Download Protein expression during exponential growth in 0.7 M NaCl medium

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FEMS Microbiology
Letters 137 (1996) 1-8
Protein expression during exponential growth in 0.7 M NaCl
medium of Saccharomyces cerevisiae
Joakim Norbeck, Anders Blomberg
*
Department of General and Marine Microbiology, Lundberg Laboratory, Uniuersio of Gb;teborg, Medicinaregatan 9C,
413 90 Giiteborg, Sweden
Received
1 December
1995; revised 22 December 1995; accepted 22 December 1995
Abstract
Saccharomyces
cereuisiae
exponentially growing in basic or 0.7 M NaCl medium were isotopically labelled with
35S-methionine, followed by protein separation and quantification by two-dimensional
polyacrylamide gel electrophoresis
(2D-PAGE) combined with computerised image analysis. The electrophoretic
separation resolved about 650 proteins of
which 13 displayed significant and at least 2-fold changes in rate of synthesis during saline growth. By sequencing of
2D-PAGE resolved proteins, one of the 8 induced spot, p42.9/5.5, was shown to correspond to the full length (containing
the N-terminal extension) product of the GPDI gene encoding the cytoplasmic glycerol 3-phosphate dehydrogenase. The
expression of the TDH3 gene, glyceraldehyde 3-phosphate dehydrogenase, and the EN02 gene, enolase, decreased during
growth in NaCl medium, declines hypothesised to have an impact on the flux to glycerol.
Keywords: Saccharomyces cereuisiae; NaCl stress; Protein synthesis;
2D-PAGE; Protein sequencing;
1. Introduction
The adaptive response of Saccharomyces
cereto NaCl involves exclusion of Na+ to maintain low intracellular concentrations
of this ion and
an osmotic potential compensatory accumulation
of
the three-carbon polyol glycerol [I]. A key protein in
the osmoregulatory
response is the cytoplasmic enzyme glycerol 3-phosphate
dehydrogenase
(GPD)
[2]. The activity and amount of GPD in cellular
extracts is increased in response to high extracellular
concentrations of NaCl [3], a regulation reportedly at
the transcriptional level [4-71.
uisiae
* Corresponding
author. Tel: +46 (31) 773 2589; fax:
(31) 773 2599; E-mail: [email protected]
0378-1097/96/$15.00
0 1996 Federation
PIZ SO378-1097(96)00006-7
of European
+46
Microbiological
GPDl;
Glycolytic
enzymes
Computer
aided analysis
of two-dimensional
polyacrylamide
gel electrophoresis (2D-PAGE) generated images of isotopically labelled protein extracts
have been performed in a quantitative investigation
of the cellular adaptation process to NaCl containing
medium [8]. It was reported that drastic expression
changes occurred during the osmotic adaptation, with
138 proteins displaying a significant and at least
2-fold change in relative synthetic rate (induction or
repression). The 20 highly responsive proteins with
more than 8-fold changes in synthesis exhibited
mainly increased rate of production; only two proteins were repressed. Responsive proteins during this
one hour osmotic adaptation could be classified into
14 different regulatory families by their temporal
response, revealing some of the intricate complexity
Societies.
All rights reserved
of the transition physiology
in S. cerer~isiur. lncreased glycerol accumulation seems to be one of the
main osmoregulatory
mechanisms even for actively
proliferating cells, however, indications of a substantially altered metabolism by salt stress has also been
presented [9]. Thus. a global study on the changes in
protein synthesis during exponential growth in NaCl
media was conducted in order to search for protein
responders indicating additional salt-instigated cellular adjustments.
2. Materials and methods
acetic acid 10% [v/v]>. The fixed gels were rinsed in
milliQ for at least 2 h in order to attain their maximum size (roughly 25 X 25 cm>, before being dried
on chromatography
paper in a gel drier. The dried
gels were exposed to Kodak X-AR film, exposure
time depending on the amount of incorporated “Smethionine in the extracts. Gels corresponding
to
cells from 0 M NaCl were exposed for 1. 3 and 7
days, those from 0.7 M NaCl for 3, 7 and 30 days.
