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Eukaryotic Cell, October 2004, p. 1297-1306, Vol. 3, No. 5
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.5.1297-1306.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Temperature-Sensitive dcw1 Mutant of Saccharomyces cerevisiae Is Cell Cycle Arrested with Small Buds Which Have Aberrant Cell Walls

Hiroshi Kitagaki, Kiyoshi Ito, and Hitoshi Shimoi*

National Research Institute of Brewing, Higashihiroshima, Hiroshima, Japan

Received 9 December 2003/ Accepted 18 June 2004

Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a {Delta}dcw1 {Delta}dfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 {Delta}dfg5). When DC61 cells were incubated at 37°C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37°C. At 37°C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37°C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37°C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 ({Delta}dcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

* Corresponding author. Mailing address: National Research Institute of Brewing, 3-7-1, Kagamiyama, Higashihiroshima, Hiroshima, Japan 739-0046. Phone: (81)824-20-0826. Fax: (81)824-20-0809. E-mail: simoi{at}nrib.go.jp.

Eukaryotic Cell, October 2004, p. 1297-1306, Vol. 3, No. 5
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.5.1297-1306.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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