SUT2 is a novel multicopy suppressor of low activity of thecAMP/protein kinase A pathway in yeast Michael Ru¨tzler*, Andre´ Reissaus, Magdalena Budzowska and Wolfhard Bandlow Ludwig-Maxi
Trang 1SUT2 is a novel multicopy suppressor of low activity of the
cAMP/protein kinase A pathway in yeast
Michael Ru¨tzler*, Andre´ Reissaus, Magdalena Budzowska and Wolfhard Bandlow
Ludwig-Maximilians-Universita¨t Mu¨nchen, Department Biologie I, Bereich Genetik, Munich, Germany
SUT2was found in a screen for multicopy suppressors of the
synthetic slow growth phenotype of a Dras2 Dgpa2 double
deletion mutant It failed, however, to cure the lethal
phenotype of a Dras1 Dras2 mutant suggesting that it acts
upstream of Ras or in a parallel pathway By testing
cAMP-dependent reactions including the accumulation of storage
carbohydrates, pseudohyphal differentiation, entry of
mei-osis as well as the measurement of FLO11 reporter activity
we show that Sut2p modulates the activity of protein kinase
A (PKA) Additionally, we demonstrate that cellular levels
of Ras2p are affected by Sut2p and that Sut2-GFPp accu-mulates significantly in the nucleus Based on the observed influence of high SUT2 gene dosage on PKA activity as well
as Sut2p’s homology to the presumptive transcription factor Sut1p, we suggest that Sut2p contributes to regulation of PKA activity at the level of transcription
Keywords: Saccharomyces cerevisiae; cAMP/PKA pathway; suppressors; genetic screen
In the yeast Saccharomyces cerevisiae two distinct
GTP-binding (G) protein systems have been found to activate
adenylate cyclase: (a) The Ras proteins Ras1p and Ras2p
[1], which are members of the highly conserved family of
small GTP-binding proteins and (b) the Gpr1p/Gpa2p
carbohydrate receptor system consisting of the G protein
coupled receptor (GPCR), Gpr1p, and its coupled
hetero-trimeric G protein composed of Gpa2 (Ga) [2] and the
atypical Gb and Gc subunits, Gpb1p, Gpb2p and Gpg1p,
respectively [3,4] ras1 ras2 Double mutants are not viable,
indicating a specific role of the Ras proteins that cannot be
complemented by Gpa2p, whereas Dgpa2 Dras2 mutants
display a very slow growth phenotype [5] cAMP activates
the three protein kinase A (PKA) catalytic subunits, Tpk1p,
Tpk2p and Tpk3p [6], via binding to the PKA regulatory
subunit, Bcy1p [7] and controls several nutrient-related
processes such as glycogen and trehalose homeostasis, entry
of meiosis and progression through the G1 phase of the cell
cycle [8] In addition, Ras2p has been found to control a
mitogen-activated protein kinase (MAPK) cascade, thereby
regulating filamentous growth [9] Both the cAMP/PKA
pathway and the MAPK cascade activity converge at the promoter of FLO11 [10], a key element in establishing filamentation, suggesting that Ras is a major switch in this process
The GPCR system has been shown to control the level of intracellular cAMP in response to glucose or sucrose [2] The exact downstream signaling events controlled by the carbohydrate receptor system are, however, as yet not well understood It has been proposed that Gpa2 acts in parallel
to the Ras proteins to activate adenylate cyclase to synthesize cAMP However, no physical interaction of Gpa2p or its recently identified b/c-like interaction partners [3,4] has been described as yet
To gain further insight into the signaling pathway downstream of Gpr1p/Gpa2p and Ras we made use of the synthetic slow growth phenotype displayed by Dgpa2 Dras2 double deletion mutants [5]: we constructed a Dgpa2 Dras2 strain in the CEN.PK2 genetic background and transformed it with a high copy yeast genomic library in order to identify high gene dosage suppressors In addition
to several known suppressors of low Ras/cAMP pathway activity, we have identified SUT2, a homologue of a presumptive transcription activator of sterol biosynthetic genes [11] and describe a possible linkage between cAMP/ PKA activity and SUT2
Experimental procedures
Strains and plasmids The S cerevisiae strains and plasmids used in this study are listed in Table 1
To construct the Dras2 strains MB1 (CEN.PK2) and MR211 (S1278b) the kanMX cassette from plasmid pUG6 [12] was amplified by polymerase chain reaction (PCR) using the primers disRAS2fwd 5¢-TAACCGT TTTCGAATTGAAAGGAGATATACAGAAAAAA AACAGCTGAAGCTTCGTACGC-3¢ and disRAS2rev
Correspondence to M Ru¨tzler, Department of Biological Sciences,
6270 Medical Research Building III, Vanderbilt University,
Station B 3582, Nashville, TN 37235–3582 USA.