Films were developed using a AGFA-CURIX
60
automatic developer. For each exposure time a calibration strip with a wide span of radioactivity was
included [ 111.
2.1. Strain and mediu
2.5. Scanning and computevised
Saccharomyes
cerer,isiae strain Y41 (ATCC
38531) was maintained,
grown and labelled in a
defined minimal medium (YNB) with 0.5% (w/v)
glucose, as reported earlier [IO].
The analysis was carried out using the PDQuest
program v. 4. I (PDI, Huntington Station, New York)
as earlier described [S]. Protein quantity was expressed as DPM in the spot relative the total amount
of radioactivity
loaded onto the first dimensional
gels.
2.2. Label&g
and sample preparation
Labelling and sample preparation was performed
in triplicates with cells grown in 10 ml of YNB
medium at 3o”C, as described earlier [ 101. lndependent of growth medium a total amount of protein in
the range 2.9-4.3
pg (average 3.4 pg) per ~1
extract was obtained.
2.3. 2D-PAGE
electrophoresis
The 2D-PAGE gels were run essentially as earlier
reported [lo], with the following minor modifications: i> the second dimensional
SDS-PAGE slab
gels were 10% acrylamide [0.80/G bis] and ii) the
effect was held constant at 1400 mW/gel
and the
gels were run overnight at 20°C. A maximum of 20
pg of total protein was loaded onto the first dimensional tubes and this amount of protein corresponded
to approximately
I .4 X IO6 DPM and 0.25 X 1Oh
DPM for the control and the salt grown cells, respectively.
2.4. Spot risualisution
l-2
After the completed run the gels were fixed for
h in fixing solution (ethanol 50% [v/v] and
2.6. Quantification
duta analysis
of dominant spots
Some of the dominant spots were saturated or
distorted in shape and could not be accurately quantified by computerised image analysis. These spots
were cut out, placed in 5 ml of Aquassure and
counted in a scintillator. The spot corresponding to
actin was included as a reference through which the
quantifications
of the dominant spots could be related to the spots quantified by image analysis.
2.7. Preparutirle 2D-PAGE
and sequencing
The preparative IEF gels were run as earlier
described [lo]. Identification of the spots were performed by sequencing of reversed phase-HPLC fractionated peptides generated by trypsin digestion in-gel
of 2D-PAGE resolved proteins [ 101. For the sequencing of Fbalp and Eno2p, proteins were visualised by
copper staining [ 121. The protein spots were electroblotted to an immobilon-psq membrane (Millipore
Inc.), using a Mini Trans-blotTM assembly (Bio-Rad),
and sent to KEBO laboratories for N-terminal sequencing on an Applied biosystems (model 473A)
protein sequencer.
J. Norbeck, A. Biomberg / FEMS Microbiology Letters 137 (19%) 1-8
2.8. Measurements
of activities of glycolytic enzymes
The enzyme activity measurements
were performed essentially as described previously [13]. The
protein extraction method however differed slightly.
1.5 g of acid washed glass beads was added to a
pellet of approximately 2.5 X lo9 cells together with
protease inhibitors (Pefabloc @ 1 mg/ml, Leupeptin
3 pg/ml,
Pepstatin 15 pg/ml
and Bestatin 40
pg/ml)
in 1 ml of buffer (Triethanolamine
10 mM,
DTT 1 mM and EDTA-Na,,
pH 7.5). After vortexing 4 X 30 s (with placement on ice > 1 min inbetween) the glass beads and debris were pelleted at
2000 X g for 3 min in a table top centrifuge. The
supematant was further centrifuged at 18 000 X g for
15 min and the supematant from this step was used
for all enzyme measurements.
Care was taken to
keep samples ice-cold at all times.
3. Results
and discussion
3.1. Salinity induced changes in global protein synthesis
Early respiratory phase cultures of strain Y41 of
Saccharomyces
cereuisiae, growing in non-saline
medium, were used as inoculum into medium without NaCl (control) or to medium supplemented with
0.7 M NaCl. After a lag phase of roughly the same
length in the two media (1.5 h) growth resumed,
however, at a slower rate for the salinity stressed
culture; the generation times were 2.2 and 3.2 h,
respectively.
