Download Transcription of multiple cell wall protein

FEMS Microbiology Letters 161 (1998) 345^349
Transcription of multiple cell wall protein-encoding genes in
Saccharomyces cerevisiae is di¡erentially regulated during
the cell cycle
L. Heleen P. Caro a , Gertien J. Smits a; *, Piet van Egmond a , John W. Chapman b ,
Frans M. Klis a
a
Institute for Molecular Cell Biology, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands
b
Unilever Research Laboratory, Vlaardingen, The Netherlands
Received 12 January 1998; accepted 16 February 1998
Abstract
The yeast cell wall consists of an internal skeletal layer and an outside protein layer. The synthesis of both L-1,3-glucan and
chitin, which together form the cell wall skeleton, is cell cycle-regulated. We show here that the expression of five cell wall
protein-encoding genes (CWP1, CWP2, SED1, TIP1 and TIR1) is also cell cycle-regulated. TIP1 is expressed in G1 phase,
CWP1, CWP2 and TIR1 are expressed in S/G2 phase, and SED1 in M phase. The data suggest that these proteins fulfil distinct
functions in the cell wall. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
Keywords : Saccharomyces cerevisiae ; Cell cycle; Transcriptional regulation; GPI protein; Cell wall
1. Introduction
The cell wall of yeast may account for up to 30%
of the dry weight of the cell, and therefore represents
a major investment of the cell [1]. The main components of the cell wall are mannoproteins and L-linked
glucans, and a small amount of chitin [2^4]. The wall
is responsible for the mechanical strength of yeast
cells. As a result, the cell wall has often been envisioned as a static structure with a constant composition. Evidence is, however, accumulating that the
cell wall may vary considerably in response to both
external and internal cues.
* Corresponding author. Tel.: +31 (20) 525 7841;
Fax: +31 (20) 525 7934; E-mail: [email protected]
For example, external cues such as heat shock or
hypo-osmotic shock, or changes in the carbon
source, a¡ect the transcription of several genes that
encode cell wall biosynthetic enzymes, as well as several genes encoding glucanase-extractable cell wall
proteins [4^12]. Also, the biosynthesis of the glucan
and chitin components of the cell wall is cell cycleregulated, both transcriptionally [5,13^15] and posttranslationally (see [4]).
We provide evidence that the transcription of ¢ve
cell wall proteins is cell cycle-regulated, and that
these genes are expressed at di¡erent stages of the
cell cycle. This suggests that these proteins, which
have so far been regarded only as general structural
cell wall proteins, have speci¢c functions in cell wall
construction.
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
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2. Materials and methods
2.1. Yeast strains and growth conditions
Saccharomyces cerevisiae strain X2180-1A was obtained from the Yeast Genetic Stock Center, Berkeley, CA, USA. Cells were grown in YPD medium.
2.2. Synchronization
Synchronization by K-factor was performed basically according to De Nobel et al. [16]. For centrifugal elutriation, a 5-ml elutriator rotor JE-6B (Beckman Instruments BV, Mijdrecht, The Netherlands)
was used. Cells were grown in batch cultures on
YPD. Elutriation was performed as described by
Woldringh et al. [17]. In both cases, samples were
taken every 15 min. For microscopic analysis, cells
were ¢xed with 0.13% (w/v) formaldehyde and sonicated brie£y to break up aggregates. For RNA isolation, 20 ml of cell suspension was centrifuged, the
cells were washed with ice-cold water, and stored in
liquid nitrogen.
2.3. RNA isolation and Northern hybridization
RNA was isolated by the hot-phenol method,
without the use of glass beads, as described in Current Protocols (Greene Publishing Associates, Inc.
and John Wiley and Sons, Inc., New York). 10 Wg
of RNA per lane was separated on a formaldehyde/
formamide-containing agarose gel system. RNA was
cross-linked to Hybond-N‡ membrane (Amersham)
by UV radiation. Northern hybridizations were performed in the presence of 50% formamide at 42³C
using 32 P-labeled gene fragments. The blots were
washed at 42³C with decreasing concentrations of
SSC, down to 0.5USSC, in the presence of 0.1%
SDS.
Hybridization signals were quanti¢ed by scanning
of autoradiograms in the linear range of the ¢lms.
Expression levels were normalized to actin levels.
3. Results
To study the cell cycle-regulated expression of cell
wall protein-encoding genes, we synchronized grow-
Fig. 1. Response of mRNA levels of cell wall protein-encoding
genes to the addition of K-factor to asynchronous cultures. All
mRNA levels were normalized to ACT1, and the initial value
was set at 100%. b : CWP1, a : CWP2, F : SED1, E : TIP1, O:
TIR1.
ing cultures with K-factor and by centrifugal elutriation. The results were essentially the same, although
with centrifugal elutriation less synchrony was
achieved. When the cells are arrested in G1 with Kfactor, it takes approximately one generation time
(100 min at 23³C) until all cells have reached G1.
