Mycology Department, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan1
Division of Life Science, Graduate School of Agricultural Science, Tohoku University,Aoba-ku, Sendai 981-8555, Japan2
Department of Hygiene, School of Medicine, Yokohama City University, 39 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan3
Author for correspondence: Hisafumi Yamada-Okabe. Tel: +81 467 47 2242. Fax: +81 467 46 5320. e-mail: hisafumi.okabe{at}roche.com
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ABSTRACT |
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Keywords: yeast, chitin synthase 3, overexpression, proteinprotein interaction
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INTRODUCTION |
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The level of Chs2p is regulated during the cell cycle. Chs2p is localized in the bud neck at the end of mitosis, and then degraded in a Pep4p-dependent manner (Chuang & Schekman, 1996 ). Chs3p is a metabolically stable protein that appears at a ring on the cell surface at bud emergence and then accumulates in the mother-bud neck of small-budded cells (DeMarini et al., 1997
; Santos & Snyder, 1997
; Ziman et al., 1996
). Chs3p function requires several cellular factors, including the products of the genes CHS4, CHS5, CHS6, MYO2 and TIG1 (Bulawa, 1992
; DeMarini et al., 1997
; Holthuis et al., 1998
; Santos & Snyder, 1997
; Santos et al., 1997
; Trilla et al., 1997
; Ziman et al., 1998
). CHS4 encodes a protein that directs the correct localization of Chs3p at the mother-bud neck by binding Bni4p as well as one of the septins, Cdc10p (DeMarini et al., 1997
). Chs5p, Myo2p and Tig1p play important roles in the polarized distribution of Chs3p (Holthuis et al., 1998
; Santos & Snyder, 1997
), and Chs6p mediates the translocation of Chs3p from the chitosome to the cell surface (Ziman et al., 1998
). Among these genes, mutation in either CHS4 or CHS5 decreases Chs3p activity and cell-wall chitin content (Bulawa, 1992
; Santos et al., 1997
; Trilla et al., 1997
).
Previously, we used mutation analyses to demonstrate the residues important for Chs2 activity. Chs2p contains two regions called con1 and con2, which are highly conserved among various chitin synthases, in the middle and at the C-terminus of the protein. Region con1 contains two amino acid sequence motifs, Glu-Asp-Arg and Gln-Arg-Arg-Arg-Trp, and con2 has one motif, His-Asp-X-X-Trp-X-Thr, where X represents variations. Because enzyme activity was severely impaired by mutations of the conserved amino acids in these motifs, they are presumed to contribute to the active site of the enzyme (Nagahashi et al., 1995 ; Yabe et al., 1998
). In fact, mutations of some of these conserved amino acids in Chs3p also diminished the activity and functionality of the enzyme (Cos et al., 1998
).
In contrast to the sequence conservation in the probable active sites among the three yeast chitin synthases, Chs3p activity is regulated by a mechanism different from those for Chs1p and Chs2p. Firstly, overexpression in yeast of CHS1 or CHS2 led to an increase in chitin synthase activity, whereas overexpression of CHS3 alone did not (Bulawa et al., 1986 ; Nagahashi et al., 1995
; Valdivieso et al., 1991
). Introduction into yeast of additional copies of CHS4, however, enhances Chs3p activity and increases the amount of cell-wall chitin (Bulawa 1993
; Trilla et al., 1997
), suggesting that Chs3p activity requires CHS4 as a positive regulator. Secondly, two types of Chs3p activities, trypsin-dependent (zymogenic) and trypsin-sensitive (non-zymogenic), are present in membranes (Choi et al., 1994
), although both Chs1p and Chs2p are known only as zymogens (Cabib et al., 1996
). The zymogenic activity of Chs3p is not detergent-extractable, and Chs3p needs to be in complex with the substrate during activation by trypsin (Choi et al., 1994
). Zymogenic Chs3p activity was detected only after the membranes were treated with detergents, and a certain level of detergent-extractable non-zymogenic Chs3p activity might be expected to be present in the membranes (Choi et al., 1994
). Such non-zymogenic Chs3p activity, however, has never been recovered and confirmed.