Protein extracts were prepared from
35S-methionine
labelled mid-exponential
cultures,
and were subjected to 2D-PAGE analysis. Irrespective of growth condition roughly 650 spots were
detected and quantified in the gels. After manual
matching of 261 spots in the different gels (leaving
out spots which by visual inspection clearly not
changed in rate of synthesis), computer comparisons
of significant and more than 2-fold quantitative differences were allocated. Only proteins which exhibited a production level of at least 100 ppm (0.01%)
in either growth condition are reported (Table 1, Fig.
1). The most salinity induced protein, p23.9/5.7,
changed expression by a factor of 4.5. This protein
was also the most highly expressed of all the respon-
Table
3
1
Proteins exhibiting significant (by log t-test criteria) and at least
2-fold changes in synthesis (induction or repression) in NaCl
grown S. cerecisiae
M, /PI
0 M NaCl
&Da/PHI
(ppm)
a
0.7 M NaCl
n-fold change
(ppm)
Induced proteins
23.9/5.7
134
25.6/6.4
87
34.9/5.3
74
42.9/5.5 h
32
35.1/6.6
60.3
48.5/6.2
63
47
25.6/6.6
5 I .4/6.0
64
+_3l
f 7
+54
532
*4l
+I0
+19
f 9
602+16
380&- 19
286+ I7
113+27
149+ 10
153f 12
106f31
129+25
4.5
4.4
3.8
3.6
2.5
2.4
2.3
2.0
Repressed proteins
59.6/6.5
157
36.6/5.2
165
27.6/5.4
211
59.4/5.5
114
53.2/6.2
122
k27
k28
i25
+25
+20
15+28
47+65
86k20
52f26
61+23
10.7
3.5
2.4
2.2
2.0
Only proteins with an expression level of at least 100 ppm in the
induced state have been included. Data represent mean of triplicate independent experiments + SD.
a Relative rate of synthesis as parts per million. DPM recorded in
the spot is related to the total amount of DPM loaded onto the first
dimensional gel.
b Identified as the product of the GPDI gene by microsequencing
(Table 2). ZD-PAGE position indicated in Fig. 2.
sive ones, and exhibited during exponential growth
in 0.7 M NaCl an expression level of about 600 ppm.
The small number of salt-responsive
proteins and
their minor expression change scored in this study
for proliferating cells strongly contrasts the earlier
reported cellular response during adaptation to salt,
which was accompanied
by a massive change in
global protein synthesis; e.g. 18 proteins being induced at least eight fold [8]. A prominent feature of
these highly NaCl-responsive
proteins for adapting
cells were their transient response, all exhibiting
maximal rate of synthesis in the middle of the adaptation period. The data presented here further supports this transient expression, since few proteins
exhibited induced levels during saline growth, and
those that did so only increased their rate of synthesis to a minor extent. The protein that displayed the
most dramatic change in expression was p59.6/6.5
which was repressed by a factor of about 10 to an
expression level in the NaCl medium of only 15 ppm
(Table 1).
Thus, the transition physiology of yeast cells display great plasticity in terms of protein synthesis,
probably responding to signals triggered by the initial osmotic dehydration [8]. Exponentially
growing
cells, on the other hand, has adapted to the new
conditions and potentially re-established almost norma1 signalling levels, indicated by their less dramatic
expression changes.
3.2. Protein ident$cation
b!t tnicrosequet~cit~g
Protein p42.9/5.5
(Fig. 1). exhibiting a 3.6-fold
salt induction (Table I ). displayed M, and p I values
similar to Gpdlp (theoretical value 42.8/5.3). Link-
age of this NaCl responsive spot to the GPDl gene
was unequivocally
confirmed
by sequencing
of
trypsin generated peptides (Table 2), and the obtained amino acid sequences clearly distinguished it
from its recently cloned isogene product, Gpd2p [6].
It was also clear that the 30 amino acid N-terminal
extension, previously believed to be cleaved off. was
present in this salt responsive Gpdlp spot, as one of
the trypsin generated peptides spanned residues 8- 16.
