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Characterization of temperature-sensitive mutations in the yeast syntaxin 1 homologues Sso1p and Sso2p, and evidence of a distinct function for Sso1p in sporulation

Jussi Jäntti², Markku K. Aalto^1,*, Mattias Öyen¹, Lena Sundqvist², Sirkka Keränen² and Hans Ronne^1,

¹ Department of Plant Biology, Uppsala Genetic Center, Swedish University of Agricultural Sciences, Box 7080, S-750 07 Uppsala, Sweden
² VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Finland
^* Present address: Department of Biosciences, Division of Genetics, Viikki Biocenter 2, PO Box 56, FIN-00014 University of Helsinki, Finland

View larger version (54K): [in a new window]   Fig. 1. Temperature sensitivity of sso1-1 sso2 (H1239) and sso1 sso2-1 (H603) and congenic wild-type (W303-1A) strains. Aliquots corresponding to 6x104 cells and three tenfold dilutions of each strain were spotted onto YPD plates which were incubated for 2 days at the indicated temperatures.

View larger version (38K): [in a new window]   Fig. 2. Secretion of Hsp150 protein in wild-type (W303-1A) and congenic sso1-1 sso2 (H1239) and sso1 sso2-1 (H603) cells. Yeast cells were grown at 24°C until they reached an A600 of 0.3. The cells were then shifted to either 24, 37 or 38°C. Samples were collected after 15, 30, 60, 120 and 240 minutes. Cells were removed by centrifugation and the amount of Hsp150p in a 10 µl aliquot of the growth medium was quantified by western blotting.

View larger version (87K): [in a new window]   Fig. 3. Electron micrographs of the sso1-1 sso2 strain H1239 at permissive and restrictive temperatures. The cells were grown in YPD at 24°C to an A600 of 0.3. The cell culture was then split into subcultures grown either at 24°C or 38°C. The cells were fixed after 3 hours of growth and samples were processed for electron microscopy. (a) The sso1-1 sso2 cells accumulate transport vesicles at 38°C. (b) They also have problems with completing cytokinesis and frequently accumulate vesicles at the septum. (c) Cells grown at 24°C do not accumulate vesicles or display incomplete septum formation, but occasionally have wider bud necks than wild-type cells. Bars, 500 nm (a); 200 nm (b); 1 µm (c).

View larger version (65K): [in a new window]   Fig. 4. Electron micrographs of sso1 sso2-1 (H902) and congenic wild-type (NY179) cells at different temperatures. The cells were first grown in YPD at 24°C until an A600 of 1.0. An aliquot of each culture was then incubated at 37°C for 3 hours. (a) sso1 sso2-1 cells at 37°C; (b) wild-type cells at 24°C; (c) wild-type cells at 37°C. The arrow in panel a points to the incomplete septum that can frequently be found in sso1 sso2-1 cells at the restrictive temperature. Bars, 1 µm (a,b); 500 nm (c).

View larger version (124K): [in a new window]   Fig. 5. Electron micrographs of sso1 sso2-1 cells (H902) grown at the permissive temperature (24°C). The panels were chosen to illustrate the following morphological defects: (a) incomplete cell separation; (b) accumulation of vesicles in the bud and unusually wide bud neck; (c-e) incomplete septum formation and accumulation of vesicles in the bud neck. The arrows point to the relevant structures. Bars, 1 µm (a,b,c); 200 nm (d,e).

View larger version (40K): [in a new window]   Fig. 6. (A) Characterisation of the anti-Sso1p and anti-Sso2p antisera. Cell lysates from wild-type (W303-1A), sso1 (H403) and sso2 (H404) cells (10 µg of total protein) and 15 ng of purified Sso1-GST and Sso2-GST fusion proteins were analysed by western blotting using the specific antisera against Sso1p or Sso2p. Molecular weight markers are shown on the right. (B) Expression of wild-type and mutant Sso1p and Sso2p at permissive and restrictive temperatures. Wild-type (W303-1A), sso1-1 sso2 (H1239), and sso1 sso2-1 (H603) cells grown at 24°C were shifted to either 24°C or 38°C. Samples were taken at 0, 15, 30, 60 and 120 minutes after the temperature shift. Cell lysates were prepared and 20 µg of total protein was analysed by western blotting using the specific antisera against Sso1p or Sso2p.

View larger version (110K): [in a new window]   Fig. 7. Suppression of the temperature-sensitive sso2-1 mutation by overexpression of other genes that are involved in secretion. H902 cells (sso1 sso2-1) containing different SEC genes on high copy number plasmids from the genomic pHR81 library were grown at 24°C, and then replicated to plates that were incubated at different temperatures, as shown in the figure. The vector control is pHR81 (Nehlin et al., 1989). Plasmid pMO9 carrying the SSO2 gene on a single copy plasmid was included as a control (SSO2 CEN in the figure).

View larger version (116K): [in a new window]   Fig. 8. Synthetic interactions between sso1, sso2 and mso1 mutations. W303 congenic strains of the indicated genotypes (Table 1) were patched onto a YPD plate and grown at room temperature. The plate was then replicated to fresh plates that were incubated at the indicated temperatures.

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