ac i e mu an alleles we e subjec ed o ex ensi e epis asis analyses. The
obse ed g ow h pheno ypes, suppo ed by measu emen s o p o ein kinase A
ac i i y and he accumula ion o ese e ca bohyd a es, indica e an ac i a o y
ole o he GTPase in he cAMP/Ras signaling pa hway, wi h Rho5 ac ing up-
s eam o Ras2.
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011858 Sep embe 9, 2025 2 / 28
In oduc ion
The yeas Saccha omyces ce e isiae has been employed by mankind since hou-
sands o yea s o making b ead and alcoholic be e ages like bee and wine [1].
I was hus con inuously selec ed o e icien suga u iliza ion, wi h glucose as he
p e e ed ca bon sou ce [2]. Besides a la ge amily o hexose anspo e s [3], his
p omp ed he e olu ion o complex signaling ne wo ks o de ec and p ope ly eac
o he suga concen a ion in he medium (see [4–7] o some selec ed e iews). As
ou lined in Fig 1, h ee majo signaling pa hways ha e been iden i ied in his con ex ,
which a e cha ac e ized by he ime ic SNF1/AMPK complex, he Rg 2/Sn 3 senso
pai , and he cAMP-ac i a ed p o ein kinase A (cAMP-PKA), espec i ely.
The SNF1 gene was o iginally iden i ied in sc eens o yeas mu an s impai ed in
ca aboli e ep ession o u iliza ion o suc ose and o he ca bon sou ces han glu-
cose (hence “suc ose non- e men e s”, also designa ed as CAT1 [8–10]). I was hen
cloned and cha ac e ized as encoding he p o ein kinase subuni o a ime ic com-
plex, comp ising an addi ional gamma-subuni (Sn 4), and one o h ee al e na i e
be a-subuni s (Gal83, Sip1, Sip2) go e ning i s subcellula localiza ion [11]. Homo-
logues o his ime ic complex in plan s and animals, known as AMP-ac i a ed p o-
ein kinase (AMPK), we e also ound o go e n ene gy me abolism, wi h mal unc ions
ha ing se e e e ec s on human heal h [12,13]. In yeas , glucose dep i a ion leads
o phospho yla ion and ac i a ion o he Sn 1 kinase subuni by one o h ee p o ein
kinases (Elm1, Sak1, Tos3), upon which he ime ic complex comp ising Gal83 as a
ß-subuni en e s he nucleus and igge s gene exp ession o he u iliza ion o al e -
na i e ca bon sou ces (see [11] and [7], and e e ences he ein, o a de ailed o e -
iew). Two o i s majo a ge p o eins a e he ansc ip ional ac i a o Ad 1 and he
ansc ip ional ep esso Mig1, wi h he la e being expo ed om he nucleus upon
i s phospho yla ion. In addi ion, SNF1 ac i i y exe s a my iad o cellula in e ac ions,
which ela ed o his wo k include he ansc ip ion ac o s Msn2/Msn4, he cAMP-
ac i a ed p o ein kinase A (PKA), nu ien signaling h ough he TORC1 complex, and
hexokinase PII [14,15]. In e es ingly, Hxk2 also unc ions as a co- ep esso oge he
wi h Mig1 in nuclea gene exp ession [16]. Mo eo e , yeas cell wall syn hesis was
ound o be egula ed by he SNF1 complex in a Mig1-dependen manne [17,18].
Glucose signaling media ed by he Sn 3/Rg 2 senso s in S. ce e isiae ac s on
he exp ession o se e al hexose anspo e genes h ough inac i a ion o a ime ic
ep esso complex wi h Rg 1 as he DNA-binding subuni (Fig 1; e iewed in [4,7]).
While ansc ip ion o he a ge HXT genes is ep essed by limi ing glucose concen-
a ions, ample glucose igge s he p o eosomal deg ada ion o he M h1 and S d1
subuni s and he eby inac i a es he ep esso complex. This pa hway is c osslinked
o SNF1 signaling indi ec ly by Mig1-media ed ep ession o genes encoding Rg 1
and i s co- ep esso M h1 [19], and di ec ly by phospho yla ion and ac i a ion o
Rg 1 [20].
Finally, Rg 1 ep ession is also alle ia ed upon i s hype phospho yla ion by p o-
ein kinase A (PKA) [21,22], he cen al componen o he hi d and p obably mos
ex ensi ely s udied ou e o yeas glucose signaling (Fig 1; see again [4,7] o gene al
o e iews). In b ie , glucose signaling in yeas was o iginally ela ed o he ac ion o
Compe ing in e es s: The au ho s ha e
decla ed ha no compe ing in e es s exis s.
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he small GTPase homologues o human Ras (Ras1 and Ras2) in s imula ing adenyla e cyclase [23,24]. Much la e , sig-
naling h ough he G p o ein coupled ecep o (GPCR) Gp 1 media ed by he GTPase Gpa2 was p oposed o be he mo e
impo an igge o adenyla e cyclase ac i i y [25]. In bo h cases he esul ing peak in cAMP concen a ion leads o disso-
cia ion o he inhibi o y Bcy1 subuni s om he e ame ic PKA complex and concomi an libe a ion o he ca aly ic sub-
uni s, wi h he h ee iso o ms Tpk1, Tpk2, and Tpk3 [26,27]. These show pa ially o e lapping bu also dis inc speci ici ies
owa ds a a ie y o cy osolic a ge p o eins and nuclea ansc ip ion ac o s ( e iewed in [28,29]). Amongs he la e ,
he edundan ansc ip ion ac o s Msn2/Msn4 a e a majo a ge . They a e inac i a ed by phospho yla ion and expo ed
om he nucleus in he p esence o high glucose concen a ions [30,31]. Upon glucose o o he nu ien limi a ions, as
well as in esponse o di e en en i onmen al s esses (ESR pa hway), hey eside in he nucleus, whe e hey ac i a e he
exp ession o genes h ough binding o s ess esponsi e p omo e elemen s (STREs, [32]). In addi ion o PKA-media ed
glucose signaling, nuclea expo o Msn2 can also be p o oked by i s phospho yla ion by he p o ein kinase Rim15, which
p o ides a link o TORC1-media ed nu ien signaling and he egula ion o au ophagy [33,34].
Fig 1. Simpli ied scheme o glucose signaling pa hways in Saccha omyces ce e isiae. Glucose (g ey pen agons) is in e nalized by hexose
anspo e s, wi h a majo impo ance o Hx 1-Hx 7. A e ac i a ion by hexokinase (p ima ily Hxk2) i is channeled in o glycolysis. A high ex acellula
glucose concen a ions he Reg1-Glc7 phospha ase complex dephospho yla es he SNF1 complex, as well as Hxk2 and Mig1, leading o ep ession o
genes equi ed o he u iliza ion o al e na i e ca bon sou ces (SNF1 pa hway, designa ed in o ange). In he cAMP/PKA pa hway (depic ed in g een)
he G-p o ein coupled ecep o Gp 1 senses ex acellula glucose and ansmi s he signal o Gpa2, which ac i a es he adenyla e cyclase Cy 1. Cy 1
can also be ac i a ed by he edundan Ras1 and Ras2 GTPases in esponse o in acellula glucose-induced changes. Ac i a ed Cy 1 p oduces cyclic
AMP which binds o he egula o y subuni s o he he e o e ame ic p o ein kinase A (PKA), igge ing i s ac i a ion. The p o ein kinases Rim15 and Mck1
a e inac i a ed by PKA-dependen phospho yla ion, as a e he edundan ansc ip ion ac o s Msn2 and Msn4. A hi d glucose- esponsi e pa hway is
ini ia ed by he Rg 2/Sn 3 senso s (shown in iole ), which pe cei e ex acellula glucose and ac i a e he yeas casein kinases Yck1 and Yck2. These
phospho yla e and he eby ma k he co ac o s o he ansc ip ion ac o Rg 1 o p o eoly ic deg ada ion, namely M h1 and S d1. The ime ic ansc ip-
ion complex Rg 1/M h1/S d1 go e ns he exp ession o se e al hexose anspo e genes (HXTs). Lines ending in ba s designa e inhibi ion, a ows
indica e ac i a o y unc ions on a ge p o eins. The p oposed ole o Rho5 as a posi i e egula o o Ras2 is also shown.