This is interesting since the purified GPD enzyme
[14] was devoid of the 30 most N-terminal amino
acids and the sequence started at position 3 1 [ 151.
This N-terminal discrepancy could be an outcome of
differences in the physiological states of the cells at
harvest (exponential versus stationary phase cultures).
70
50
30
610
PI (PW
Fig. I. 2D-PAGE analysis of [ “S]methionine
labelled proteins fol- salinity instigated protein expression changes in S. cerer~i.siw. The
central portion of the gels (autoradiogram)
is displayed, covering a pl range of 4.9-6.5 and a M, range of 24 to 100 kDa. (A) Cells growing
exponentially in 0 M NaCl medium. (B) Cells growing exponentially in 0.7 M NaCl medium. Protein spots that exhibited a significant and
more than 2-fold expression change in saline cultures compared to the control are indicated. Arrows mark position of spots with increased
synthesis in 0.7 M NaCl medium and squares indicate spots with decreased synthesis in 0.7 M NaCl medium. Displayed are the raw scan of
the longest exposures.
J. Norbeck, A. Blomberg/
FEMS Microbiology Letters 137 (1996) 1-8
tentatively indicating novel intricate regulation of the
Gpdl protein. The other salt-responsive spots were
in our preparative gels not found at their predicted
amounts (Table l), a discrepancy potentially caused
by differential methionine contents. However, a
number of other proteins in the 2D pattern of S.
cereuisiue were identified by the sequencing procedures (Table 2) and these together with some of our
previous identifications are depicted in Fig. 2. An
identified protein that displayed an almost 2-fold
change in expression during NaCl growth was
Samlp, which was repressed 1.8 fold in salt (629 to
352 ppm). Samlp catalyses the synthesis of Sadenosylmethionine (AdoMet) from methionine and
5
ATP. AdoMet is an important methyl donor for
transmethylation and is also the propylamino donor
of polyamin biosynthesis [ 161. The Samlp down
regulation in salt indicates somewhat decreased activities in these processes. In this context it is of
interest that salt dependent differential regulation of
the different SAM isogenes have been observed in
plants, hypothesised to reflect NaCl imposed changes
in cell wall synthesis [17]. The Pglucanase Bgl2p
involved in cell wall synthesis in yeast was expressed to the same level in salt grown or control
cultures. The biosynthetic proteins Met6p and Lys9p
displayed salt invariant expression, indicating minor
alterations by NaCl treatment to amino acid biosyn-
610
PI (PHI
Fig. 2. Identified proteins in the ZD-PAGE pattern of S. cereuisiae. All spots indicated are identified by microsequencing,
and sequences are
reported in Table 2 or in [lo]. The following gene designations are used: ACTl, actin; ATP2, F, -subunit of the mitochondrial ATPase;
ADHI, alcohol dehydrogenase I; BGL2, pglucanase;
EFBl, translational elongation factor 1 p; EN01 and EN02, enolase A and Ef; FBAl,
fructose bisphosphate dehydrogenase;
GDHl, glutamate dehydrogenase
(NADP+); GPDl, glycerol-3-phosphate
dehydrogenase;
HXK2,
hexokinase PII; LYS9, saccharopine dehydrogenase;
MET6, methionine synthetase; MET17, O-acetyl (homolserine sulthydrylase; PDCl,
pyruvate decarboxylase;
IPPI, inorganic pyrophosphatase;
SAMl, S-adenosylmethionine
synthetase I; SSA1/2,
SSBl, SSB2 and SSEl,
HSP 70 isogenes; TDH3 and TDH2, glyceraldehyde
3-phosphate dehydrogenase.
For those who prefer the other orientation of 2D yeast
patterns (acidic side to the right) the flipped version of this image is available at our WWW-2DPAGE
database server: http://yeastZDPAGE.gmm.gu.se.