In this paper we show that the non-zymogenic activity of Chs3p can be extracted with CHAPS and cholesteryl hemisuccinate from membranes prepared from cells overexpressing CHS4, and that the association of Chs3p and Chs4p is important for non-zymogenic Chs3p activity. In addition, the region of Chs4p required for the activity of, and binding to, Chs3p is demonstrated by truncation of CHS4 and yeast two-hybrid analysis.
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METHODS |
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To examine the functionality of CHS4, the haploid S. cerevisiae strain YMS1 (MATa ade2 trp1 leu2 ura3 his3 chs4::ADE2), in which the 4·2 kb AflIINheI region of the chromosomal CHS4 was replaced by ADE2, was transformed by pYES-CHS4 carrying the wild-type or a truncated CHS4 and selected by uracil auxotrophy in the presence of galactose. The resulting Ura+ transformants were collected and spread on plates containing galactose and 2 mg Calcofluor white ml-1.
Membrane preparation and chitin synthase assay.
Total membranes were prepared from yeast cells as described previously (Nagahashi et al., 1995 ). To extract Chs3p, 20 mg protein from total membranes was suspended in 2 ml of a buffer containing 20 mM Tris/HCl (pH 7·6), 160 mM NaCl, 20 µM GTP-
S, 5 mM dithiothreitol, 160 mM NaCl, 33% (v/v) glycerol, 0·5% (v/v) CHAPS, and 0·1% (w/v) cholesteryl hemisuccinate, and left on ice for 30 min. After centrifugation at 200000 g for 30 min, the supernatants were collected and used as the membrane extracts (S100). The precipitates were also suspended in an equal volume of the same buffer and used as the extracted membranes (P100). In some experiments, the total membranes and membrane extracts were incubated with the indicated concentrations of trypsin in the presence or absence of 1 mM UDP-N-acetylglucosamine (UDP-GlcNAc). After incubation at 30 °C for 15 min, trypsin was inactivated by the addition of soybean trypsin inhibitor at a concentration twice that of the trypsin added to the reaction mixture.
Unless otherwise specified, chitin synthase assays were performed in a standard 100 µl reaction mixture containing 30 mM Tris/HCl, pH 7·5, 5 mM MgCl2, 32 mM GlcNAc, 1·0 mM [3H]UDP-GlcNAc (specific activity 10000 d.p.m. nmol-1), the indicated amount of total membranes or membrane extract at 30 °C for 60 min, and acid-insoluble radioactivity was measured using a scintillation counter.
Western blotting.
The indicated amounts of total membranes or membrane extract were suspended in a loading buffer for SDS-PAGE (Laemmli, 1970 ) and heated at 90 °C for 2 min. The proteins were then fractionated by SDS-PAGE (Laemmli, 1970
), transferred electrophoretically to a PVDF membrane (Sambrook et al., 1989
), and reacted with an anti-c-Myc monoclonal antibody (clone 9E10, Oncogene Science) or an anti-Chs3p polyclonal antibody, and then with horseradish-peroxidase conjugated protein A (Amersham). The proteins which hybridized with the anti-c-Myc antibody or the anti-Chs3p antibody were detected by using an ECL-protein detection kit (Amersham). To generate the anti-Chs3p antibody, the Chs3p fragment that encompasses amino acids 255434 was expressed in Escherichia coli as a fusion with glutathione S-transferase (GST). Rabbits were immunized with the purified protein, and the IgG fractions that contained the anti-Chs3p antibody were purified by affinity column chromatography using protein A Sepharose CL-4B (Pharmacia). To remove anti-GST antibodies from the IgG fractions, the purified IgG fractions were repeatedly loaded onto GST columns in which GST was bound to glutathione Sepharose beads, and the flow-through fractions, which contained no detectable anti-GST antibody, were pooled and used as the anti-Chs3p antibody preparation.
Yeast two-hybrid analysis.
The entire ORF of CHS3 was cloned between the EcoRI and BamHI sites of pGAD424, generating a plasmid that would express Chs3p as a fusion protein with the transactivation domain of Gal4p. Regions of the CHS4 ORF encoding truncated Chs4 proteins were also cloned between the BamHI and SalI sites of pGBT9 (Clontech) to express these products as fusion proteins with the DNA-binding domain of Gal4p. The resulting plasmids were then transformed into S. cerevisiae strain HF7c (MATa ura3-52 his3-200 lys2-801 ade2-101 trp1-901 leu2-3,112 gal4-542 gal80-538 LYS2::GAL1HIS3 URA3::(GAL4 17-MERS)3CYC1LACZ), where HIS3 gene expression was dependent on both the DNA-binding and transactivation domains of Gal4p (Feilotter et al., 1994 ). After the transformation of HF7c, the Leu+ Trp+ transformants were collected and tested for their ability to grow in the absence of histidine.