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Despi e he ac ha his in ica e glucose signaling ne wo k has been ex ensi ely s udied in he model yeas S.
ce e isiae o e he pas i e decades, e idence o new c oss alks is cons an ly a ising. Thus, we ound ha he small
GTPase Rho5 may also ake pa in nu ien signaling, as ho5 mu an s show s ong syn he ic de ec s wi h gpa2, gp 1,
o sch9 dele ions [35]. His o ically, Rho5 was o iginally iden i ied as a nega i e egula o o cell wall in eg i y (CWI)
signaling [36], and also ound o egula e he opposing high osmola i y glyce ol (HOG) pa hway [37]. Bo h pa hways
a e equi ed o p ope yeas mi ophagy [38]. Mo eo e , upon exposu e o oxida i e s ess Rho5 apidly ansloca es
om he plasma memb ane o mi ochond ia, and igge s mi ophagy and apop osis [39,40]. In e es ingly, he oles
o Rho5 in ene gy me abolism and mi ochond ial unc ions appea o be conse ed in i s human homologue Rac1,
whose mal unc ion is associa ed wi h se e al diseases, including diabe es, cance , and neu odegene a i e diso de s
( e iewed in [41]).
In his wo k, bo h he ansc ip ome and p o eome we e analyzed in ho5 mu an s and compa ed o wild- ype cells,
which subs an ia ed he no ion ha he small GTPase pa icipa es in yeas glucose signaling. We hen emba ked on mo e
de ailed epis asis analyses wi h dele ion mu an s in selec ed componen s o he h ee majo signaling pa hways. Resul s
ob ained a e consis en wi h Rho5 ac ing p ima ily h ough he cAMP-PKA pa hway.
Resul s
RNAseq and p o eome analyses ela e Rho5 o glucose signaling and gene al s ess esponse
As Rho5 has been ound o be in ol ed in he egula ion o a la ge numbe o signaling p ocesses [42], we decided o
apply wo global assays o assess he e ec o ho5 dele ion mu an s on yeas physiology. Fi s , da a on he ansc ip ome
we e ob ained by RNAseq o he wild- ype and he dele ion mu an unde s anda d g ow h condi ions in syn he ic medium
wi h 2% glucose, bo h in he absence and p esence o 0.8 mM hyd ogen pe oxide. A o al o 99 genes showed signi ican
up egula ion in hei exp ession unde s anda d g ow h condi ions when he dele ion mu an was compa ed o he wild-
ype, whe eas 95 genes appea ed o be down egula ed (Fig 2A; cu -o s applied a p- alues less han 0.05 and a leas a
wo old change in exp ession; see S1 Table o a comple e lis o up- and down egula ed genes).
As would be expec ed om he es ablished Rho5 unc ions, he up egula ed genes included hose encoding cell wall
and mi ochond ial p o eins (Table 1). In addi ion, a numbe o genes ela ed o ca bohyd a e me abolism we e up eg-
ula ed, including se e al hexose anspo e genes (HXTs) and he glucose- ep essed genes HXK1 and GLK1, which
encode hexokinase I and glucokinase, equi ed o suga consump ion in he la e phase o wine e men a ions. Mo eo e ,
genes encoding key enzymes o he pen ose phospha e pa hway, s ess p o ec ion and ese e ca bohyd a e me abolism
we e ound o be up egula ed in he ho5 dele ion as compa ed o he wild ype. O no e, many o hese genes and hose
placed in o he o he me abolic g oups ca y s ess- esponsi e elemen s (STREs) in hei p omo e s (Table 1).
Unde oxida i e s ess, i.e., exposu e o 0.8 mM H2O2 o six hou s, candida e genes in ol ed in he gene al o en i on-
men al s ess esponse (ESR) we e up egula ed bo h in he wild- ype and he ho5 dele ion s ains (Fig 2B and S2 Table).
Di e en ial egula ion be ween he wo gene ic backg ounds was obse ed o 120 genes (up egula ion) and o 276
genes (down egula ion; S2 Table).
In o de o con i m he alidi y o he RNAseq esul s, exp ession o a subse o genes om he di e en g oups lis ed
in Table 1 was in es iga ed by eal- ime RT-PCR (CCW22, CYC1, ECM4, GPH1, GSY2, HXK1, HXT6/7, OM45, PGM2,
TPS2; S1 Fig). Compa ison be ween wild- ype and he ho5 dele ion g own unde s anda d condi ions con i med he
inc ease in exp ession le els o all genes up egula ed in RNAseq. We a ibu e di e ences in he exac old-changes
de ec ed by he wo me hods o he ela i ely mode a e inc eases in gene exp essions obse ed, and o he e o ma gins
associa ed wi h he eal- ime RT-PCR me hod, e.g., in de e mining he exac concen a ion o he empla e cDNA. In con-
as o wha was obse ed in RNAseq, CYC1 exp ession inc eased in he RT-qPCR on he ho5 dele ion as compa ed o
he wild- ype. Besides he e o ma gins jus men ioned, his could be explained by he o e all low exp ession o he gene,
wi h mino changes in g ow h condi ions leading o s ong e ec s on i s ep esen a ion in he cDNA pool.
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011858 Sep embe 9, 2025 5 / 28
Fig 2. T ansc ip ome and p o eome analyses o ho5 dele ions as compa ed o wild- ype cells. A) Volcano plo o RNAseq da a compa ing a ho5
dele ion (FSO62-7A) o i s isogenic wild- ype s ain (HD56-5A). Cells we e g own on syn he ic comple e medium wi h 2% glucose (SCD) and gene al
cu -o s o di e en ially exp essed genes we e applied a p- alues less han 0.05 and an a leas wo old change in ansc ip abundance, wi h h ee
biological eplica es o each s ain. Signi ican exp ession changes a e depic ed in ed. T ansc ip s no mee ing he s ingen cu -o c i e ia a e desig-
na ed as ollows: Blue colou indica es exp ession changes wi h p- alues below 0.05 bu a old-change o less han 2. Shown in g een a e exp ession
changes wi h a old-change o a leas 2, bu a p- alue highe han 0.05. G ey ansc ip s we e deemed less signi ican , as hey ha e p- alues highe
han 0.05 and a less han wo old change. B) Volcano plo o RNA-sequencing esul s in which ho5 dele ion cells (FSO62-7A) we e compa ed o wild
ype cells (HD56-5A) a e g ow h on SCD in he p esence o 0.8 mM H2O2. Cu -o s and colou codes we e applied as in A), again wi h h ee biological
eplica es o each s ain. C) Volcano plo o p o eins de ec ed in mass spec ome y, compa ing a ho5 dele ion (FSO62-7A) o wild ype cells (HD56-5A)
a e g ow h on SCD. Cu -o s we e se a p- alues less han 0.05 and a leas a wo old change in p o ein abundance. Th ee biological eplica es we e
used o each s ain. Shown in g een a e all signi ican ly down- egula ed p o eins, while signi ican ly up- egula ed p o eins a e shown in ed. All p o eins
depic ed in g ey ei he ha e ei he a p- alue highe han 0.05 o a old-change o less han 2.
h ps://doi.o g/10.1371/jou nal.pgen.1011858.g002

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Table 1. Selec ed genes/p o eins di e en ially exp essed in a ho5 dele ion as compa ed o wild ype.