Fax: + 1 6159360129, Tel.: +1 6153433718,
E-mail: m.ruetzler@vanderbilt.edu
Abbreviations: DAPI, 4¢,6-diamidino-2-phenylindole; ECL, enhanced
chemiluminescence; FOA, 5-fluoroorotic acid; GFP, green fluorescent
protein; GPCR, G protein coupled receptor; MAPK,
mitogen-activated protein kinase; PKA, protein kinase A; PVDF,
poly(vinylidene difluoride).
*Present address: Department of Biological Sciences, 6270 Medical
Research Building III, Vanderbilt University, Station B 3582,
Nashville, TN 37235–3582 USA.
(Received 8 December 2003, revised 2 February 2004,
accepted 9 February 2004)
Trang 2ATTTCCTTTTTATTAGCATAGGCCACTAGTGGAT
CTG-3¢ and both wild-type strains were transformed with
the DNA fragment For disruption of SUT2 we utilized the
loxP-S pombe his5+-loxP cassette [13] (the
Schizosaccaro-myces pombe his5gene can complement a S cerevisiae his3
mutation, but due to sequence divergence integration is
preferred at the intended disruption locus) The cassette was
amplified from the plasmid pUG27 [13] using the primers
disSUT2fwd 5¢-TGACGCTCACCAAGCTATTGGTTT
GTTTGGATCAATCGTCAGATATGAAGGCATAG
GCCACTAGTGGATCTG-3¢ and disSUT2rev 5¢-TAT
TAATATTCCTATATTTTACATAGGAGGAAATTA
CATGCATGAAACCTACAGCTGAAGCTTCGTAC
GC-3¢, respectively The plasmid pFL38-RAS2 was
con-structed by ligating the 3 kb HindIII/EcoRI-RAS2
frag-ment from plasmid YCplac22-RAS2 [14] to the respective
sites of pFL38 Plasmid p426MET25-RAS2 was a gift from
B Klebl (Functional Genomics Center Martinsried, Aventis
Pharma Deutschland GmbH, Martinsried, Germany) The plasmid was used to transform MB1 Dras2 ura3 prior to disruption of GPA2 in order to reduce the appearance
of spontaneous second site suppressors after the disruption
of GPA2 GPA2 was disrupted with a TRP1-containing construct allowing deletion of basepairs 237–870 of the GPA2open reading frame to yield TG1 For construction
of MR349 (CEN.PK2 Dras1 Dras2), MB1 [p426MET25-RAS2] was deleted for RAS1 utilizing the S pombe his5+ construct amplified from plasmid pUG27 as described above using the primers disRAS1fwd 5¢-TTCACGATTGAACAGGTAAACAAAATTTTCC CTTTTTAGAACGACATGCAGCTGAAGCTTCGTA CGC-3¢ and disRAS1rev CAAAACCATGTCATAT CAAGAGAGCAGGATCATTTTCAACAAATTATGC ATAGGCCACTAGGGATCTG-3¢ YEp351-SUT2 was constructed to contain SUT2 as the only open reading frame present in the plasmid in order to confirm the role of SUT2as a high copy suppressor of the synthetic phenotype
Table 1 S cerevisiae strains and plasmids used in this study.