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RESULTS |
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Region of Chs4p that is essential for binding to Chs3p
It has been shown that Chs4p is physically associated with Chs3p and Bni4p and that this interaction is necessary for the recruitment of Chs3p to the plasma membrane (DeMarini et al., 1997 ). Therefore, we wondered whether the association of Chs3p and Chs4p is also important for the stimulation of Chs3p activity. The ability of Chs4p to interact with Chs3p was confirmed by yeast two-hybrid analysis using the truncated CHS4 genes listed in Fig. 3
. As shown in Fig. 5
, the ability of the Chs4p fragments to interact with Chs3p correlated well with their capacity to stimulate Chs3p activity in the YMS120 cells and to confer Calcofluor sensitivity on the YMS1 cells (Fig. 4
). Thus, Chs4(214445)p and Chs4(269445)p, which failed to enhance Chs3p activity and restore Calcofluor-white-sensitive growth, were unable to directly interact with Chs3p (Fig. 5
). In contrast, all of the other fragments of Chs4p which elicited these responses interacted directly with Chs3p. These results strongly support the idea that Chs4p stimulates the non-zymogenic activity of Chs3p by binding with it.
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DISCUSSION |
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Chs4p contains the amino acid sequence motifs characteristic of EF-hand calcium-binding sites and prenylation sites in the middle and at the C-terminus, respectively (Kawamoto et al., 1992 ). Experiments involving the truncation of Chs4p revealed that neither the EF-hand motif nor the prenylation site of Chs4p was essential for Chs3p-binding and stimulation of non-zymogenic Chs3p activity. The real roles of these sequence motifs in Chs4p function remain to be elucidated; they may be required for other roles of Chs4p such as resistance to Kluyveromyces lactis toxin (Kawamoto et al., 1992
; Takita & Castilho-Valvicius, 1993
).
DeMarini et al. (1997) also demonstrated the physical association of Chs3p and Chs4p by yeast two-hybrid analysis. According to their results, the C-terminal 86 amino acids of Chs4p play an important role in binding to Chs3p because Chs4p lacking the C-terminal 86 amino acids interacted only weakly with Chs3p (DeMarini et al., 1997
). It was also demonstrated that the C-terminal region of Chs4p was crucial for the association with Bni4p, which directs the correct localization of Chs3p at the bud neck through interaction with Cdc10p (DeMarini et al., 1997
). In this study, we have shown that the C-terminal 134 amino acids of Chs4p are not essential for Chs3p activity, cellular chitin synthesis or binding to Chs3p. Because we judged association of the Chs4p fragments with Chs3p based on the histidine auxotrophy of transformants, it was difficult to determine the binding affinities of the truncated Chs4 proteins with Chs3p quantitatively. Nevertheless, our results and those of DeMarini et al. (1997)
imply that a weak association of Chs4p with Chs3p is sufficient to elicit Chs3p activity.
The mechanism underlying the stimulation of Chs3p activity by Chs4p remains unclear. One possibility is that Chs4p stabilizes and/or increases the amount of Chs3p, although this is rather unlikely for the following reasons. Firstly, Chs3p is a stable protein with a half-life in excess of 10 h in vivo (Chuang & Schekman, 1996 ). Secondly, overexpression of CHS3 alone increased the amount of Chs3p in membranes, yet Chs3p activity remained very low. Another possibility is that Chs4p affects the conformation of Chs3p and converts an inactive form of Chs3p into an active form. This seems more likely, because the simultaneous overexpression of CHS3 and CHS4 did not significantly increase the chitin synthase activity detected as compared to that of cells overexpressing CHS4 alone (data not shown). Thus, the expression of non-zymogenic Chs3p activity would appear to depend directly on the amount of Chs4p present.
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ACKNOWLEDGEMENTS |
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Received 19 July 1999;
revised 27 October 1999;
accepted 16 November 1999.