Gene name P o ein unc ion RNA Seq [ old
change]
RNA Seq
[p- alue]
Mass Spec
[ old change]
Mass Spec
[p- alue]
STREb
Glucose up ake and ac i a ion
HXK1 Hexokinase isoenzyme 1 1.05 1.10 x 10–2 1.57 1.80 x 10–3 Yes
HXT11 Hexose anspo e -6.65 1.96 x 10–6 nd nd
HXT13/15/16/17aPu a i e ansmemb ane polyol anspo e n c n c 1.18 4.91 x 10–2
HXT5 Hexose anspo e wi h mode a e glucose a ini y 2.33 1.03 x 10–5 nd nd Yes
HXT6aHigh-a ini y glucose
anspo e
1.95 1.05 x 10–6 3.57 1.05 x 10–3
HXT7aHigh-a ini y glucose anspo e 2.75 1.53 x 10–10 3.57 1.05 x 10–3
Pen ose Phospha e Pa hway
GND2 6-phosphoglucona e dehyd ogenase 1.74 3.70 x 10–4 1.63 3.68 x 10–6
NQM1 T ansaldolase o unknown unc ion 1.20 4.70 x 10–3 1.79 4.60 x 10–4 Yes
SOL4 6-phosphoglucono lac onase 1.47 7.18 x 10–5 1.06 1.10 x 10–4
TKL2 T anske olase 2.69 7.35 x 10–7 1.39 1.19 x 10–3 Yes
S ess p o ec ion and ese e ca bohyd a es
GDB1 Glycogen deb anching enzyme (deg ada ion) 1.36 7.52 x 10–3 1.87 3.40 x 10–3
GLC3 Glycogen b anching enzyme (accumula ion) 1.53 5.33 x 10–6 n c n c
GPH1 Glycogen phospho ylase (mobiliza ion) 1.81 7.79 x 10–5 1.65 6.20 x 10–4 Yes
GSY1 Glycogen syn hase isoenzyme 1 1.62 6.68 x 10–5 1.43 4.65 x 10–2
GSY2 Glycogen syn hase isoenzyme 2 1.09 1.62 x 10–3 1.06 1.30 x 10–4 Yes
PGM2 Phosphoglucomu ase 1.35 1.35 x 10–3 1.34 1.94 x 10–2 Yes
TFS1 Inhibi o o Ras GAP (I a2p) and ca boxy-pep idase Y (P c1p) 1.40 5.05 x 10–5 1.58 1.06 x 10–3 Yes
TPS2 Phospha ase subuni o T-6-P syn hase/phospha ase 1.27 3.00 x 10–4 1.32 3.90 x 10–4 Yes
TSL1 La ge subuni o he T-6-P syn hase/phospha ase 1.31 1.36 x 10–3 1.41 3.20 x 10–4 Yes
Cell su ace and cell wall a chi ec u e
ECM4 S-glu a hionyl-(chlo o) hyd oquinone educ ase 1.02 8.96 x 10–5 1.32 9.89 x 10–3 Yes
EIS1 Eisosome componen equi ed o assembly n c n c 1.01 1.93 x 10–2 Yes
LSP1 Eisosome co e componen n c n c 1.20 1.60 x 10–4 Yes
PIR3 Cell wall p o ein -1.41 8.02 x 10–6 nd nd
SCW10 Cell wall p o ein -1.15 2.61 x 10–3 n c n c
YLR042C Cell wall p o ein -1.91 1.88 x 10–3 nd nd
Mi ochond ial unc ions
ALD4 Aldehyde dehyd ogenase n c n c 1.07 1.42 x 10–3 Yes
CYB5 Cy och ome b5 -1.13 7.13 x 10–4 n c n c
CYC1 Cy och ome c, iso o m 1 -2.14 3.78 x 10–13 n c n c
GUT2 Glyce ol-3-phospha e dehyd ogenase ns ns 1.14 1.36 x 10–5
NCA3 P o ein in ol ed in mi ochond ion o ganiza ion -1.55 5.10 x 10–4 nd nd
NDE2 Ex e nal NADH dehyd ogenase 1.16 1.89 x 10–2 nd nd
OM14 Mi ochond ial ou e memb ane ecep o o cy osolic ibosomes ns ns 1.06 1.94 x 10–3
OM45 Mi ochond ial ou e memb ane p o ein o unknown unc ion 1.56 8.08 x 10–5 1.78 5.60 x 10–4
PIC2 Coppe /phospha e ca ie 1.14 2.44 x 10–6 nd nd
SFC1 Succina e- uma a e anspo e 1.07 2.66 x 10–2 nd nd
YMC2 Pu a i e mi ochond ial inne memb ane anspo e -1.12 2.37 x 10–3 n c n c
n c = no ele an changes; nd = no de ec ed.
aDue o high sequence simila i y, hese yeas hexose anspo e s canno be di e en ia ed om he pep ides de ec ed in mass spec ome y. Da a could
hus e lec he concen a ion changes in ei he one o hese anspo e s o any combina ion o hem.
bWhe e s ess esponsi e elemen s (STREs) ha e ei he been shown o unc ion in gene exp ession, o a leas we e de ec ed in bioin o ma ic su eys
[43], hei p esence is indica ed as “yes”.
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In a complemen a y app oach o he gene exp ession analyses, di e en ial p o ein concen a ions we e assessed
o he same s ains g own unde s anda d g ow h condi ions using mass spec ome y. A o al o 64 p o eins inc eased
signi ican ly in hei amoun when he dele ion mu an was compa ed o he wild ype, whe eas only 14 p o eins showed a
dec eased concen a ion (Fig 2C; wi h cu -o s applied a p- alues less han 0.05 and a leas a wo old change in p o ein
abundance; see S3 Table o a comple e lis o a ec ed p o eins). Again, p o eins mos s ongly a ec ed by he ho5 dele-
ion included hose in ol ed in ca bohyd a e me abolism (Table 1), wi h a numbe o hem o e lapping wi h and con i ming
he RNAseq da a (Fig 3).
Epis asis analyses e eal gene ic in e ac ions o RHO5 wi h glucose signaling h ough he SNF1 complex and
hexokinase
The global exp ession da a sugges ed a ela ionship be ween Rho5 and ca bohyd a e me abolism. We he e o e p o-
ceeded by assessing he pheno ypes o ei he a ho5 dele ion o he hype -ac i e RHO5G12V allele in combina ion wi h
di e en mu an s in he majo glucose signaling pa hways. Fo his pu pose, classical gene ic c osses we e pe o med, he
esul ing diploids we e subjec ed o e ad analyses, and g ow h was i s moni o ed by de e mina ion o colony sizes o
he di e en mu an and wild- ype seg egan s on ich medium pla es wi h 2% glucose as a ca bon sou ce.
As shown in Fig 4A, seg egan s wi h a ho5 dele ion o m sligh ly smalle colonies han hose wi h he wild- ype allele.
Howe e , he g ow h a ea is educed by app oxima ely h ee old in seg egan s lacking ei he he kinase subuni o he
SNF1 complex (sn 1Δ) o he Reg1 subuni o i s phospha ase ( eg1Δ), which is equi ed o i s inac i a ion (no e ha in
lack o a hype -ac i e Sn 1 kinase de i a i e eg1 dele ions a e commonly employed o cons i u i ely ac i a e he SNF1
complex [44]). In e es ingly, an addi ional ho5 dele ion agg a a es he g ow h de ec o s ains lacking Reg1, while i does
no al e g ow h o he sn 1 dele ions (Fig 4A). The ac ha he slow g ow h o ho5 eg1 s ains is es o ed back o ha
o a single eg1 dele ion by an addi ional lack o Sn 1, i.e., in ho5 eg1 sn 1 iple dele ions, sugges s ha Rho5 nega-
i ely a ec s he ac i i y o he SNF1 complex. These g ow h impai men s de i ed om colony sizes we e also con i med
by eco ding g ow h cu es in liquid syn he ic medium, sugges ing ha hey a e indeed owed o di e en ial g ow h a es,
a he han a delay in spo e ge mina ion (S2A Fig).
Fig 3. Venn-Diag am o o e lapping da a ob ained om RNA-sequencing and mass spec ome y om compa ison o a ho5 dele ion
(FSO62-7A) o a wild- ype s ain (HD56-5A) a e g ow h on SCD. Cu -o c i e ia o signi ican di e ences we e he same as desc ibed in he legend
o Fig 2.
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Fig 4. Epis asis analyses based on g ow h o seg egan s om e ad analyses on ich medium (YEPD). Pla es we e incuba ed o h ee o i e
days a 28°C, depending on he c osses o be analyzed. Only ou exempla y e ads a e shown o each c oss, wi h colo ed ci cles designa ing di e en
combina ions o gene dele ions as indica ed. Colony sizes o each combina ion (de e mined om pixel a ea and gi en as pe cen age om wild ype se
a 100%) we e de e mined om a leas 40 e ads om each c oss and quan i ied in he columns o he diag am a he igh (n = o al numbe o seg e-
gan s ob ained o each geno ype; e o ba s a e indica ed o each da a se , h ee as e isks indica e highly signi ican di e ences wi h p- alues below
0.001; n.s. = no signi ican ). Diploids analyzed we e ob ained om he ollowing c osses: A) A s ain ca ying a ho5 sn 1 double dele ion (FSO67-2C)
wi h a eg1 sn 1 double dele ion (FSO66-3C). B) A s ain ca ying a ho5 dele ion (FSO71-2A) wi h a eg1 mig1 double dele ion s ain (FSO79-8C). C) A
s ain ca ying a ho5 dele ion (FSO43-1D) wi h one ca ying a hxk1 hxk2 double dele ion (HOD257-2B).
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As he ansc ip ion ac o Mig1 is a majo downs eam a ge o SNF1 signaling in ca bohyd a e me abolism, epis a ic
ela ionships we e also in es iga ed wi h a mig1 dele ion. Su p isingly, he g ow h e a da ion obse ed in a ho5 eg1
double dele ion, amoun ing o less han 10% o wild- ype seg egan s, was no alle ia ed in a iple ho5 eg1 mig1 dele ion
(Figs 4B and S2B), demons a ing ha he obse ed Rho5- and Sn 1-dependen g ow h e ec s a e no media ed by Mig1.
By con as , he g ow h de ec caused by a lack o Sn 1 equi es a unc ional Mig1, as i is elie ed in s ains wi h a sn 1
mig1 double dele ion (S3 Fig).
