Cell wall morphology of pan1-4, end3Δ, and sla1Δ mutants. Electron microscopy analysis of the cell wall ultrastructure in YMC437 (sla1Δ; A), YHT167 (end3Δ; B), and YHT99 (pan1-4; C) cells grown in YPD liquid medium at 24°C. (D) Electron micrograph showing the damaged cell wall of strain YHT99 after 3 h of incubation at 37°C. Such a phenotype was also observed in sla1Δ and end3Δ mutants at 37°C (data not shown). Bars, 500 nm.

Cell wall defects in various act1 mutants. (A) Calcofluor white sensitivity. Strains CRY1 (wild type [WT]), YHT99 (pan1-4), YHT167 (end3Δ), YMC437 (sla1Δ), DDY335 (act1-1), DDY338 (act1-101), DDY342 (act1-113), DDY346 (act1-119), and DDY349 (act1-124) were patched onto a YPD plate as indicated and then replica plated onto a YPD plate supplemented with calcofluor white (CFW); 1 mg/ml). Both plates were incubated at 24°C. (B) Electron micrographs of act1-101 (left) and act1-124 (right) mutants grown in YPD liquid medium at 24°C. The middle panel shows the act1-101 mutant after 2 h of incubation at 37°C. The arrow in the right-hand panel indicates a cell with defective septum. Bars, 500 nm. (C) Fluorescence micrographs of act1-101 (left) and act1-124 (right) mutants grown in YPD liquid medium at 24°C and stained with rhodamine-phalloidin. The middle panel shows the actin distribution of the act1-101 mutant after 2 h of incubation at 37°C. Bar, 5 μm.

Genetic interactions between PAN1, END3, and SLA1. (A) The end3Δ sla1Δ heterozygous diploid, YMC439, was sporulated, dissected, and grown for 5 days at 24°C and 30°C. Letters to the right of each panel indicate the identity of each colony determined from the segregation of the disruption markers: w, wild type; e, end3Δ; s, sla1Δ; d, end3Δ sla1Δ. (B) Suppression of the pan1-4 sla1Δ synthetic lethality. YMC438 (pan1-4 sla1Δ, pRS316-PAN1) cells transformed with pRS314 (a), pRS314-SLA1 (b), pRS424-END3 (c), and pRS314-PAN1 (d), were patched onto an SC-Trp plate and replica plated onto a 5-fluoro-orotic acid plate. All plates were incubated at 24°C. (C) Effects of SLA1 overexpression in the pan1-4 mutant. YHT99 (pan1-4) cells doubly transformed with pRS425 plus pRS424-SLA1 (a), pRS425-END3 plus pRS424 (b), pRS425-END3 plus pRS424-SLA1 (c), pRS425-END3 plus pRS424-SR (d), pRS425-END3 plus pRS424-SH3 (e), and pRS425-END3 plus pRS424-SLA1ΔSR (f) were patched onto an SC-Leu,-Trp plate at 24°C and replica plated at 37°C. As a control, the wild-type CRY1 was also transformed with pRS425 plus pRS424-SR (g) and analyzed in parallel.

