Fig. 3.

Localization and chitin synthase activity of phosphomutant forms of Chs2. A–P. Yeast cells of the chs2^S222A-YFP (A–D) and chs2^S222E-YFP strain (I–L) were grown in YPD+uri at 30 °C for 4 h, and hyphae of the chs2^S222A-YFP (E–H) and chs2^S222E-YFP strain (M–P) were grown on 20% FCS agar pads with uridine at 37 °C for at least 2 h before imaging. Chs2^S222A-YFP and Chs2^S222E-YFP was observed at septation sites in yeast cells as a bar (A and B, I and J) and as a spot (C and D, K and L). In hyphae, Chs2^S222A-YFP and Chs2^S222E-YFP were observed as two spots at the middle of septa (E–H, M–P). Chs2^S222E-YFP but not Chs2^S222A-YFP was observed at hyphal tips (O and P, G and H). Scale bars are 2 μm. Q. Chitin synthase activity of membrane preparations from yeast and hyphal forms of the wild-type (BWP17), CHS2/chs2Δ0 heterozygous mutant, chs2Δ0 null mutant, and chs2^S222A and chs2^S222E phosphomutant strains. Measurements were made following trypsin treatment of the membrane preparations for two independent biological replicates measured in triplicate (n = 6). Error bars are SD. R. The average fluorescence intensity of YFP spots at septa of yeast and hyphae quantified using ImageJ. Error bars are SEM. n = 50–80 spots for each measurement. ^∗ P < 0.05 by ANOVA.