In pa allel o he SNF1 complex, hexokinase PII was among he i s componen s ound o pa icipa e in yeas glucose
ep ession [45,46]. Besides i s associa ion wi h Mig1 in he nucleus a high ex e nal glucose concen a ions, i s inhibi-
o y ac ion on he SNF1 complex is p obably associa ed wi h i s ca aly ic ac i i y, bu s ill somewha enigma ic ( e iewed
in [47]). HXK2 encodes one o h ee yeas isozymes capable o glucose phospho yla ion, oge he wi h a glucokinase
encoded by GLK1 and ano he hexokinase encoded by HXK1. Exp ession o he la e wo is subjec o glucose ep es-
sion and only hxk1 hxk2 glk1 iple dele ions canno g ow on glucose as a sole ca bon sou ce [48,49]. As expec ed, we
ound only a mode a e dec ease in colony sizes a e e ad analyses o hxk2 dele ions compa ed o wild- ype seg e-
gan s, whe eas hose o hxk1 hxk2 double dele ions we e educed by app oxima ely 80% (Fig 4C). In e es ingly, his
pheno ype could be pa ially alle ia ed by an addi ional ho5 dele ion, which es o ed g ow h o he iple mu an s o
app oxima ely 30% o ha o he wild- ype colonies. Again, hese indings we e subs an ia ed by eco ding g ow h cu es
o seg egan s wi h he di e en mu an combina ions (S2C Fig).
We a ibu e he sligh posi i e e ec o he ho5 dele ions o an inc ease in espi a o y capaci y (S4 Fig), which may
coun e ac he educed ene gy supply caused by he hxk1 hxk2 dele ions. In his case, he hype -ac i e Rho5G12V a ian
also caused a mino , hough less signi ican inc ease in espi a ion, ins ead o he expec ed dec ease, indica ing ha lack
o he GTPase a ec s ene gy me abolism mo e s ongly han i s s imula ion.
The slow g ow h pheno ype o he eg1 dele ion agg a a ed by he addi ional lack o Rho5 desc ibed abo e appea ed
o be in iguing. As demons a ed in he ollowing sec ion o esul s, we ound e idence o Rho5 ac ing ups eam o he
Ras2-GTPase in cAMP signaling (see also Fig 1). The e o e, colony sizes we e also de e mined in epis asis analyses
in ol ing mu an alleles o hese h ee genes. As e iden om S5A Fig, a lack o Ras2 also agg a a es he pheno ype o a
eg1 dele ion simila o he ho5 eg1 double dele ion. Vice e sa, in oduc ion o he hype -ac i e RAS2G19V allele sup-
p esses he slow g ow h de ec o ho5 eg1 seg egan s (S5B Fig; no e ha RAS2G19V p e en s spo ula ion o yeas dip-
loids h ough i s inhibi o y ac ion on he mas e ansc ip ional egula o Ime1. The e o e, we o e exp essed IME1 in he
espec i e diploids by in oducing a high-copy numbe plasmid wi h he gene unde he con ol o he s ong yeas PFK2
p omo e o ci cum en he p oblem, es o e ascus o ma ion and allow e ad analysis).
RHO5 gene ically in e ac s wi h cAMP-PKA signaling
Nex , we add essed he ela ionship be ween Rho5 and he cAMP/PKA signaling pa hway. Since g ow h o mu an s lack-
ing componen s o ha pa hway is gene ally no impai ed unde s anda d g ow h condi ions, we employed hei sensi i i y
owa ds hyd ogen pe oxide as a eadou o epis asis analyses wi h ho5 dele ions. Al hough PKA also phospho yla es
se e al cy osolic enzymes in ol ed in ca bohyd a e me abolism, i s majo e ec on nuclea gene exp ession unde hese
condi ions is media ed by he edundan ansc ip ion ac o s Msn2/Msn4 (Fig 1; e iewed in [4,50]). As hey also media e
he yeas s gene al s ess esponse, i is no su p ising ha msn2 msn4 double dele ions display an inc eased sensi i i y
owa ds oxida i e s ess exe ed by hyd ogen pe oxide (Fig 5A). This pheno ype canno be escued by an addi ional ho5
dele ion, which on i s own shows hype - esis ance, indica ing ha Rho5 ac s ups eam o Msn2/Msn4 in he signaling
cascade.
To u he iden i y a which s age Rho5 in e e es wi h he signaling cascade, we consecu i ely dele ed genes encoding
o he ups eam componen s. Besides being a di ec a ge o inhibi ion by PKA-media ed phospho yla ion, Msn2/Msn4
can be ac i a ed by he p o ein kinases Rim15, Mck1, and Yak1, which a e inhibi ed by PKA-media ed phospho yla ion
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mu an s, also indica ing a conce ed ac ion o Rho5 h ough PKA and TORC1 signaling [35]. And he ac i a ed SNF1
complex unde glucose limi a ion also ac i a es TORC1, ul ima ely p omo ing exp ession o Msn2/Msn4-dependen genes
[86]. These include ATG8 and ATG39, which encode componen s o he au ophagic machine y [87,88]. Ano he au oph-
agy componen , A g21, was shown o in e ac di ec ly wi h Rho5 [63]. As Rho5 apidly localizes o mi ochond ia unde
oxida i e s ess oge he wi h i s dime ic GEF Dck1/Lmo1, i has been p oposed o di ec ly igge mi ophagy [83,89].
Whe he o no his is ela ed o i s e ec on Ras2 obse ed he ein emains o be de e mined, gi en ha Ras2 and PKA in
i s inac i e e ame ic o m a e also ec ui ed o mi ochond ia by Hsp60 and egula e hei u no e [28].
Ma e ials and me hods
Yeas s ains and g ow h condi ions
Yeas s ains used in his wo k a e lis ed in Table 2 and we e de i ed om HD56-5A, one o he pa en al s ains o he
common CEN.PK se ies [90], o i s isogenic diploid DHD5 [91]. Fo cloning pu poses and plasmid ampli ica ion, E. coli
s ain DH5α was used (In i ogen, Ka ls uhe, Ge many). S anda d p ocedu es we e ollowed o gene ic manipula ions o
yeas and plasmid cons uc ions [92]. Comple e sequences o all plasmids, modi ied ch omosomal loci, and oligonucle-
o ides employed a e a ailable upon eques .
Rich medium (YEPD) was based on yeas ex ac (1% w/ ) and pep one (2% w/ ), supplemen ed wi h 2% glucose
(w/ ). Syn he ic media (SC) con ained 0.67% yeas ni ogen base (w/ ) supplemen ed wi h ammonium sul a e, amino
acids and bases as equi ed [92], wi h 2% glucose (w/ , SCD) as a ca bon sou ce. His idine concen a ion was aised
om 2 o 4 mg/L, i necessa y, o eco d g ow h cu es. E. coli cells we e g own in LB medium (yeas ex ac a 0.5%
w/ , yp one a 1% w/ , and sodium chlo ide a 1% w/ ), wi h he addi ion o 50 mg/L ampicillin o 25 mg/L kanamycin as
equi ed o plasmid selec ion.
Gene ic manipula ions and epis asis analyses
Dele ion mu an s we e ob ained by one-s ep gene eplacemen s, using PCR p oduc s ob ained wi h p ime s gene a ing
40–50 bp o homology lanking he genomic a ge sequences, wi h selec ion o gene ic ma ke s as desc ibed [93]. Fo
complemen a ion o auxo ophic ma ke s wi h KlURA3 (pJJH1286) o KlLEU2 (pJJH1287) om Kluy e omyces lac is,
modi ied plasmids we e used, which ca ied he ma ke genes lanked by he TEF2 p omo e and he TEF2 e mina o
om Ashbya gossypii and by wo loxP si es. Alleles encoding hype -ac i e a ian s o he GTPase (RHO5G12V o RAS2G19V)
we e inse ed a he na i e gene ic loci by subs i u ion o he espec i e dele ion ma ke s, using SkHIS3 inse ed in o he
espec i e 3’ non-coding egions as selec ion ma ke .
S ains wi h di e en dele ion o mu an alleles we e c ossed by s anda d yeas gene ic echniques [92], spo ula ed on
pla es wi h 1% po assium ace a e (w/ ), and subjec ed o e ad analyses on YEPD pla es using a Singe MSM400 mic o-
manipula o (Singe Ins umen s, Some se , UK). Pla es we e incuba ed o 3–4 days a 30°C and scanned o documen-
a ion. The images we e adjus ed o b igh ness and con as using ImageJ wi h he same se ings o he en i e pla e, and
colony sizes we e de e mined wi h he analyze pa icles unc ion o he p og am. A leas 50 e ads we e sepa a ed o
each c oss and used o compa e colony sizes a e assigning he geno ypes om ma ke analyses. The a e aged sizes o
wild- ype seg egan s om each c oss we e se o 100% and he ela i e sizes o mu an seg egan s we e a e aged and
calcula ed. S a is ical analyses we e ob ained using he T.TEST unc ion o Excel.
In s ains ca ying he hype -ac i e RAS2G19V allele, spo ula ion does no occu due o he inhibi o y ac ion o PKA on
he mas e ansc ip ional egula o Ime1, which is equi ed o p ope meiosis [94]. The e o e, we cons uc ed a yeas 2
µm plasmid based on YEp181 wi h LEU2 as a selec i e ma ke [95]. The coding sequence o IME1 was ampli ied by PCR
om a wild- ype s ain (HD56-5A) wi h he p ime pai 25.028/25.029 (5’-gc cgga cc-ATGCAAGCGGATATGCATGG-3’ and
5’-gcgaaccgg aagcTTAAGAATAGGTTTTACT-AAACTTG-3’; unde lined sequences designa ing he BamHI and HindIII
es ic ion si es used o cloning, le e s in small p in a e no homologous o he a ge sequence) and cloned unde he

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Table 2. Yeas s ains employed in his wo k.
