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Supporting Information

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Figure S1: Pob1 function is required for polarized elongation of growing S. pombe cells. Differential interference contrast images of typical phenotypes of the indicated strains are shown. pob1-664 mutant cells have a lemon-like shape at the restrictive temperature, perhaps caused by defective growth while the polarity cue remains functional. WT, wild type.

Figure S2: Cortical localization of Pob1. A) Pob1Y719C is detached from the cell cortex upon high temperature shift. Cells expressing red fluorescent protein mCherry-fused Pob1 and Pob1Y719C instead of Pob1 growing exponentially in YEA at 25°C were shifted to 36°C for the indicated time (min). Wild-type protein (left column) partially strayed away from cell ends to other cortical area upon temperature shift from 25°C to 36°C (arrowheads). This perturbation of Pob1-mCherry localization was repaired within 50 min. On the other hand, mutant protein Pob1Y719C-mCherry, displaying fainter signal of cortical localization even at 25°C, was detached from the cell cortex upon the temperature shift (right column). In additional incubation, polarized localization of Pob1Y719C-mCherry was never returned, and many spots of its fluorescence signal were detected in a cytoplasm (small arrows). Size and intensity of spots were gradually increased (medium arrows). Finally, signal was detected in a vacuole-like structure after around 140 min (large arrows). B) Localization of Pob1 in Δfor3 and Δrho3 null cells. Strains expressing Pob1-mCherry growing exponentially in YEA at 25°C were shifted to 36°C for the indicated time. C) Localization of Pob1 is maintained after Lat-A treatment. Fluorescence microscopy images of F-actin and Pob1-HA are shown. Wild-type cells expressing the triple HA-tagged protein and growing exponentially in YEA at 25°C were treated with 10 µM Lat-A for 10 min. The cells were fixed and stained with anti-HA to detect Pob1 (right) and with Bodipy-phallacidin to detect F-actin (left). Bars: 10 µm.

Figure S3: Pob1 genetically and physically interacts with Cdc42. A) Two-hybrid assay between Pob1 and Rho3. A two-hybrid experiment was carried out using the indicated combination of genes in Y190 strain. Colony formation on a plate lacking histidine, in the presence of 20 mM 3-aminotriazole for suppressing false-positive growth, was diagnostic of a positive interaction between the proteins indicated to the bottom. Cdc42 is used as a positive control (31). Unlike Cdc42, Rho3 seems not to strongly bind to Pob1. B) Cdc42 may also function with Pob1. Overexpression of either Rho3 or Cdc42 can compensate for the growth defect of pob1-664 cells. JX584 was transformed with the empty vector (pREP41), pR81pob1, pR41rho1, pR41rho2, pR41rho3, pR41rho4, pR41rho5 or pR1cdc42. The transformed cells were then streaked onto EMM plates and incubated at the indicated temperature for 5 days.

Figure S4: Pob1 controls the subcellular localization of For3. A) Delocalization of For3 in pob1-664 mutant cells. The expression of YFP-For3 is shown in wild-type (WT) and pob1-664 cells. Bar: 5 µm. B) Pob1 binds to the N-terminus of For3. A two-hybrid experiment was carried out using the indicated combination of gene fragments in HF7c. Colony formation on a plate lacking histidine was diagnostic of a positive interaction between the proteins indicated to the right.

Figure S5: Pob1 functionally interacts with exocyst. A) Genetic interaction between Pob1 and Exo70. The indicated cells were streaked onto YEA plates and incubated at 25°C or 30°C for 4 days. B) Mislocalization of the exocyst complex in pob1-664 mutant. Indicated GFP-fused proteins were expressed in wild-type cells and pob1-664 cells. Cells growing at 25°C in mid-log phase were incubated at 36°C for 2 h and then observed. Bar: 10 µm.

Table S1: List of strains used in this study.

Table S2: Plasmids used in this study.

FilenameFormatSizeDescription
TRA_1190_sm_FigS1.tif786KSupporting info item
TRA_1190_sm_FigS2.tif759KSupporting info item
TRA_1190_sm_FigS3.tif619KSupporting info item
TRA_1190_sm_FigS4.tif964KSupporting info item
TRA_1190_sm_FigS5.tif880KSupporting info item
TRA_1190_sm_TableS1.doc71KSupporting info item
TRA_1190_sm_TableS2.doc46KSupporting info item

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