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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. PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 9 3 - 7 FEMSLE 8103 31-3-98 346 L.H.P. Caro et al. / FEMS Microbiology Letters 161 (1998) 345^349 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 FEMSLE 8103 31-3-98 L.H.P. Caro et al. / FEMS Microbiology Letters 161 (1998) 345^349 347 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. FEMSLE 8103 31-3-98 348 L.H.P. Caro et al. / FEMS Microbiology Letters 161 (1998) 345^349 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). References [1] Fleet, G.H. (1991) Cell walls. In: The Yeasts, Vol. 4, 2nd edn., FEMSLE 8103 31-3-98 L.H.P. Caro et al. / FEMS Microbiology Letters 161 (1998) 345^349 [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] Yeast Organelles (Rose, A.H. and Harrison, J.S., Eds.), pp. 199^277. Academic Press, London. Klis, F.M. (1994) Cell wall assembly in yeast. Yeast 10, 851^ 869. Cid, V.J., Duraèn, A., Del Rey, F., Snyder, M.P., Nombela, C. and Sanchez, M. (1995) Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae. Microbiol. Rev. 59, 345^386. Orlean, P. (1997) Biogenesis of yeast cell wall and surface components. 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