Characteristics Source
Yeast strains
MB1 CEN.PK2; MATa ras2::kanMX ura3–52 leu2–3, 112 his3-D1 trp1–289 MAL2–8cSUC2 This study TG1 CEN.PK2; MATa gpa2::TRP1 ras2::kanMX ura3–52 leu2–3, 112 his3-D1 trp1–289 MAL2–8 c
SUC2 [p426MET25-RAS2]
This study MR349 CEN.PK2; MATa ras1::S.pombe his5+ras2::kanMX ura3–52 leu2–3, 112 his3-D1 trp1–289
MAL2–8 c SUC2 [pFL38-RAS2]
This study AR1 S1278b; MATa ura3–52 his3:hisG leu2::hisG sut2:: S.pombe his5 + This study YHUM214 S1278b; MATa ura3–52 his3:hisG trp1::hisG H.U Moesch YHUM216 S1278b; MATa ura3–52 his3:hisG leu2::hisG H.U Moesch MR161 S1278b; MATa/a ura3–52/ura3–52 his3:hisG/his3::hisG trp1::hisG/trp1::hisG This study MR211 S1278b; MATa ura3–52 his3:hisG leu2::hisG ras2::kanMX
MR298 S1278b; MATa/a ras2::kanMX/ras2::kanMX ura3–52/ura3–52 his3:hisG/his3:hisG
leu2::hisG leu )2::hisG
This study AR2 S1278b; MATa/a sut2::S.pombe his5 +
/sut2::S.pombe his5+ura3–52/ura3–52 his3:hisG/his3:hisG leu2::hisG/LEU2 TRP1/trp1::hisG
This study MR287 S1278b; MATa ura3–52 his3:hisG leu2::hisG FLO11::lacZ This study AR3 S1278b; MATa ura3–52 his3:hisG leu2::hisG ras2::kanMX FLO11::lacZ This study AR4 S1278b; MATa ura3–52 his3:hisG leu2::hisG sut2::S.pombe his5 +
FLO11::lacZ This study Plasmids
p426MET25-RAS2 High copy number, URA3 marker, RAS2 – ORF in p426MET25[32] B Klebl YEp351-GPA2 High copy number, LEU2 marker, 1.5 kb genomic Sau3A fragment containing
full length GPA2
This study YEp351-RAS1 High copy number, LEU2 marker, 4 kb genomic Sau3A fragment containing full length RAS1 This study YEp351-RAS2 High copy number, LEU2 marker, 1.5 kb genomic Sau3A fragment containing
full length RAS2
This study pFL38-RAS2 Low copy number, URA3 marker, RAS2 – ORF plus endogenous regulatory regions This study YEp 351-SUT2 High copy number, LEU2 marker, 1.9 kb genomic ScaI – PstI SUT2 fragment,
SmaI – PstI in YEp351
This study YEp 351-SUT2-GFP High copy number, LEU2 marker, SUT2 with in-frame C-terminal yEGFP fusion This study YEp 351-TPK2 High copy number, LEU2 marker, 5 kb genomic Sau3A fragment
containing full length TPK2
This study pYLZ-6int-Flo11 integration plasmid, URA3 marker, contains 950 bp of FLO11 upstream region
in pYLZ-6[15]8]
This study GPA2DTRP plasmid, containing a SmaI-SmaI fragment for deletion of bp 237–870
of the GPA2 open reading; sequence see supplementary material
This study YEp351-library BamHI-Sau3A yeast genomic library, LEU2 marker, insert size range 0.5–5 kb in YEp351 E Bogengruber
Trang 3of Dgpa2 Dras2 strains (Results) Hence, a 1.9-kb PstI-ScaI
genomic fragment containing SUT2 was ligated into
YEp351 (PstI-SmaI) To construct an SUT2-GFP fusion,
yEGFP was amplified from pUG35 (U Gu¨ldener & J H
Hegemann, Institut fu¨r Mikrobiologie, Heinrich Heine
Univ., Du¨sseldorf, Germany; unpublished results; plasmid
information available online at http://mips.gsf.de/proj/
yeast/info/tools/hegemann/gfp.html) using the primers
SUT-GFPfwd 5¢-GACTGTCGATGATTATGGTTGCC
CGCTGGCTTCCAAACCCTTATCGATACCGTCGA
CCC-3¢ and SUT-GFPrev 5¢-AACAATTTCACACACA
GGAAACAGCTATGACCATGATTACGCTATAGG
GCGAATTGGGTA-3¢, respectively YEp351-SUT2
was linearized with SphI and co-transformed with the
yEGFP PCR fragment into YHUM216 Both SUT-GFP
primers provide fragments overlapping with SUT2 and
YEp351, respectively, thereby allowing recombination
resulting in restoration of a replicating plasmid Positive
recombination was identified by selection for LEU2 and
GFP fluorescence In frame recombination of GFP
C-terminal to SUT2 was verified by DNA nucleotide
sequence analysis of isolated plasmids To generate
Flo11-b galactosidase reporter strains, a 950 bp fragment
upstream of the FLO11 open reading frame was amplified
using the primers Flo11_lacZ_fwd 5¢-GTTTAGAA
TTCGATTGTAGGCAGAA-3¢ and Flo11_lacZ_rev
5¢-AGGATCCAAATAAGCGAGTAGAAAT-3¢,
respec-tively Plasmid pYLZ-6 was converted to an integration
plasmid, as suggested [15], and the amplified
FLO11-fragment was ligated to the resulting plasmid pYLZ-6int
via EcoRI/BamHI-sites, subsequently The resulting plasmid
pYLZ-6int-Flo11 was linearized with XbaI and
subse-quently used to transform YHUM216, creating MR287
Two individual transformants were then used to obtain the
corresponding Dras2 (AR3) and Dsut2 (AR4) reporter