Designa ion Geno ype Re e ence
HD56-5A MATalpha u a3–52 his3–11,15 leu2–3,112 MAL3 SUC2 GAL [90]
HLBO37-4D MATalpha u a3–52 his3–11,15 leu2–3,112 RHO5G12V-SkHIS3 [83]
FSO105-1B MATalpha u a3–52 leu2–3,112 his3–11,15 bcy1::KlLEU2 his wo k
FSO105-1C MATalpha u a3–52 leu2–3,112 his3–11,15 ho5::kanMX his wo k
FSO105-1C MATalpha u a3–52 leu2–3,112 his3–11,15 ho5::kanMX his wo k
FSO105-1D MATa u a3–52 leu2–3,112 his3–11,15 ho5::kanMX bcy1::KlLEU2 his wo k
FSO105-4A MATa u a3–52 leu2–3,112 his3–11,15 bcy1::KlLEU2 his wo k
FSO105-7C MATalpha u a3–52 leu2–3,112 his3–11,15 ho5::kanMX bcy1::KlLEU2 his wo k
FSO105-7D MATa u a3–52 leu2–3,112 his3–11,15 ho5::kanMX his wo k
FSO105-7D MATa u a3–52 leu2–3,112 his3–11,15 ho5::kanMX his wo k
FSO105-7D MATa u a3–52 leu2–3,112 his3–11,15 ho5::kanMX his wo k
FSO16-3B MATa u a3–52 his3–11,15 leu2–3,112 his wo k
FSO35-1A MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::kanMX his wo k
FSO35-2A MATa u a3–52 his3–11,15 leu2–3,112 his wo k
FSO35-4A MATalpha u a3–52 his3–11,15 leu2–3,112 his wo k
FSO36-11A MATalpha u a3–52 his3–11,15 leu2–3,112 his wo k
FSO36-11B MATa u a3–52 his3–11,15 leu2–3,112 msn2::KlLEU2 msn4::KlLEU2 his wo k
FSO36-11C MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::kanMX msn2::KlLEU2 msn4::KlLEU2 his wo k
FSO36-11D MATa u a3–52 his3–11,15 leu2–3,112 ho5::kanMX his wo k
FSO36-5A MATalpha u a3–52 his3–11,15 leu2–3,112 msn2::KlLEU2 msn4::KlLEU2 his wo k
FSO36-5B MATa u a3–52 his3–11,15 leu2–3,112 his wo k
FSO36-5C MATa u a3–52 his3–11,15 leu2–3,112 ho5::kanMX msn2::KlLEU2 msn4::KlLEU2 his wo k
FSO36-5D MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::kanMX his wo k
FSO43-1D MATa u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 his wo k
FSO55-9A MATa u a3–52 his3–11,15 leu2–3,112 his wo k
FSO55-9B MATalpha u a3–52 his3–11,15 leu2–3,112 his wo k
FSO56-1A MATa u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 his wo k
FSO56-1B MATalpha u a3–52 leu2–3,112 his3–11,15 hxk1::KlLEU2 his wo k
FSO56-1C MATa u a3–52 leu2–3,112 his3–11,15 hxk2::kanMX his wo k
FSO56-1D MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 hxk1::KlLEU2 hxk2::kanMX his wo k
FSO56-2A MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 his wo k
FSO56-2B MATa u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 hxk1::KlLEU2 his wo k
FSO56-3A MATa u a3–52 leu2–3,112 his3–11,15 hxk1::KlLEU2 his wo k
FSO56-3C MATalpha u a3–52 leu2–3,112 his3–11,15 hxk2::kanMX his wo k
FSO56-4A MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 hxk1::KlLEU2 his wo k
FSO56-4B MATa u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 hxk2::kanMX his wo k
FSO56-4D MATalpha u a3–52 leu2–3,112 his3–11,15 hxk1::KlLEU2 hxk2::kanMX his wo k
FSO56-6D MATa u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 hxk1::KlLEU2 hxk2::kanMX his wo k
FSO56-8D MATa u a3–52 leu2–3,112 his3–11,15 hxk1::KlLEU2 hxk2::kanMX his wo k
FSO56-9C MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 hxk2::kanMX his wo k
FSO62-7A MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 his wo k
FSO66-3C MATa u a3–52 his3–11,15 leu2–3,112 eg1::kanMX his wo k
FSO67-2C MATalpha u a3–52 leu2–3,112 his3–11,15 ho5::KlURA3 sn 1::SpHIS5 his wo k
FSO71-10B MATa u a3–52 his3–11,15 leu2–3,112 eg1::kanMX his wo k
FSO71-15A MATalpha u a3–52 leu2–3,112 his3–11,15 ho5::KlURA3 his wo k
(Con inued)
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Designa ion Geno ype Re e ence
FSO71-15B MATa u a3–52 leu2–3,112 his3–11,15 eg1::kanMX sn 1::SpHIS5 his wo k
FSO71-15D MATalpha u a3–52 leu2–3,112 his3–11,15 ho5::KlURA3 eg1::kanMX his wo k
FSO71-1A MATa u a3–52 his3–11,15 leu2–3,112 his wo k
FSO71-1B MATa u a3–52 leu2–3,112 his3–11,15 ho5::KlURA3 sn 1::SpHIS5 his wo k
FSO71-1D MATalpha u a3–52 his3–11,15 leu2–3,112 eg1::kanMX his wo k
FSO71-2A MATa u a3–52 leu2–3,112 his3–11,15 ho5::KlURA3 his wo k
FSO71-2C MATalpha u a3–52 leu2–3,112 his3–11,15 eg1::kanMX sn 1::SpHIS5 his wo k
FSO71-5D MATa u a3–52 his3–11,15 leu2–3,112 sn 1::SpHIS5 his wo k
FSO71-6B MATalpha u a3–52 leu2–3,112 his3–11,15 ho5::KlURA3 sn 1::SpHIS5 his wo k
FSO71-7B MATalpha u a3–52 his3–11,15 leu2–3,112 his wo k
FSO71-7C MATa u a3–52 leu2–3,112 his3–11,15 ho5::KlURA3 eg1::kanMX sn 1::SpHIS5 his wo k
FSO71-9B MATa u a3–52 leu2–3,112 his3–11,15 ho5::KlURA3 eg1::kanMX sn 1::SpHIS5 his wo k
FSO71-9C MATalpha u a3–52 his3–11,15 leu2–3,112 sn 1::SpHIS5 his wo k
FSO71-9D MATalpha u a3–52 leu2–3,112 his3–11,15 ho5::KlURA3 eg1::kanMX his wo k
FSO75-4D MATa u a3–52 leu2–3,112 his3–11,15 ho5::kanMX his wo k
FSO75-7D MATalpha u a3–52 leu2–3,112 his3–11,15 ho5::kanMX his wo k
FSO78-14B MATa u a3–52 leu2–3,112 his3–11,15 RHO5G12V::SkHIS3 as2::SkHIS3 his wo k
FSO78-6C MATalpha u a3–52 leu2–3,112 his3–11,15 RHO5G12V::SkHIS3 as2::SkHIS3 his wo k
FSO79-4C MATa u a3–52 leu2–3,112 his3–11,15 eg1::kanMX mig1::SkHIS3 his wo k
FSO79-7C MATa u a3–52 leu2–3,112 his3–11,15 eg1::kanMX his wo k
FSO79-8C MATalpha u a3–52 leu2–3,112 his3–11,15 eg1::kanMX mig1::SkHIS3 his wo k
FSO84-10B MATalpha u a3–52 his3–11,15 leu2–3,112 im15::KlLEU2 mck1::KlLEU2 his wo k
FSO84-10D MATa u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 im15::KlLEU2 mck1::KlLEU2 his wo k
FSO84-3D MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 im15::KlLEU2 mck1::KlLEU2 his wo k
FSO84-4A MATa u a3–52 his3–11,15 leu2–3,112 im15::KlLEU2 mck1::KlLEU2 his wo k
FSO86-1C MATalpha u a3–52 his3–11,15 leu2–3,112 im15::KlLEU2 his wo k
FSO86-2A MATalpha u a3–52 his3–11,15 leu2–3,112 his wo k
FSO86-2C MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::kanMX im15::KlLEU2 his wo k
FSO86-6B MATa u a3–52 his3–11,15 leu2–3,112 im15::KlLEU2 his wo k
FSO86-7A MATa u a3–52 his3–11,15 leu2–3,112 his wo k
FSO86-7C MATa u a3–52 his3–11,15 leu2–3,112 ho5::kanMX im15::KlLEU2 his wo k
FSO88-1C MATalpha u a3–52 leu2–3,112 his3–11,15 RHO5G12V::SkHIS3 yak1::KlLEU2 his wo k
FSO88-2A MATa u a3–52 leu2–3,112 his3–11,15 RHO5G12V::SkHIS3 yak1::KlLEU2 his wo k
FSO88-2B MATalpha u a3–52 leu2–3,112 his3–11,15 RHO5G12V::SkHIS3 his wo k
FSO88-2C MATa u a3–52 leu2–3,112 his3–11,15 yak1::KlLEU2 his wo k
FSO88-3A MATa u a3–52 leu2–3,112 his3–11,15 RHO5G12V::SkHIS3 his wo k
FSO88-6A MATalpha u a3–52 leu2–3,112 his3–11,15 yak1::KlLEU2 his wo k
FSO90-1A MATa u a3–52 his3–11,15 leu2–3,112 ho5::KlURA3 mig1::SkHIS3 his wo k
FSO90-2A MATalpha u a3–52 his3–11,15 leu2–3,112 mig1::SkHIS3 his wo k
FSO90-3D MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::KlURA3 eg1::kanMX mig1::SkHIS3 his wo k
FSO90-6D MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::KlURA3 eg1::kanMX mig1::SkHIS3 his wo k
FSO90-7A MATa u a3–52 his3–11,15 leu2–3,112 mig1::SkHIS3 his wo k
FSO90-8B MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::KlURA3 mig1::SkHIS3 his wo k
FSO98-3A MATalpha u a3–52 his3–11,15 leu2–3,112 as2::SkHIS3 his wo k
FSO98-6B MATa u a3–52 his3–11,15 leu2–3,112 as2::SkHIS3 his wo k
Table 2. (Con inued)
(Con inued)
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011858 Sep embe 9, 2025 19 / 28
con ol o a ailo ed PFK2 p omo o employed p e iously [61], o yield plasmid pJJH3540 (comple e sequence and a se
o simila exp ession ec o s a ailable upon eques ). I s inhe i ance in s ains HOD675/IME1 and HOD677/IME1 was
ensu ed by selec ion o leucine p o o ophy and he espec i e diploids we e g own o e nigh in ich medium (YEPD),
and spo ula ed on po assium ace a e pla es as desc ibed abo e.