Physical interactions between Pan1p, End3p, and Sla1p. (A) In vivo coimmunoprecipitation of Pan1p-HA, Myc-Sla1p, and End3p. Equal amount of yeast extracts prepared from YMC442 (lanes 2, 4, 6, and 8), YMC443 (lane 5), and YMC444 (lanes 1, 3, and 7) were subjected to anti-HA or anti-Myc immunoprecipitations (IP) as indicated and analyzed, after electrophoresis, by immunoblotting using anti-HA (left), anti-Myc (middle), and anti-End3p (right) antibodies. To detect the directly immunoprecipitated proteins (lanes 1, 2, 5, and 6) 4 μl of each sample was loaded. All other lanes were loaded with 25 μl. (B) Binding of GST-SR fusion protein to HA-Pan1p and End3p. Equal amounts of yeast extracts derived from YMC440 (sla1Δ, pRS316-HA-PAN1; lanes 1 to 3), YMC441 (sla1Δ end3Δ, pRS316-HA-PAN1; lane 4), and YMC440 transformed with pRS424-SLA1 (lanes 5 and 6) were incubated with anti-HA, GST-SH3, or GST-SR as indicated. The precipitates were separated by SDS-PAGE and immunoblotted with anti-HA (top) and anti-End3p (bottom) antibodies to visualize HA-Pan1p and End3p, respectively. The lesser amount of HA-Pan1p observed in lane 4 is due to the slower growth of the YMC441 cells, as less HA-Pan1p was also detected in the cell extract with anti-HA antibody (data not shown). (C) Interaction between Pan1p long repeats and Sla1p C-terminal repeats. CRY1 cells containing pGAL-HA-LR1 (lanes 1 and 4), pGAL-HA-LR2 (lanes 2 and 5), and pGAL-HA-LR2 together with pGAL-END3 (lanes 3 and 6) were grown in galactose-containing liquid medium to early log phase, and extracts were prepared. Equal amounts of the extracts were incubated with anti-HA (lanes 1 to 3) or GST-SR (lanes 4 to 6) as indicated. The precipitates were subjected to SDS-PAGE and immunoblotted with anti-HA to detected the HA-tagged constructs. (D) Formation of the Pan1p-End3p-Sla1p complex. The diagram shows the structural domains of Pan1p, Sla1p, and End3p and the interactions among them. Pan1p LR2 is involved in the interaction with the End3p C-terminal repeats (ER), whereas LR1 of Pan1p and the N-terminal EH domain of End3p bind to the Sla1p C-terminal repeats (SR). Based on this interaction pattern, Pan1p, End3p, and Sla1p are able to exist as a heterotrimeric complex. N, N terminus.

Effects of Sla1p C-terminal repeat overexpression. (A) Wild-type (CRY1) cells transformed with pRS316 (a), pGAL-SR (b), and pGAL-SH3 (c) were patched onto a galactose-containing SC-Ura plate at 24°C and then replica plated to fresh plates, with or without 1 M sorbitol, at 37°C. (B) YHT99 (pan1-4) and YHT167 (end3Δ) cells transformed with pRS316 (a), pGAL-SR (b), and pGAL-SH3 (c) were patched onto a glucose-containing SC-Ura plate and replica plated to a galactose-containing SC-Ura plate. Both plates were incubated at 24°C.

Cell wall morphology of cells overexpressing the Sla1p C-terminal repeats. Wild-type (CRY1) cells, with or without plasmid pGAL-SR, were grown in galactose-containing liquid medium at 24°C and processed for electron microscopy analysis as described in Materials and methods. In cells without pGAL-SR, both the mother and bud, from small budded (A) to large budded (C) and until septation had occurred (E), had a single thin cell wall layer. A slight thickening of the cell wall was observed only at the mother-bud junction. In cells overexpressing the Sla1p C-terminal repeats (B, D, and F), the mother cell displayed an aberrant cell wall that was thicker and consisted of more than one layer. The bud, however, remained as a monolayer through out the cell cycle. Bars, 500 nm.

(A) Fluorescence micrographs of wild-type CRY1 cells containing pRS316 (WT) or pGAL-SR (GAL-SR) grown in galactose-containing liquid medium at 24°C and stained with rhodamine-phalloidin (top). The arrowhead indicates an example of the enlarged cells. The cells were also stained with lucifer yellow and visualized by phase contrast (middle) to highlight the vacuole and with fluorescein isothiocyanate fluorescence optics (bottom) to visualize lucifer yellow. (B) Rhodamine-phalloidin staining of YHT99 (pan1-4) cells and YHT99 cells containing pRS424-END3 incubated for 3 h at 37°C. Bars, 5 μm.