strains by standard genetic methods
High copy suppressor screen
The Yep351-based yeast genomic library used for the
suppressor screen was a gift from E Bogengruber (Institute
for Genetics and General Biology, University of Salzburg,
Austria) The insert size ranges from 0.5 to 5 kb All yeast
transformations were performed by a modified lithium
acetate method [12] Transformation efficiency was
opti-mized to yield 500–1000 colonies per plate to facilitate
subsequent identification of suppressors After 2–3 days of
growth on selective medium, colonies where replica-plated
onto 5-fluoroorotate (FOA) containing medium (0.075%,
BioVectra, Canada) to select against plasmid
p426MET25-RAS2 URA3 After an additional 2 days of growth, plasmids
where isolated from colonies that had formed To distinguish
from spontaneous genomic suppressor mutants, plasmids
that accelerated growth of Dgpa2 Dras2 cells were identified
after re-transformation into TG1 and FOA-selection against
the RAS2 and URA3-harboring plasmid
Yeast culture, sample preparation, biochemical analysis,
immunoblots and invasive growth assay
For determination of endogenous glycogen or trehalose
levels, yeast strains were cultivated overnight in SC
medium (0.17% yeast nitrogen base, ammonium free; 0.5% ammonium sulfate; 2% glucose) with required amino acid supplements (0.002%) to a final D600 of 6 (± 0.5) A fraction of these cultures (equaling approx
50 mg of wet weight cells) was collected to determine the level of storage carbohydrates after entry of stationary phase The remainder of the cultures was used to inoculate fresh SC-medium to an D600of 0.5 Aliquots from these cultures were collected at the time points indicated in Fig 3 and the storage carbohydrate levels were determined
as described by Lillie and Pringle [16] Culture conditions for immunoblots were as described for storage carbohy-drate determination Cells equal to 5 D600units (1 unit¼
1 D600ÆmL)1) were harvested by centrifugation, washed once in ice-cold water and resuspended in 1.5% SDS, 1M
2-mercaptoethanol and disrupted with acid-washed glass beads (0.45–0.55 mm) by vortexing for 3· 1 min between 1-min intervals of cooling at 0C Samples were centri-fuged for 1 min at 800 g, and subsequently supernatants were assayed for protein content by determining A280 All samples were then diluted to an A280 of 20, 1/2 volume sample buffer (15% glycerol, bromophenol blue, 66 mM
Tris/HCl, pH 6.8) was added, and 30 lL of each sample was subjected to SDS/PAGE and transferred to a poly(vinylidene difluoride) (PVDF)-membrane Ras pro-teins were detected using ECL after incubation of the membranes with monoclonal anti-H-Ras antibody (259) and peroxidase-labeled chicken anti-rat antibody (both from Santa Cruz Biotechnology, Heidelberg, Germany) Each blot was re-incubated with chicken anti-Aky2p Ig [17] and peroxidase coupled anti-chicken secondary Ig (Sigma-Aldrich, Taufkirchen, Germany) as a loading control
To determine sporulation efficiency, diploid yeast cells were cultured in YPD medium overnight Aliquots were washed and transferred to sporulation medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose) Formation of asci was monitored after 3 days in a Thoma cell counting chamber Invasive growth was assayed after 3 days of growth on YPD medium at
30C by washing nonadhering cells from the plates with
a squeeze bottle
b-Galactosidase assays Yeast cells were grown in YPD, harvested at an D600 of 2.5–3, disrupted with glass beads (diameter: 0.5–0.75 mm, Braun, Melsungen, Germany) and total protein concentra-tion was determined as described by Bradford [18] For b-galactosidase assays, an appropriate amount of pro-tein was incubated in Z-buffer (5· Z-buffer ¼ 300 mM
Na2HPO4, 200 mMNaH2PO4, 50 mMKCl, 5 mMMgSO4,
250 mM 2-mercaptoethanol) with 0.7 mgÆmL)1 o-nitro-phenyl-b-D-galactopyranoside (ONPG) as substrate After 30–40 min of incubation at 30C, the reaction was terminated by adding 1M Na2CO3 [19] The amount of hydrolyzed ONPG was determined (A420) and activity of b-galactosidase (as U per mg protein) calculated as follows:
DA420· 1000/0.0045 · total protein (lg) · incubation time (min) For statistical analysis of sporulation efficiency and lacZ reporter expression, the SPSS 11.0 software package (S PS S Inc., Chicago, IL, US A) was used