G ow h cu es o yeas s ains unde s anda d and oxida i e s ess condi ions we e eco ded wi h a Va ioscan Lux
pla e eade (The moFishe Scien i ic) as de ailed in [83].
De e mina ion o ese e ca bohyd a es and p o ein kinase A ac i i y
Fo he de e mina ion o glycogen and ehalose con en he me hod o Pa ou and F ancois [96] was adap ed. Thus, cells
we e g own o e nigh in 5 ml o YEPD o la e loga i hmic phase, inocula ed o an OD600 o 0.3 in 10 ml o o esh syn he ic
medium wi h 2% glucose (SCD) and incuba ed o ano he 22 h a 28°C wi h shaking a 180 pm in 100 mL E lenmeye
lasks. The OD600 was de e mined o ange be ween 5–8 and cells om 5 mL o each cul u e we e ha es ed by cen i u-
ga ion (3 min a 5000 g a oom empe a u e), d ained and suspended in 250 µL o sodium ca bona e (250 mM). Suspen-
sions we e ans e ed o sc ew-capped Eppendo ubes, igh ly closed and incuba ed o 4 h wi h shaking (800 pm) a
95°C. The pH was adjus ed by addi ion o 150 µL o 1 M ace ic acid and 600 µL sodium ace a e (0.2 M, pH 5.2), yielding
a o al o 1 mL suspension. Samples we e di ided in o wo 500 µL aliquo s. Fo assessmen o he glycogen con en , 1
U o amyloglucosidase om Aspe gillus nige (Sigma/Ald ich, A7420) was added and incuba ed o e nigh a 57°C wi h
shaking a 800 pm. The o he hal o each sample was incuba ed wi h 0.05 U o ehalase (Sigma/Ald ich, T8778) a 30°C,
also o e nigh wi h cons an agi a ion. P io o de e mina ion o he glucose libe a ed in bo h assays, ubes we e cen i-
uged o 10 min in a mic o uge a ull speed and he supe na an was ca e ully emo ed in o s anda d 1.5 mL Eppendo
ubes. Depending on he expec ed glycogen and ehalose con en , 10–100 µL we e employed o enzyma ic de e mi-
na ion o glucose. This was done wi h he glucose de e mina ion ki o Roche (Boeh inge Mannheim, p oduc numbe
10716251035), which is based on he educ ion o NADP measu ed a 340 nm wi h hexokinase and glucose-6-phospha e
dehyd ogenase as ancilla y enzymes, basically ollowing he manu ac u e s ins uc ions. Di e en om hose ins uc ions,
Designa ion Geno ype Re e ence
HOD257-2B MATalpha u a3–52 leu2–3,112 his3–11,15 hxk1::KlLEU2 his wo k
HOD320-2D MATa u a3–52 his3–11,15 leu2–3,112 ho5::kanMX as2::SkHIS3 his wo k
HOD320-6A MATalpha u a3–52 his3–11,15 leu2–3,112 as2::SkHIS3 his wo k
HOD343-2A MATalpha u a3–52 his3–11,15 leu2–3,112 mck1::KlLEU2 his wo k
HOD343-2C MATa u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 mck1::KlLEU2 his wo k
HOD343-4A MATalpha u a3–52 his3–11,15 leu2–3,112 mck1::KlLEU2 his wo k
HOD343-4C MATalpha u a3–52 his3–11,15 leu2–3,112 ho5::SpHIS5 mck1::KlLEU2 his wo k
HOD610-2C/ RAS2G19V MATalpha u a3–52 his3–11,15 leu2–3,112 RAS2G19V-KlURA3 his wo k
HOD610-1D/ RAS2G19V MATa u a3–52 his3–11,15 leu2–3,112 RAS2G19V-KlURA3 his wo k
HOD k21 MATa u a3–52 his3–11,15 leu2–3,112 RAS2G19V-SkHIS3 ho5::kanMX his wo k
HOD k22 MATa u a3–52 his3–11,15 leu2–3,112 RAS2G19V-SkHIS3 ho5::kanMX his wo k
HOD675/
IME1
MATa/MATalpha u a3–52/u a3–52 his3–11,15/his3–11,15 leu2–3,112/leu2–3,11 RHO5/ ho5::KlURA3
REG1/ eg1::kanMX RAS2/RAS2G19V-SkHIS3
pJJH3540 (2 µm-LEU2-PFK2p-IME1)
his wo k
HOD677/
IME1
MATa/MATalpha u a3–52/u a3–52 his3–11,15/his3–11,15 leu2–3,112/leu2–3,11 RHO5/ ho5::KlURA3
REG1/ eg1::kanMX RAS2/RAS2G19V-SkHIS3
pJJH3540 (2 µm-LEU2-PFK2p-IME1)
his wo k
All s ains we e de i ed om HD56-5A [90] and a e isogenic excep o he ma ing ype and he gene ic manipula ions indica ed.
h ps://doi.o g/10.1371/jou nal.pgen.1011858. 002
Table 2. (Con inued)
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011858 Sep embe 9, 2025 20 / 28
assays we e pe o med by adding he samples o a olume o 700 µL es mix con aining all ancilla y enzymes and NADP.
Abso p ions a 340 nm we e de e mined p io o sample addi ion and again 15 min a e incuba ion a oom empe a u e.
Glucose concen a ions we e calcula ed om he obse ed di e ences in A340 wi h an ex inc ion coe icien o NADPH o
6.223 and no malized assuming ha 1 OD600 in he o iginal cul u e equals 0.4 mg/mL o d y weigh .
P o ein kinase A (PKA) ac i i y was de e mined in c ude ex ac s using a luci e ase-coupled assay. Cells we e g own
in 2.5 mL YEPD p ecul u es o e nigh , added o 12.5 mL esh YEPD medium in an E lenmeye lask and incuba ed wi h
shaking o ano he 4–5 h a 28°C. C ude ex ac s we e p epa ed om ha es ed cells by b eaking wi h glass beads in
50 mM po assium phospha e bu e , pH 7.0 and p o ein con en was de e mined by Mic obiu e , as desc ibed in de ail in
[97]. PKA ac i i ies we e de e mined wi h componen s o a ki o iginally designed o measu e cAMP concen a ion (cAMP-
Glo Assay, P omega p oduc numbe V1501). As a modi ica ion, only he Kemp id pep ide subs a e and he luci e ase
coupling was used om he ki , omi ing he addi ion o cAMP and ancilla y PKA. Sample olumes we e adjus ed o 25
µL in bu e , o which 120 µl o es mix we e added in a black 96-well mic o i e pla e. Ligh gene a ion was hen eco ded
o 10 min a 30°C using a Va ioscan Lux pla e eade (The moFishe Scien i ic). Ini ial eloci ies in he linea ange o he
cu es we e used o calcula e ela i e ligh emissions pe min and no malized o he samples p o ein con en . The alue
o he wild- ype was se o 100% and he ela i e ac i i y o he o he s ains was calcula ed independen ly o each
biological and echnical eplica e. I is impo an o no e ha PKA ac i i y compe es wi h luci e ase o he ATP subs a e
p oducing an in e se ela ionship, i.e., he highe he ela i e ligh uni s, he lowe is he PKA ac i i y.
