Variation at Pea2p residue 409 impacts invasive growth, budding, and polarisome interactions.

<p>A) Invasive growth of the wild-type Σ1278b strain, <i>pea2</i>Δ strain, and a strain containing the integrated <i>pea2</i>-M409L point mutation in the Σ1278b background was assayed by plate washing (++, strong invasive growth; +, invasive growth; −, lack of invasive growth). Invasive growth levels were quantified by determining the ratio of pixel intensity of images of the spotted cultures before and after plate washing; these ratios are indicated to the lower right of each spotted culture image post-washing. B) Cell morphology was assessed by determining the length-to-width ratio of 200 cells each for the indicated strains. Prior to imaging, cells grown on YPD medium were scraped into a suspension in liquid media. For length-width calculations, the longest cell axis was selected for cell length measurements. C) Activity of MAPK and PKA signaling pathways in <i>pea2</i> mutants. The bar graphs indicate activity of plasmid-based cAMP-dependent PKA and pseudohyphal growth MAPK pathway-responsive <i>P</i>_flo11-<i>lacZ</i> reporters. The PKA pathway-responsive <i>P</i>_flo11 reporter contains a fragment of the <i>FLO11</i> promoter (<i>P</i>_flo11-6/7) 21.0–21.4 kb from the <i>FLO11</i> start codon, and the Kss1p MAPK-responsive <i>P</i>_flo11 reporter contains a promoter fragment (<i>P</i>_flo11-9/10) 21.6–22.0 kb upstream of the <i>FLO11</i> start codon. β-galactosidase activity was measured from cells growing exponentially in SC-Ura medium. Error bars indicate standard deviations of three independent measurements. D) Budding patterns were analyzed for the indicated strains by calcofluor white staining of invasive cells recovered from the agar. Sample images of the observed budding patterns are presented. Budding patterns of the <i>pea2</i>Δ and <i>pea2</i>-M409L mutants are distinct from wild type, with <i>p</i>-values of <0.01 and <0.001, respectively, by Pearson's chi-squared test. E) Western blots indicate co-immunoprecipitation of Spa2p by Pea2p variants in the Σ1278b background. For this analysis, chromosomal <i>PEA2</i> and <i>pea2</i>-M409L were tagged at the carboxy terminus by integration of a cassette encoding ProA. Spa2p tagged at its amino terminus with the hemagglutinin (HA) epitope was expressed from a plasmid using a copper-inducible promoter. The binding affinity between Pea2p and Spa2p was revealed by IgG pull-down using Pea2p-ProA as bait. Pea2p and Spa2p levels were normalized against levels observed in total cell lysate. The ratio of recovered Spa2p by the S288C-encoded Pea2p-L409 variant is presented relative to the ratio of Spa2p recovered by Σ1278b-encoded Pea2p-M409; thus, the boxed ratios indicate that Pea2p-L409 recovered 60% the amount of Spa2p co-immunoprecipitated with Pea2p-M409.