Clg1p and Pef1p interact and regulate CLS through the same pathway.

<p>(A) The two-hybrid assay of Clg1p and Pef1p. Cells expressing both Gal4 DNA binding domain (DBD)- and Gal4 activation domain (AD)-fusion proteins were analyzed as described in Materials and Methods. Blue color is indicative of a positive interaction whereas white color means no detectable protein interaction. Clg1(N) p is the N-terminal fragment predicted to be made in the <i>clg1</i> insertion mutation, and encodes the first 590 bps of the <i>clg1</i>⁺ ORF. (B) Clg1p co-immunoprecipitates with Pef1p in <i>S. pombe</i> cells. Lysates prepared from cells expressing Pef1p-3HA and FLAG-Clg1p, or Pef1p-3HA alone, were immunoprecipitated with anti-FLAG antibody. The precipitated complexes and 100 µg of input lysate were analyzed by SDS-PAGE and Western blotting with anti-HA or anti-FLAG antibodies. (C) Deletion of <i>pef1</i>^<i>+</i> extends CLS in the same pathway as <i>clg1</i>^<i>+</i>. The CLS of the <i>pef1</i>Δ single deletion mutant and the <i>clg1</i>Δ <i>pef1</i>Δ double deletion mutant were determined in SD + 3% glucose medium along side the wild type and <i>clg1</i>Δ mutant strain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069084#pone-0069084-g002" target="_blank">Figure 2C</a>). The lifespans of the two <i>pef1∆</i> mutants were longer than that of the wild type (p = 0.0045 for <i>pef1</i>Δ, p = 0.0029 for <i>clg1</i>Δ <i>pef1</i>Δ). The lifespan curve of the <i>clg1</i>Δ <i>pef1</i>Δ double deletion mutant was indistinguishable from the <i>pef1</i>Δ single deletion mutant, and the <i>clg1</i>Δ <i>pef1</i>Δ curve significantly overlapped with the <i>clg1</i>Δ mutant (p > 0.1 for all comparisons). Survival curves comparing each individual mutant with the wild type with error bars are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069084#pone.0069084.s005" target="_blank">Figure S5</a>.