6
.I. Norbeck. A. Blomherg/
Table 2
Experimentally
obtained protein or peptide sequence:,
2D-PAGE resolved proteins (listed in alphabetic order)
M,
/pI
40.0/5.5
46.1/5.3
28.3/-.29.5/-.42.8/6.2
35.8,‘6.1
49.0/5.6
42.9/5.5
Residues in Gene
protein
assigned
AGFAXDD
XIXELGIYP
LLDAXVVCQ
IGELAFNL
IXESTVAGFL
(QJLNASLADK
WFNHIA
AVSKVYARSVYDSRGNPTVE
GVEQILKRKTGVIVGEDVHN
FHPXVNLXIL
EIGYLFGAY
VIELGGTVVXL
LNLTXXHLN
19- 25
371-379
390-398
24- 31
111-120
13- 21
50- 55
2- 21 h
2- 20h
79- 88
157-165
236-246
x- 16
160-173
45.4/5.2
ILXGFAWLGLF.
FDXNYH(Y)VRP
AXTYF(D)EQSN
NYPNHI(I)L
FVEGDNPEEF
DLPNAD(K)ETDPF
GIDLTNVXLPD
DXXLAW
314-324
122-131
234-243
692-699
130-139
350-361
239-249
157-162
IDXVSSAQH
187-196
70.0/5.
74.5/5.3
IIVDAYGKEEHVLIFD
LXAEEVDFXE
253-258
187-196
330-339
3 I .3/5.5
I
ACTI
A TP?
BGL2
EFBI
EN02
FBAl
GDHl
tiPI)/
L
95- IOX
GVQyLXX?ITEELi
44.2/6.4
for the
Amino acid sequence in
one-letter notation a
YLPGIXLPDNLVAN
45.1/5.3
74.3/6.1
FEMS Microbiology
LYSY
MET6
MET1 7
IPPl
SAMI’
SSAI/Z
Letters 137 (IYY61 I-K
expression of these proteins. Proper expression of
actin has previously been shown to be of importance
during NaCl growth, since act mutants displayed
impaired growth in NaCl media [ 181. Actin is slightly
regulated at the transcriptional
level during adaptation to salt [7]. However, the expression of actin did
not display any significant change during exponential growth in saline media. This might indicate
cellular independence of actin levels during osmotic
stress as long as certain threshold requirements are
met. The two proteins Ssal p and Ssa2p (belonging to
the HSP 70 family in S. cerevisiae) [19] displayed a
slight salt response during adaptation to NaCl [8],
while we here observed no difference in expression
during exponential growth. The HSP70 homologues
Ssblp and Ssb2p have been shown to be involved in
the translational process and to be associated with
ribosomes [20]. The two isogene products have earlier been identified [lo], and both proteins displayed
salt invariant expression. The constancy of the SSB
proteins and the translational elongation factor 1p
(Efblp) indicate a non-affected protein synthesis during salt growth.
Taken together, the constant expression of proteins involved in a number of cellular functions
indicate a robust and only slightly altered overall
metabolism during growth in salt.
SSEI ’
The 2D position of the sequenced proteins are indicated in Fig. 2.
’ Sequences obtained from peptides generated by trypsin digestion and subsequent HPLC fractionation
of 2D-PAGE resolved
spots, except for the sequences from Fbalp and Eno2p which are
from N-terminal sequencing on whole proteins blotted onto PVDF
membranes.
h The N-terminal methionine in Fbalp and Eno2p was post-translationally removed.
’ Underlined residues are unique to GPDl compared to GPD2.
’ Underlined residues are unique to SAM1 compared to SAM2.
’ Underlined residues are unique to SSEI compared to SSE2.
thesis, and the average expression levels of these two
proteins were 754 and 222 ppm, respectively. The F,
@subunit of the mitochondrial ATPase, Atp2p. also
exhibited constant expression (about 290 ppm), indicating a non effected level of mitochondria and/or
ATP producing capacity. Assimilation of ammonia
and sulfate mediated by Gdhlp and Met1 7p, respectively, also seemed unaffected from the constant
3.3. Levels of glycolytic enzymes during saline growth
Studies on cells of S. cerel~isiae grown aerobically in chemostat have indicated rather drastic
changes in overall carbon flow under saline conditions [9]. It was reported that (i) the fractional carbon
distribution into respiration or fermentation changed,
and (ii) up to 16% of the consumed glucose at high
dilution rates could not be accounted for by the
analysed products. This indicates rather diverse
changes in catabolic activities during NaCl stress.
