The Osw3 active site serine is essential for function.

<p>(A) YS478 (<i>OSW3</i>), and YS479 (<i>osw3-S365A</i>) 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 <i>OSW3-3HA</i> or <i>osw3-S365A-3HA</i> (pRS424- P_TEF2-OSW3-3HA or pRS424- P_TEF2-osw3-S365A-3HA). The tagged <i>OSW3</i> genes were expressed constitutively under the <i>TEF2</i> promoter and analyzed in vegetative cells. (D) Sporulation time course of AN120 (wild type) carrying high copy plasmids expressing <i>OSW3-3HA</i> or <i>osw3-S365A-3HA</i> under the <i>OSW3</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007184#pone.0007184-VandeVen1" target="_blank">[28]</a>. Western blot analysis using anti-HA antibodies was used to examine the processing of Osw3-HA. <i>OSW3-HA</i> was expressed from the constitutive <i>TEF2</i> 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007184#pone-0007184-g008" target="_blank">Figure 8C</a>). 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.