Trang 4For fluorescence microscopy, YHUM216
[YEp351-SUT2-GFP] cells were grown in selective medium to an D600of 1
Cell suspension (100 lL) were added to 1 mL of 70%
ethanol ()20 C), mixed, spun down and re-suspended in
mounting solution (0.1M Pipes/KOH, pH 6.9, 5 mM
EGTA, 5 mM MgCl2, 50% glycerol, 0.01 mg DAPI)
Images were taken with a Zeiss Axioscop equipped with a
Spot RT Monochrome CCD camera (Diagnostic
Instru-ments Inc., USA) and evaluated by using the SPOT 3.02
software (Diagnostic Instruments Inc., USA)
Results
SUT2 is a high copy suppressor of synthetic slow growth
in Dgpa2 Dras2 strains
To identify high copy suppressors of the synthetic slow
growth phenotype of Dgpa2 Dras2 we produced the
double deletion genotype in a CEN.PK2 background To
avoid emergence of spontaneous genomic suppressors, the
parental strain TG1 contained plasmid
p426MET25-RAS2 URA3 carrying a RAS2 wild-type copy to allow
propagation after disruption of the genomic RAS2 copy
and a URA3 marker After transformation with a yeast
genomic YEp351-based DNA library, p426MET25-RAS2
URA3 was removed by selection against the URA3
marker using FOA [20] Using this experimental setup we
screened a total of 35 000 colonies and thereby identified
a set of plasmids that contained several suppressors
(RAS1, RAS2, GPA2, TPK2, SCH9) (Fig 1A and data
not shown) whose relation to the cAMP/PKA pathway
has been described before [1,6,21–23] In addition, we
identified a plasmid containing the SUT2 gene, which
after sequence analysis was putatively characterized as a
homologue of the sterol uptake, biosynthesis and
traf-ficking regulator SUT1 [11] In the present work, we
investigate how SUT2 might be linked to the Ras/cAMP
pathway
High SUT2 gene dosage does not suppress lethality
of a Dras1 Dras2 strain
In order to investigate the interaction of Sut2p with the
Ras/cAMP pathway, we constructed a strain (denoted as
MR349) that was deleted for both RAS genes This
genetic combination is lethal To test whether SUT2 is
able to complement the lethal phenotype of Dras1 Dras2
the Dras2 strain was transformed with a low copy
URA3-selectable plasmid construct encoding RAS2, prior to
deletion of RAS1 In addition, this mutant strain carried
SUT2 on a LEU2 plasmid Again, we used selection
against the RAS2 URA3 plasmid with FOA to test for
complementation of lethality by SUT2 (see Fig 1B) In
contrast to TPK2 or RAS2, high copy SUT2-containing
plasmids were incapable of rescuing the lethality caused
by the Dras1 Dras2 double mutant Because SUT2 was
able to suppress the Dgpa2 Dras2, but not the Dras1
Dras2 phenotype, we concluded that SUT2 action
requires at least one of the Ras proteins to sustain its
effect on the cAMP pathway
SUT2 modulates PKA-dependent processes
We then addressed the question as to whether high SUT2 gene dosage suppresses the slow growth phenotype of Dgpa2 Dras2 cells by increasing PKA activity Low activity of the PKA pathway leads to accumulation of glycogen and trehalose, arrest of the cell cycle in G0 phase and alleviates entry of meiosis in diploids Upon partial nutrient limitation, invasive growth may occur in haploids [24] which in the wild type requires high PKA activity These consequences of PKA-pathway activity were tested subsequently in order to examine the influence
of high copy SUT2 on PKA activity We found that high copy SUT2 significantly reduced glycogen and trehalose levels in a Dras2 mutant background, whereas there was apparently no influence on carbohydrate content in wild type CEN.PK2 cells (Fig 2, top) We did not investigate the influence of high SUT2 gene dosage on PKA-dependent processes in Dgpa2 Dras2 cells, as it was not possible to propagate these cells without a suppressor-plasmid, such as p426MET25-RAS2 When Dgpa2 Dras2 cells were grown without a suppressor-plasmid, we noticed frequent appearance of spontaneous mutations that accelerated growth and consequently might have affected PKA activity
As CEN.PK2 has been previously reported to have a mutation in the adenylate cyclase gene that renders it largely insensitive to stimulation by Gpa2p and Ras2p [25], we
