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. 2012 Oct 3;7(10):e46386. doi: 10.1371/journal.pone.0046386

Figure 5. Mutations of D19 and E21 result in intracellular retention of Chs3p.

Figure 5

(A) Ten-fold serial dilutions of chs3Δ (RSY1699) or chs3Δ AP-1Δ (SPY21) cells expressing N-terminal deletion mutants of CHS3, with and without the W391R substitution, were spotted onto synthetic complete medium -Ura plates +100 ug/ml calcofluor (-Ura + CF). (B) Ten-fold serial dilutions of chs3Δ AP-1Δ cells expressing alanine substitution mutants from residue 16 to residue 25 of Chs3-W391R were spotted onto - Ura + CF. (C) Subcellular fractionation of Δ15Chs3-W391R, Δ25Chs3-W391R and Chs3-D19AE21AW391R expressed in chs3Δ AP-1Δ cells on step sucrose/EDTA gradients. Total membranes from spheroplasts were separated on a step sucrose/EDTA gradient. The protein composition of fractions obtained from the sucrose gradients was analyzed by SDS/PAGE and immunoblotting (PM marker: Pma1p; Golgi/EE markers: Tlg1p).