AN120 (wild-type), AN262 (chs3Δ), and AN264 (dit1Δ) harboring high copy plasmids expressing SPR1-GFP or ssGFP (pRS424-SPR1-GFP or pRS424-ssGFP) were examined by fluorescence microscopy 8 hours or 20 hours after transfer to sporulation medium. Arrows indicate asci displaying fluorescence in the ascal cytoplasm. Bar = 5 µm.

AN120 (wild-type), AN262 (chs3Δ), and AN264 (dit1Δ) harboring a high copy plasmid expressing SPR1-GFP (pRS424-SPR1-GFP) were transferred to sporulation medium and, at the indicated time points after transfer, cells were collected and the percentage of asci displaying Spr1-GFP fluorescence throughout the ascus was determined. More than 200 cells were analyzed at each time point. Values shown are the averages of three independent experiments. The vertical lines indicate the range of the values.

YS28 (fks3Δ) and ER309 (gas2Δ gas4Δ) harboring a high copy plasmid expressing SPR1-GFP (pRS424-SPR1-GFP) were examined by fluorescence microscopy 8 hours or 20 hours after transfer to sporulation medium. Arrows indicate abnormalities in the GFP fluorescence from the spore wall. Bar = 5 µm.

For each mutant identified in the visual screen, the distribution of Spr1-GFP was analyzed by fluorescent microscopy 20 hours after shift to sporulation medium. Data shown are the averages of three experiments. The vertical lines indicate the range of the values.

(A) Analysis of dityrosine fluorescence by patch assay. Each mutant was sporulated on a nitrocellulose filter and dityrosine fluorescence was visualized under UV light. AN120 (wild type) and AN264 (dit1Δ) were analyzed as positive and negative controls, respectively. (B) Quantitation of dityrosine fluorescence in each mutant strain. Each strain was sporulated and dityrosine fluorescence was quantified at the spore periphery by fluorescence microscopy. For each strain, fluorescence was measured in ten different spores. Mean values (in arbitrary units) are shown. Bars indicate standard error.

AN120 (wild type) and the indicated osw mutant strains were sporulated and the survival of the spores assessed after exposure to Zymolyase. Percent survival for each strain was normalized to the average survival of the wild type strain. Data shown are the averages of at least four experiments. The vertical lines indicate one standard deviation.

(A) Schematic of Osw3: SS = signal sequence; INHB = inhibitory pro-domain; PROT = protease catalytic domain. (B) AN120 (wild-type) and AN264 (dit1Δ) carrying a high copy OSW3-GFP plasmid (pRS424-OSW3-GFP) were analyzed by fluorescence microscopy at 5, 8, and 20 hours after transfer to sporulation medium. Arrow indicates fluorescence in the ascal cytoplasm of the dit1Δ mutant. Bar = 5 µm.

(A) YS478 (OSW3), and YS479 (osw3-S365A) were sporulated for 20 h and analyzed by fluorescence microscopy. Bar = 5 µm. (B) Distribution of Spr1-GFP in the strains in (A) was analyzed by fluorescence microscopy after 20 h. Data shown are the averages of three independent experiments. The vertical lines indicate the range of the values. (C) Western blot analysis of AN120 (wild type) carrying high copy plasmids expressing OSW3-3HA or osw3-S365A-3HA (pRS424- P_TEF2-OSW3-3HA or pRS424- P_TEF2-osw3-S365A-3HA). The tagged OSW3 genes were expressed constitutively under the TEF2 promoter and analyzed in vegetative cells. (D) Sporulation time course of AN120 (wild type) carrying high copy plasmids expressing OSW3-3HA or osw3-S365A-3HA under the OSW3 promoter (pRS424-OSW3-3HA or pRS424-osw3-S365A-3HA). Samples were removed at the indicated times and examined with and without PNGase treatment. Subtilisin proteases are often expressed as zymogens that are activated by cleavage of the pro region [28]. Western blot analysis using anti-HA antibodies was used to examine the processing of Osw3-HA. OSW3-HA was expressed from the constitutive TEF2 promoter and the mobility of the protein was analyzed in vegetative cells. Under these conditions the protein migrated as two distinct bands after SDS-PAGE fractionation (Figure 8C). To test whether these two bands were due to different glycosylated forms of the protein, extracts were treated with PNGase F to remove N-linked glycosylations before western analysis. After PNGase treatment, both the upper and lower Osw3-HA bands displayed increased mobility. This result demonstrates that both forms of Osw3-HA are glycosylated and that differential glycosylation does not create the two different mobilities.