Some of the glycolytic enzymes have been shown to
be good indicators of metabolic changes, exhibiting
metabolism specific expression levels for both transcripts [21] and protein products [22]. Thus, quantitative studies on levels of glycolytic enzymes might
reveal overall catabolic changes during saline growth
(Fig. 3). One of th e isoenzymes of glyceraldehyde
3-phosphate dehydrogenase
(Tdh3p) exhibited the
highest level of expression of the glycolytic enzymes
J. Norbeck,
A. Blomberg
/ FEMS MicrobiologJl
A
PDCI
ADHl
ACT1
0
500
loo0
M&mine
1500
zoo0
25Oil
normalized PPM
HxK2
FBAl
GPDl
TDH3
0
loo
300
200
Percent
400
500
Fig. 3. Quantification
of 2D-PAGE resolved glycolytic enzymes
(indicated in Fig. 3). Actin (ACTI) is included as a point of
reference. (A) Quantity expressed in ppm divided by number of
methionines in protein, enabling stoichiometric
comparison. (B)
Quantity expressed as percent, with values in 0 M NaCl medium
being set at 100%. Data represent mean of triplicate independent
experiments + S.D.
in either growth condition and roughly 1% of the
incorporated 35S-methionine ended up in this subunit. The incorporation
values were normalised to
the somewhat different number of methionines in the
proteins, and these methionine normalised ppm values revealed Tdh3p to be roughly 30 times more
abundant on a mole basis than Actlp (Fig. 3A).
During non-saline
growth Gpdlp
was found in
roughly 100 fold lower amounts in relation to Tdh3p,
reflecting under these conditions the minor metabolic
flux into glycerol compared to into ethanol and
biomass. Gpdlp was the only protein of the identified central catabolic enzymes that exhibited a major
salt response (Fig. 3B). The enzymes upstream from
the triosephosphates, Hxk2p and Fhalp, displayed no
significant
changes, while downstream
especially
Tdh3p and Eno2p exhibited decreased expression
values. The isoproteins Tdh2p and Enolp displayed
no significant change (data not shown). The data on
Letters 137 (19961 1-8
I
salt induced alterations in protein expression was
supported by enzyme activity measurements,
where
both the glyceraldehyde
3-phosphate dehydrogenase
and enolase activities decreased in salt grown cells,
the former from 6199 to 5085 mU/ng protein (18%
reduction) and the latter from 1048 to 792 (25%
reduction). Other determined enzyme activities as
those for hexokinase, aldolase, triosphosphate
isomerase, and pyruvate decarboxylase
exhibited no
significant salt dependent variation in activities (data
not shown).
The rate of glycerol synthesis was earlier indicated to be under the control of the amount of
Gpdlp, and a high flux control coefficient was reported for this enzyme [2]. However, the determined
flux control coefficient for GPD might be overestimated if additional metabolic changes occur in parallel, especially those alterations in close vicinity to
the glycerol pathway [23]. Recent results indicate
that at least under certain conditions Gpdlp has low
controlling power over the flux to glycerol, since
overproduction
of the enzyme had no apparent impact on glycerol formation [5]. Apparently,
additional enzyme activities have to be regulated besides
Gpdlp in order to obtain the massive osmoregulatory
production of glycerol. It is conceivable
that the
decreased amounts of glyceraldehyde
3-phosphate
dehydrogenase
and enolase here reported would be
prerequisites for diverting an increasing amount of
triosephosphate monomers into the glycerol pathway.
It is thus hypothesised that the controlling power
over glycerol formation under saline conditions will
be shared between Gpdlp, Tdh3p and Eno2p.
Acknowledgements
This work was financially
supported by grants
01758-304 and 01758-305 from NFR. We are indebted to Lennart Adler and Lena Gustafsson for
valuable comments on the manuscript.
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