Fig 1 Complementation of CEN.PK2 Dgpa2 Dras2 (A)or CEN.PK2 Dras1 Dras2 (B)by different high copy plasmids Droplets containing the indicated cell numbers (top) of high-copy transformants were applied onto FOA medium Plasmid inserts are indicated on the left margin e.p., empty plasmid (YEp351) Note that the parental strains CEN.PK2 Dgpa2 Dras2 and CEN.PK2 Dras1 Dras2 contained plasmid p426MET25-RAS2 (2l plasmid) and pFL38-RAS2 (centromeric plasmid), respectively, prior to FOA selection.
Trang 5re-examined storage carbohydrate levels in the independent
genetic background of strain S1278b In this case, high
SUT2 dosage conferred a reduction in PKA activity as
judged from storage carbohydrate levels in S1278b cells
during vegetative growth on glucose (Fig 2, bottom)
indicating that the strong, nutrient responsive PKA activity
ofS1278b [26] is modulated by Sut2p
It is reasonable to suppose that the observed differences
between the two strains both containing high dosage of
SUT2 could be due to the reported difference in PKA
pathway activity On this basis, we hypothesized that (a)
high SUT2 gene dosage might increase PKA activity only
when it is low, but that (b) it acts inhibitory when PKA
activity is high To gain further insight into SUT2 function
we generated a sut2D strain for subsequent analyses To
challenge the first part of the above hypothesis, we used
diploidS1278b cells and determined sporulation efficiency,
which is inversely correlated with PKA activity [27] We
found that high SUT2 gene dosage drastically diminished
sporulation efficiency and, thus, mimics a phenotype of
increased PKA activity inS1278b wild type and slightly less
in Dras2 cells While the deletion of SUT2 in wild-type cells
slightly decreased sporulation efficiency the difference was
not statistically significant (Fig 3)
High SUT2 gene dosage reduces invasive growth
To address the assumption that a high level of SUT2
reduces PKA activity only when PKA activity is high we
investigated the influence of SUT2 on invasive growth,
which requires high PKA activity We found that after three
days of growth on YPD medium,S1278b cells carrying a
high copy SUT2 plasmid did not at all adhere to the agar
medium in agreement with low cellular PKA activity On
the other hand,S1278b wild-type control cells showed the
normal, strong invasive growth In fact, the extent of the
reduction of invasive growth by high copy SUT2 was
similar to a Dras2 strain (Fig 4A) To quantify this
observation, we generated a set of FLO11::lacZ reporter strains, which allowed us to study expression of FLO11, the major indicator of invasive growth In agreement with the observed effect during invasive growth, high SUT2 gene dosage reduced b-galactosidase expression in wild type to a level similar to Dras2 cells Consistently, Dsut2 displayed an increase in reporter gene expression, suggesting that Sut2p,
in fact, negatively regulates high PKA activity (Fig 4B) Based on the genetic evidence that high SUT2 gene dosage is sufficient to sustain growth in Dgpa2 Dras2 but not Dras1 Dras2 cells and considering that Sut2p has initially been identified as a homologue of the putative transcription factor Sut1p [11], we reasoned that Sut2p might act to modify the transcription of one or both RAS genes Therefore, we determined the level of total Ras protein in extracts of cells grown as described for the carbohydrate determination experiments using Western blots with an anti-H-RAS(259) antibody which detects both, yeast Ras1p and Ras2p [14] We found that high SUT2 gene dosage had only a limited effect on Ras protein levels when cells were harvested in stationary phase However, in cells shifted to fresh glucose medium and re-grown for an additional 4 h, Ras protein levels were strongly reduced by high SUT2 gene dosage (Fig 4C), thus providing a possible explanation for the observed phenotype of reduced invasive growth sug-gestive of low PKA activity
Sut2p-GFP localizes to the nucleus
In order to support the possibility that SUT2 encodes a transcription factor, like its anaerobically expressed isozyme Sut1p [11], we determined the subcellular localization of GFP fused to the C-terminus of Sut2p Expression was controlled by the authentic SUT2 promoter on a high copy
