PMC

A network of protein-protein and genetic interactions among glc7-E101Q SSL genes. Genes showing SSL interaction with glc7-E101Q (nodes) were grouped in modules based on their physical interaction profiles (blue edges). The genetic interactions interconnecting modules or connecting modules and single nodes are depicted as red edges.

Biological processes overrepresented in glc7-E101Q SSL genes. Indicated are the frequencies of high-level GO terms represented in the set of SSL genes (closed bars) that were significantly enriched (p < 0.01, **; p < 0.05, *) when compared to those represented in the set of array genes (open bars). The number of genes identified in each GO process is shown in parentheses.

glc7-E101Q mutant is impaired in glucose metabolism. (A) Growth of wild type (GLC7), mutant (glc7-E101Q) and rescue (glc7-E101Q/GLC7-res) strains was examined by spotting assays (5-fold serial dilutions of 1.0 × 10⁶ cells/ml; 5 μl/spot) grown in YPAD or YPAG for 48 hr at 30°C and 37°C. (B) Glycogen content was assessed in wild type (GLC7), mutant (glc7-E101Q) and rescue (GLC7-res) strains by iodine staining. Brown color correlates with glycogen content.

Structure of PP1 catalytic motif and the glc7-E101Q mutant. (A) PP1 protein phosphatases have highly conserved catalytic domains, consisting of residues acting as an acidic catalyst for phosphotransfer (CD1 and CD2) from the substrate and a phospho acceptor hisidine residue (CD3). Residues in the catalytic pocket are highly conserved between human (PP1), yeast PPP-type PPases (Glc7 and others) and the bacterial PPase, λ-PPASE. (B) Left panel: The Glc7 catalytic pocket (pink) was modeled using the tertiary structure of human PP1C-γ (PDB Model 1jk7A; blue). Right panels: The E101Q mutation is predicted to inhibit the binding of a metal cation required to accelerate phospho-transfer to H124, but to not alter the shape/size of the catalytic pocket relative to wild type Glc7.

glc7-E101Q mutant exhibits slow growth but no appreciable delay in the cell cycle or chromosome segregation defect. (A) The growth of wild type (GLC7) vs glc7-E101Q strains in rich medium (YPAD) was determined from early log-phase cultures. (B) FACs analysis for DNA content (propidium iodide staining) of log-phase wild type and glc7-E101Q cells. Cell counts are shown plotted against fluorescence intensity (PI). (C) Budding index of wild type and glc7-E101Q mutant strains. (D) Fluorescence microscopy of anaphase cells (mitotic spindle >2.5 μm) wild type and glc7-E101Q cells expressing a chromosome [GFP-LacI (green) and centromeric (CEN15) repeats of the lactose operon (lacO)] and spindle pole body (SPB) [Spc29-CFP (red)] markers. The percentage of cells with chromosomes localized to both SPBs is indicated at the bottom (n = 3, 300 cells/experiment). (E) Test for chromosome segregation using a sectoring assay. Wild type GLC7, glc7-E101Q and ame1-6 strains lacking a functional ADE2 gene (ade2Δ-101) and transformed with a Circle III/SUP11 were plated on to medium lacking adenine (YPD) and scored for appearance of red sectors after 4-day incubation. (F) Spotting assay of glc7-E101Q vs. wild type on DMSO control or benomyl plates (5-fold serial dilutions of 1.0 × 10⁶ cells/ml; 5 μl/spot). Benomyl-sensitive (tub1-1) and resistant (tub2-104) strains were plated as controls.