Fo all assays desc ibed in his sec ion, wo biological and wo echnical eplica es we e eco ded and mean alues and
s anda d de ia ions we e compu ed.
High- h oughpu analyses
RNA p epa a ion, RNA seq and bioin o ma ic analyses we e pe o med by S a Seq (Mainz, Ge many). Fo his pu pose,
cells we e g own in 50 mL SCD in he absence o p esence o 0.8 mM hyd ogen pe oxide, inocula ed om esh o e nigh
cul u es o an OD600 o 0.2 and g own o ano he wo gene a ions o an OD600 o 0.8 a 28°C wi h shaking a 180 pm.
Cells we e collec ed by cen i uga ion, ozen in liquid ni ogen and shipped on d y ice.
P o eomes we e ob ained om mass spec ome y analyses. The e o e, yeas cells we e g own in 25 mL SCD, which
we e inocula ed om a esh o e nigh cul u e o an OD600 o 0.2 and allowed o g ow o ano he wo gene a ions a
28°C and shaking a 180 pm. Samples we e ex ac ed using he iST ki acco ding o he ins uc ions o he manu ac u e
(P eomics GmbH, Ma ins ied, Ge many). Mass spec ome y using label- ee quan i ica ion (LFQ) was pe o med a he
Mass Spec ome y Equipmen Cen e o he Depa men o Biology/Chemis y a he “CellNanOS” esea ch cen e o he
Uni e si y o Osnab ück. Fo his pu pose, d ied pep ides we e esuspended in 10 µL LC-Load bu e and 2 µL we e used
o pe o m e e sed-phase ch oma og aphy on a The mo Ul ima e 3000 RSLCnano sys em connec ed o a TimsTOF HT
mass spec ome e (B uke Co po a ion, B emen) h ough a Cap i e Sp ay Ion sou ce. Pep ides we e sepa a ed on a
Au o a Gen3 C18 column (25 cm x 75 µm x 1.6 µm) wi h CSI emi e (Ionop ics, Aus alia) a empe a u e o 40°C. Pep-
ides om he column we e elu ed ia a linea g adien o ace oni ile om 10-35% in 0.1% o mic acid ( / ) o 44 min a a
cons an low a e o 300 nL/min ollowing a 7 min inc ease o 50%, and inally, 4 min o each 85% bu e B. Elu ed pep-
ides we e hen di ec ly elec o sp ayed in o he mass spec ome e a an elec osp ay ol age o 1.5 kV and 3 L/min d y
gas.
The MS se ings o he TimsTOF we e adjus ed o posi i e Ion pola i y wi h a MS ange om 100 o 1700 m/z. The
scan mode was se o PASEF. The ion mobili y was amped om 0.7 Vs/cm2 o 1.5 in 100 ms. The accumula ion ime was
also se o 100 ms. 10 PASEF amps pe cycle esul ed in a du y cycle ime o 1.17 s. The a ge in ensi y was adjus ed o
14,000, he in ensi y h eshold o 1,200. The dynamic exclusion ime was se o 0.4 min o a oid epea ed scanning o he
p ecu so ions, hei cha ge s a e was limi ed om 0 o 5. The esul ing da a we e analyzed wi h PeaksOnline (BSI, Can-
ada) e sion 11, employing he co esponding Yeas FASTA da abases. P ecu so s we e anging om 600 o 6,000 Da. As
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011858 Sep embe 9, 2025 21 / 28
modi ica ions ca bamidome hyla ion (C) and oxida ion (M) we e chosen. DDA-MBR we e pe o med wi h MS ole ance o
10 ppm and IM ole ance o 0.05 (1/k0).
The ob ained da a om RNA-sequencing and mass spec ome y we e p ocessed using he s anda d Excel p og am.
Cu -o s we e applied a p- alues less han 0.05 and a leas a wo old change in exp ession. P o eins de ec ed by mass
spec ome y we e only conside ed, i a leas one unique pep ide was epea edly ound. I should be no ed ha while
some p o eins could no be dis inguished in he mass spec ome y da a due o hei high amino acid simila i ies (e.g.,
some membe s o he hexose anspo e amily), hei encoding RNAs we e clea ly dis inguished by RNAseq, based
p ima ily on di e ences in he 5’- and 3’-noncoding egions.
De e mina ion o espi a o y capaci y
A Seaho se analyze (Agilen Technologies Deu schland GmbH, Waldb onn, Ge many) was employed o de e mine he
espi a o y capaci y o he di e en yeas s ains wi h he “Seaho se XF Cell Mi o S ess Tes ” wi h a modi ica ion o he es
employed o mammalian cells [98]. I allows he measu emen o he oxygen consump ion a e in li e cells. Fo he assay o
yeas cells, cul u es we e g own o e nigh in SCD a 28°C wi h shaking (180 pm). A e dilu ion o an OD600 o 0.4 hey we e
again incuba ed o each an OD600 o 0.8. Th ee biological eplica es and a leas wo echnical eplica es we e de e mined,
wi h he excep ion o a ho5 dele ion, whe e only one echnical eplica e was ob ained o one o he h ee biological eplica es.
Real- ime RT-PCR
Real- ime RT-PCR (RT-qPCR) was employed o con i m he da a o RNAseq analyses o a subse o ep esen a i e
genes. Fo his pu pose, 5 ml o wo o e nigh cul u es o each s ain whe e used o inocula e 50 ml o YEPD and g own
o an op ical densi y a 600 nm o 0.8 a 28°C. RNA was isola ed acco ding o [99] and pu i ied using he “DNA- ee” ki
(The mo Fische Scien i ic, Schwe e, Ge many). The RNA was ansc ibed in o cDNA using he “iSc ip Re e se T an-
sc ip ion Supe mix o RT-qPCR” (Bio-Rad Labo a o ies GmbH, Feldki chen, Ge many) by e e se ansc ip ase poly-
me ase chain eac ion (RT-PCR). To iden i y o exclude possible con amina ions o he cDNA, a nega i e con ol was
pe o med o each measu emen . The e o e, he “iSc ip No-RT Con ol Supe mix” (Bio-Rad Labo a o ies GmbH, Feld-
ki chen, Ge many) was used in he cDNA syn hesis, which does no con ain e e se ansc ip ase. HPLC-pu i ied p ime s
designed wi h he so wa e “P ime 3” we e used o qPCR s udies ( [100]; S1 Fig). The qPCR was pe o med using he
“iTaq Uni e sal SYBR G een Supe mix” (Bio-Rad Labo a o ies GmbH, Feldki chen, Ge many) in a qTowe 2.0 by Analy ik
Jena (Jena, Ge many) wi h wo echnical eplica es o each o he wo biological eplica es. All ki s and eac ion mix u es
we e used o p epa ed acco ding o he manu ac u e ’s ins uc ions. Fo each qPCR, he h eshold cycle (C alue) was
calcula ed by he machines in eg a ed so wa e. Da a we e no malized o ac in as a housekeeping con ol using he
2-ΔΔC
T me hod acco ding o [101] and analyzed wi h eely accessible s a is ical so wa e (R Co e Team 2021 a h ps://
www.R-p ojec .o g/ and RS udio Team 2015 a : h p://www. s udio.com/).
Suppo ing in o ma ion
S1 Fig. Quan i a i e eal- ime RT-PCR analysis o he exp ession o some selec ed genes. Rela i e Fold-changes
( FC) o exp ession compa ed o he wild- ype con ol was calcula ed o he indica ed genes based on 2-ΔΔC analysis om
he C alues o RT q-PCR using he ac in gene (ACT1) as a housekeeping e e ence. Means and s anda d e o s o he
mean (e o ba s) we e calcula ed om wo echnical and wo biological eplica es. P ime s used o RT q-PCR a e lis ed
below. S ains used we e HD56-5A as a wild- ype and FSO62-7A o he ho5 dele ion.
