Correction: Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast
Fig 2
C2 domain of Inn1 directly binds to and regulates the catalytic activity of Chs2.
(A) INN1-TAP CHS2-9MYC (YMF38), C2-TAP CHS2-9MYC (YMF88) and control (YAD382) strains were grown at 24°C in YPD medium, arrested in G1 phase by the addition of alpha factor, and then released in YPD medium for 105 minutes. Cell extracts were made and Inn1-TAP or C2-TAP were immunoprecipitated on IgG beads before detection of the indicated proteins by immunoblotting (i). After induction with IPTG, pairs ofE. coli cultures expressing 6His-tagged-Inn1-C2 and Strep-tag-Chs2-215-629 were mixed and used to purify putative protein complexes following scheme in S1A (see Materials and Methods). The final purified fractions were analysed by SDS-PAGE and tagged proteins were detected with anti-Streptag or anti-His antibodies (ii). Pairs of E. coli cultures expressing 6His-tagged-Inn1-C2 or 6His-tagged-Inn1-C2-K31A were mixed with Strep-tag-Chs2-215-629 and used to purify putative protein complexes as in (ii). The final purified fractions were analysed by SDS-PAGE and tagged proteins were detected with anti-Streptag or anti-His antibodies (iii). (B) chs3Δ control (YMF505) and GAL-C2 chs3Δ (YRK3) cells were grown in YPRaff medium at 24°C and synchronised in G1 with alpha factor. Subsequently, cells were released in YPGal for 135 minutes from G1 block in the presence of calcofluor to visualise primary septum deposition. 100 cells with primary septum for each sample were examined and we found that 15% of GAL-C2 chs3Δ cells had clearly higher intensity at the primary septum than the average intensity in control cells. Examples of these cells are shown in (i). Scale bars correspond to 2μm. The relative signal intensity of primary septum was measured for 100 cells and compared to control cells, where signal intensity was set to 100% (ii). (C) C2 domain of Inn1 increases the catalytic activity of Chs2. The protein levels of overexpressed Chs2 and C2 proteins (i) and percentage of active chitin synthase (ii) in chs3Δ control cells and cells lacking Chs3 and overexpressing either GAL-CHS2 (YMF687) or GAL-CHS2 GAL-C2 (YMF581) were determined in membranes isolated from asynchronous cultures (see Materials and Methods). Control, GAL-CHS2 (YMF687) and GAL-CHS2 GAL-C2 (YMF581) were grown as in (B) and stained with calcofluor to visualise primary septum deposition. 100 cells with primary septum for each sample were examined and examples of these cells are shown in (iii) and the relative signal intensity of primary septum was measured and compared to control cells, where signal intensity was set to 100% (iv). Scale bars correspond to 2μm. (D) The chitin synthase activity in chs3Δ cells expressing CHS2 (YMF191), C2-CHS2 (YMF172), C2-K31A-CHS2 (YMF174) orCHS2-V377I (YMF192) was determined as in (C) (i). Cells were grown in YPD containing 0.1mM CuSO4 since CHS2 and CHS2 fusions were under the control of the CUP1 promoter and protein expression levels of Chs2 and its fusions were determined (ii). Note that CHS2 is highly expressed in (C), under the control of the GAL1-10 promoter, whereas CHS2 levels are much reduced in (D), under the CUP1 promoter control.