Fig 2 Levels of glycogen and trehalose in CEN.PK2 Dras2 (top)and
R1278b Dras2 (bottom)transformed with different plasmids Cultures
where grown to stationary phase in selective glucose medium over
night (D 600 5.5) and shifted to fresh glucose medium (SC 2% glucose)
at time point 0 (j) YEp351 + pFL38; (d) YEp351 +
RAS2; (h) YEp351-SUT2 + pFL38; (s) YEp351-SUT2 +
pFL38-RAS2 Experiments were performed three times with similar results. Fig 3 Sporulation efficiency ofR1278b SUT2 carried on a high copy
plasmid reduces sporulation efficiency in wild-type (P < 0.001) and Dras2 (P < 0.05) cells (Tukey HSD; n ¼ 4) Error bars show ± 1.0 SD; columns show mean values WT, MR161; h.c SUT2, YEp351-SUT2; Dras2, MR298; Dsut2, AR2.
Trang 6plasmid We found that Sut2p-GFP localizes to the entire
cytoplasm of the cell with some accumulation in the nucleus
of most cells (Fig 5) To verify that the observed fluorescent
signal was, indeed, mainly localized to the nucleus, we
carried out DAPI staining of ethanol fixed cells Cells which
retain GFP fluorescence in this procedure show a clear
colocalization of DAPI and green fluorescence in agreement
with a possible involvement of Sut2p in transcription
regulation
Discussion
In this report we describe the isolation of SUT2 in a screen
for high copy suppressors of the synthetic slow growth
phenotype of Dgpa2 Dras2 In addition to SUT2, a number
of other suppressors were identified that have been
impli-cated to function in the RAS/cAMP pathway These include
the two disrupted genes, GPA2 and RAS2, the second RAS
gene RAS1, SCH9, a protein kinase A homologue, which
previously has been described as a high copy suppressor of a number of defects in the RAS/cAMP pathway [23] and TPK2, one of the three catalytic subunits of PKA [6] Interestingly, this screen did not yield any plasmids that contained TPK1 or TPK3, the other two genes encoding catalytic subunits of PKA Plasmids that contained either RAS1, RAS2 or SUT2 were isolated frequently in the screen (50–100 times each), whereas GPA2, SCH9 and TPK2 were isolated < 10 times This may indicate that the yeast genomic library utilized in this study did not contain a perfectly random array of DNA fragments and hence the screen was probably not comprehensive
Sut2p has been described previously as a homologue
of the putative transcriptional activator Sut1p When expressed under the control of a strong heterologous promoter both proteins enhance uptake of sterols and, at least Sut1p, also increases the biosynthesis of sterol precursors [11] In contrast to the exclusively anaerobically expressed SUT1, expression of SUT2 is apparently not controlled by oxygen [28] In order to establish an epistatic relationship between SUT2 and elements of the RAS/cAMP pathway we determined the effect of high SUT2 gene dosage on the lethal double-deletion of both RAS-genes In contrast to concomitant deletion of GPA2 and RAS2, high SUT2 gene dosage did not rescue the RAS1 RAS2-double deletion, suggesting that SUT2 either acts upstream or, alternatively, in a parallel pathway to RAS To further investigate the relation between SUT2 and the RAS/cAMP pathway we studied the influence of high SUT2 gene dosage on storage-carbohydrate homeo-stasis, which is controlled by PKA Consistent with SUT2’s function as a high copy suppressor of the syn-thetic Dgpa2 Dras2 phenotype, high SUT2 gene dosage resulted in decreased storage-carbohydrate levels in a CEN.PK2 background However, this effect was only observed in Dras2 mutants
Surprisingly, re-evaluation of this result in an independent genetic background (S1278b) yielded a different result: high SUT2 gene dosage led to increased storage carbohydrate levels, which suggests reduced PKA activity PKA activity in CEN.PK2 is reduced due to a mutation in adenylate cyclase [25] whereas S1278b is known to contain a particularly strong PKA pathway [26] We therefore hypothesized that Sut2p may represent a new element in PKA feedback-regulation and, hence, affects these two strains differently: increased SUT2 gene dosage stimulates low PKA activity but
Fig 5 Localization of the Sut2-GFPp chimeric protein Cells were fixed
in 70% ethanol for DAPI staining and imaged as described in Experimental procedures.