(PDF)
S2 Fig. Epis asis analyses based on he g ow h o s ains ca ying mu a ions in genes encoding componen s o
he SNF1 signaling pa hway in combina ion wi h RHO5 a ian s. G ow h cu es we e eco ded on syn he ic medium

PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011858 Sep embe 9, 2025 22 / 28
wi h 2% glucose (SCD; supplemen ed wi h 4 mg/L his idine o s ains wi h a his idine auxo ophy) as indica ed. E o ba s
gi e he s anda d de ia ions a each ime poin ob ained om a leas wo biological and wo echnical eplica es om
pa allel measu emen s o each cu e (i.e., wo independen isogenic seg egan s we e measu ed, wi h wo independen
inocula es, each). Geno ypes o he o he wise isogenic s ains a e lis ed in Table 2 (main ex ). S ains employed we e
A) wild ype (FSO71-1A and FSO71-7B) ho5 (FSO71-2A and FSO71-15A) eg1 (FSO71-10B and FSO71-1D) sn 1
(FSO71-5D and FSO71-9C) ho5 eg1 (FSO71-9D and FSO71-15D) ho5 sn 1 (FSO71-1B and FSO71-6B) eg1 sn 1
(FSO71-15Band FSO71-2C) ho5 eg1 sn 1 (FSO71-7C and FSO71-9B). B) wild ype (FSO86-7A and FSO86-2A) ho5
(FSO75-4D and FSO75-7D) eg1 (FSO71-10B and FSO71-1D) mig1 (FSO90-7A and FSO90-2A) ho5 eg1 (FSO71-9D
and FSO71-15D) ho5 mig1 (FSO90-1A and FSO90-8B) eg1 mig1 (FSO79-4C and FSO79-8C) ho5 eg1 mig1
(FSO90-3D and FSO90-6D). C) wild ype (FSO55-9A and FSO55-9B) ho5 (FSO56-1A and FSO56-2A) hxk1 (FSO56-3A
and FSO56-1B) hxk2 (FSO56-1C and FSO56-3C) ho5 hxk1 (FSO56-2B and FSO56-4A) ho5 hxk2 (FSO56-4B and
FSO56-9C) hxk1 hxk2 (FSO56-8D and FSO56-4D) ho5 hxk1 hxk2 (FSO56-6D and FSO56-1D).
(PDF)
S3 Fig. Epis asis analyses based on g ow h o seg egan s om e ad analyses on ich medium (YEPD). Fou
exempla y e ads a e shown, wi h colo ed ci cles designa ing di e en combina ions o gene dele ions as indica ed.
Colony sizes o each combina ion (de e mined om pixel a ea and gi en as pe cen age om wild ype se a 100%)
we e de e mined om 29 e ads and quan i ied in he columns o he diag am a he igh (n = o al numbe o seg egan s
ob ained o each geno ype. E o ba s a e indica ed o each mu an combina ion. Th ee as e isks indica e highly signi i-
can di e ences wi h p- alues below 0.001; n.s. = no signi ican ). Diploids analyzed we e om he c oss o a s ain ca y-
ing a eg1 mig1 double dele ion (FSO79-8C) wi h one ca ying a sn 1 dele ion (HOD201-2D).
(PDF)
S4 Fig. Respi a ion measu emen o a wild- ype s ain (HD56-5A), a ho5 dele ion s ain (FSO62-7A) and a
RHO5G12V mu an s ain (HLBO37-4D). Signi icance is indica ed by one as e isk, while h ee as e isks indica e a e y
high signi icance. No e ha di e ences be ween each s ain wi h and wi hou hyd ogen pe oxide a e also highly signi ican
bu no highligh ed wi h as e isks he e o he sake o cla i y.
(PDF)
S5 Fig. Epis asis analyses o mu an s in REG1, RHO5 and RAS2 based on g ow h o seg egan s om e ad
analyses on ich medium (YEPD). Fou exempla y e ads a e shown, each, wi h colo ed ci cles designa ing di e en
combina ions o gene dele ions as indica ed. Colony sizes o each combina ion (de e mined om pixel a ea and gi en as
pe cen age om wild ype se a 100%) we e de e mined and quan i ied in he columns o he diag am a he igh (n = o al
numbe o seg egan s ob ained o each geno ype. E o ba s a e indica ed o each mu an combina ion. Th ee as e isks
indica e highly signi ican di e ences wi h p- alues below 0.001; n.s. = no signi ican ). Diploids analyzed we e: A) F om
he c oss o a s ain ca ying a eg1 dele ion (FSO79-7C) wi h one ca ying a as2 dele ion (HOD320-6A); and B) om he
he e ozygous diploid s ain o eg1 ho5 RAS2G19V ans o med wi h an IME1 exp ession plasmid (HOD666/IME1).
(PDF)
S6 Fig. Epis asis analyses o ho5 and yak1 mu an combina ions based on hei sensi i i y owa ds hyd ogen
pe oxide. G ow h cu es we e eco ded on syn he ic medium wi h 2% glucose (SCD), as desc ibed in ma e ial and me h-
ods o he main ex , wi h o wi hou hyd ogen pe oxide as indica ed. E o ba s gi e he s anda d de ia ions a each ime
poin ob ained om a leas wo biological and wo echnical eplica es. Geno ypes o he o he wise isogenic s ains a e
lis ed in Table 2. S ains employed we e wild ype (FSO35-2A and FSO35-4A), RHO5G¹²V, (FSO88-3A and FSO88-2B),
yak1 (FSO88-2C and FSO88-6A), RHO5G¹²V yak1 (FSO88-2A and FSO88-1C).
(PDF)
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011858 Sep embe 9, 2025 23 / 28
S1 Table. Sou ce da a on RNA sequencing compa ing gene exp ession in a ho5 dele ion o ha o a wild- ype
g own in syn he ic comple e medium (SCD). Th ee biological eplicas we e eco ded wi h s ains and g ow h condi ions
explained in he legend o Fig 2 and in he Ma e ial and Me hods sec ion. Fo a quick e e ence, genes ha e been o de ed
acco ding o he log2-Fold Change (column D). Gene names a e gi en in column B, s a is ical signi icance o changes
(p- alue) in column G and p o ein unc ions acco ding o he Saccha omyces Genome Da abase (SGD; h ps://www.
yeas genome.o g; las accessed on July 3, 2025) in column L.
(CSV)
S2 Table. Sou ce da a on RNA sequencing compa ing gene exp ession in a ho5 dele ion o ha o a wild- ype
g own in syn he ic comple e medium (SCD) in he p esence o 0.8 mM hyd ogen pe oxide. Th ee biological epli-
cas we e eco ded wi h s ains and g ow h condi ions explained in he legend o Fig 2 and in he Ma e ial and Me hods
sec ion. Fo a quick e e ence, genes ha e been o de ed acco ding o he log2-Fold Change (column D). Gene names a e
gi en in column B, s a is ical signi icance o changes (p- alue) in column G and p o ein unc ions acco ding o he Saccha-
omyces Genome Da abase (SGD; h ps://www.yeas genome.o g; las accessed on July 3, 2025) in column L.
(CSV)
S3 Table. Sou ce da a on mass spec ome y analysis compa ing p o ein amoun s in a ho5 dele ion o ha
o a wild- ype g own in syn he ic comple e medium (SCD). Th ee biological eplicas we e eco ded wi h s ains
and g ow h condi ions explained in he legend o Fig 2 and in he Ma e ial and Me hods sec ion. Fo a quick e e ence,
gene names a e gi en in column B, ela i e changes in p o ein abundance a e gi en in column C, and p o ein unc ions
acco ding o he Saccha omyces Genome Da abase (SGD; h ps://www.yeas genome.o g; las accessed on July 3,
2025) in column U.
(CSV)
Acknowledgmen s
We hank Rosau a Rodicio o c i ical eading o he manusc ip and sha ing he expe ise in he egula ion o yeas
ca bohyd a e me abolism. In addi ion, we hank Sand a Ba els o excellen echnical assis ance in he RT-PCR
expe imen s.
Au ho con ibu ions
Concep ualiza ion: Jü gen J. Heinisch.
Da a cu a ion: S e an Wal e , Hans-Pe e Schmi z.
Fo mal analysis: F anziska Schwei ze , Linne Bischo , S e an Wal e , Silke Mo is, Hans-Pe e Schmi z.
In es iga ion: F anziska Schwei ze , Linne Bischo , Jü gen J. Heinisch.
Me hodology: F anziska Schwei ze , S e an Wal e , Silke Mo is, Jü gen J. Heinisch.
P ojec adminis a ion: Jü gen J. Heinisch.
So wa e: Hans-Pe e Schmi z.
Supe ision: Jü gen J. Heinisch.
Valida ion: F anziska Schwei ze , Jü gen J. Heinisch.
Visualiza ion: F anziska Schwei ze .
W i ing – o iginal d a : Linne Bischo , Jü gen J. Heinisch.
W i ing – e iew & edi ing: F anziska Schwei ze , Jü gen J. Heinisch.
PLOS Gene ics | h ps://doi.o g/10.1371/jou nal.pgen.1011858 Sep embe 9, 2025 24 / 28
Re e ences
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o g/10.1534/gene ics.114.173633 PMID: 25657346
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