During this period the expression of the cell wall
proteins changed (Fig. 1): the mRNA levels of
CWP1, CWP2, and TIR1 decreased, whereas those
of SED1 and TIP1 increased. The increase in mRNA
levels of SED1 and TIP1 indicates that the arrest by
K-factor takes e¡ect at a time when transcription of
both genes is induced, whereas this is not the case for
CWP1, CWP2, and TIR1 (see also Fig. 2B). Alternatively, K-factor may have a direct e¡ect on cell
wall protein expression, but since the induction is
slow, and no consensus sequences for binding of
Ste12p are present in the promoter regions of
SED1 and TIP1, the former possibility seems more
likely.
The e¡ect of G1 arrest on cell wall protein expression was corroborated when the transcription of the
cell wall protein-encoding genes was followed during
several division cycles in K-factor-synchronized cultures (Fig. 2A). The transcription of CWP1 peaked
slightly later than H2A, at the time when most cells
had small buds, which is estimated to be late in
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Fig. 2. A: Messenger RNA levels of various cell wall protein-encoding genes in synchronized cultures. All mRNA levels were normalized
to ACT1 levels, and the highest value was set at 100%. The percentage of budded cells (b), small budded cells (F) and the mRNA levels
of CLN2 and HTA1/2 (H2A) serve as cell cycle progression and synchronicity markers. Probes were used against CWP1, CWP2, TIR1,
SED1, and TIP1 mRNA. Thin vertical lines indicate the times at which CLN2 mRNA is at its maximum. B : Transcriptionally active periods of cell wall protein-encoding genes. Depicted is a normalized cell cycle, which is the average of all three cycles from A. Averages of
the start and end of activated transcription of each gene were calculated relative to the peaks in CLN2 transcription.
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S phase or early in G2. About 10 min later, the
mRNA level of CWP2 reached its maximum, which
coincided with the peak in the number of budded
cells. It can therefore probably be placed in G2.
Although the peaks of CWP1 and CWP2 are slightly
separated, the induction of transcription of these
genes occurs around the same time, and might therefore be brought about by similar mechanisms. Approximately 30 min after CWP2, SED1 transcription
peaked. Almost all visible buds were large buds at
that time, indicating that this is M phase. TIP1 transcription reached its maximum after SED1, but before CLN2. This seems to occur early in G1, around
the time of cell separation, since the percentage of
budded cells rapidly decreased at the time when
TIP1 transcription was highest. The transcription
of TIR1 was somewhat more di¡use, but we estimate
TIR1 transcription to occur mainly in the same period as CWP1 and CWP2 (see Fig. 2B), based on the
periodic increases in the mRNA level of this gene.
Assuming that mRNA levels start to increase
when transcription is induced, and start to level or
decrease (depending on the mRNA stability) when
transcription ceases or is shifted down, we can determine the periods during which the genes are transcriptionally activated. A graphic representation of
this is shown in Fig. 2B, in which dark areas represent the periods of increased transcriptional activity.
4. Discussion
Since yeast is a unicellular organism, it has to
continuously respond to changes in the environment.
The cell wall not only functions as a barrier between
the cell and the environment, but is also the ¢rst
contact with the outside. Therefore, it has to constantly adapt, to provide optimal protection from,
and optimal interaction with the outside.
Several of the ¢ve (out of a total of almost 40 [18])
cell wall protein-encoding genes we have studied are
known to be activated under speci¢c conditions.
TIP1 expression, for example, is induced at high
and low temperatures [7] and during anaerobic
growth [11]. TIR1 expression occurs under conditions of fermentation [9,11] and at low temperatures
[10]. Surprisingly, no clear phenotypes are found
when any of these genes are deleted; only deletion
of CWP2 results in an increased sensitivity to calco£uor white, Congo red, and zymolyase [19], whereas
deletion of SED1 results in a somewhat increased
tolerance for calco£uor white and Congo red (M.J.
van der Vaart, personal communication). Deletion of
EGT2, which encodes another cell wall protein [18],
does result in a clear phenotype, namely a delayed
cell separation [20].
We studied the transcription of these cell wall protein-encoding genes during the cell cycle. We were
expecting to ¢nd a certain amount of cell cycle regulation, since incorporation of mannoproteins into
the cell wall is cell cycle regulated and is highest in
M phase [16]. Surprisingly, transcription of all ¢ve
genes was cell cycle regulated, and occurred in at
least three di¡erent phases of the division cycle: in
S/G2 (CWP1, CWP2 and TIR1), in M (SED1) and
in early G1 phase (TIP1 (and EGT2 [20])). By studying transcription levels, one cannot draw de¢nite
conclusions about the protein expression levels.
However, recent data show that GFP-fusion proteins
of Cwp1p and Cwp2p are incorporated in distinct
but separate regions of the cell wall (A.F.J. Ram,
unpublished results). This asymmetric distribution
is consistent with cell cycle-regulated expression.
Possibly, asymmetric deposition of cell wall proteins
provides the cells with landmarks for, for example,
bud site selection. In addition, the fact that various
cell wall proteins are di¡erentially expressed under
di¡erent growth conditions makes it highly likely
that they have speci¢c (structural) functions.
Acknowledgments
We would like to thank Dr. M.J. van der Vaart
who supplied us with several probes. We also thank
Dr. A.F.J. Ram for sharing data prior to publication. Furthermore, we are much obliged to P. Huls,
who performed the centrifugal elutriation. This research was supported by the Dutch Ministry of Economic A¡airs and the Life Science Foundation
(SLW).
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