Fig 4 Influence of Sut2p on invasive growth, FLO11 expression and
Ras protein level in R1278b (A) Cells were grown on YPD medium for
3 days at 30 C (top, left) and then washed off the plates with a squeeze
bottle to determine invasive growth (top, right) Genotypes are as
indicated (center, left) (B) To quantify the influence of Sut2p on
invasive growth, Flo11-b galactosidase reporter assays were carried
out as detailed in Experimental procedures YEp351-SUT2 reduces
lacZ reporter expression in WT (P < 0.01) In contrast, deletion of
SUT2 increased reporter expression (P ¼ 0.001; Tukey HSD; n ¼ 9
and 3 for Dras2, respectively Error bars show ± 1.0 SD Columns
represent mean values (C) Immunoblot with H-Ras (259) or
anti-Aky2 Ig: Total protein was prepared from cells grown overnight in SC
medium (stat.) or after shift to fresh SC medium (growth) as indicated
in Experimental procedures WT, YHUM216 and MR287 (lacZ);
Dras2, MR211 and AR3 (lacZ); Dsut2, AR1 and AR4 (lacZ); h.c.
SUT2, YEp351-SUT2.
Trang 7inhibits high PKA activity Subsequent experiments on
sporulation efficiency and invasive growth of S1278b
supported this hypothesis Sporulation in S cerevisiae is
facilitated by starvation-conditions that result in low PKA
activity High SUT2 gene dosage yielded diminished
spor-ulation efficiency, which indicates increased PKA activity
relative to wild type In contrast, invasive growth in haploids
requires strong PKA activity and is assayed on rich medium
[24] Under these growth conditions, high SUT2 gene dosage
resulted in a strong reduction of agar invasion in a
RAS2-wild-type strain, and this reduction was similar to the
phenotype of Dras2 mutants We consistently found that
during growth in rich medium, high SUT2 gene dosage
resulted in diminished expression of FLO11, a surface
flocculin whose expression is stimulated by PKA and that
is essential for invasive growth [10] As it has been proposed
that Sut1p and Sut2p act as transcription factors [11] we
investigated if SUT2 affects expression of elements of the
RAS/cAMP pathway Indeed we found that high SUT2 gene
dosage reduced Ras2p expression in cells that had been
grown in rich medium Importantly, no reduction in Ras2p
was observed in cultures approaching stationary phase,
which correlates with a reduction in PKA-activity
Interestingly, a link between sterol-biosynthesis and RAS
has previously been established: if yeast cells were starved
for mevalonate, an early precursor of isoprenoids and
sterols, levels of both RAS-mRNAs were decreased [29]
One possible explanation for the effect of high SUT2 gene
dosage on the RAS/cAMP pathway is that Dgpa2 Dras2
and possibly additional mutants reducing PKA activity are
simultaneously down-regulated for isoprenoid/sterol
bio-synthesis, thereby reducing Ras1p abundance and, hence,
impairing the residual G-protein stimulating adenylate
cyclase High copy expression of SUT2 then could, in
analogy to its homologue SUT1, relieve this sterol
precur-sor-starvation by increasing sterol-biosynthesis Therefore it
will be interesting to determine if genes of the sterol
biosynthetic pathway are subject to regulation by Sut2p,
which may yield a better understanding of the proposed
connection between the nutrient-sensitive activity of PKA
and sterol biosynthesis
Acknowledgements
This work was supported by a grant from the Deutsche
Forschungs-gemeinschaft to W B (Ba415/24–1) We thank H U Mo¨sch,
Go¨ttingen, for the gift of strains and plasmids We are also indebted
to B Klebl, Martinsried, for the gift of plasmid p426MET25-Ras2 M.
Angermayr and G Strobel are acknowledged for their help with yeast
genetics and valuable discussions We also acknowledge L J Zwiebel
for reading the manuscript Finally, we thank T Grimm for
construction of strain CEN.PK2 Dgpa2 Dras2.
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