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. 2012 May 1;22(7):1155–1168. doi: 10.1038/cr.2012.71

Figure 4.

Figure 4

S-acylation of three cysteins in the N-terminus directs vacuolar membrane targeting of CBL2. (A) Different versions of CBL2, which contain either single cysteine to serine exchanges (C4S, C12S, C18S), two cysteine to serine exchanges (C12, 18S), or three cysteine to serine exchanges (C4,12,18S) were generated. HA tagged versions were transiently expressed in N. benthamiana leaves for 2 days. Native proteins were extracted and sub-cellularly fractionated by a 1 h 100 000× g centrifugation step. The soluble (S) and pellet (P) protein fractions were analyzed by western blotting as described in the Materials and Methods section. Mock infiltrated leaves (expressing a GFP protein) were used to prepare a control sample (C). (B) Different versions of CBL2, which contain either single cysteine to serine exchanges (C4S, C12S, C18S), two cysteine to serine exchanges (C12,18S), or three cysteine to serine exchanges (C4,12,18S) were fused with GFP. Proteins were transiently expressed for 2 days in N. benthamiana leaves together with a soluble cytosolic OFP marker protein, and analyzed microscopically. Bars in the merged pictures represent 20 μm. (C) Different mutated versions of the CBL2 N-terminal peptide fused to GFP (CBL2nC4S and CBL2nC4,12,18S) transiently expressed in N. benthamiana leaves, together with the cytosolic OFP control protein. Epidermal cells were microscopically analyzed. Bars in the merged pictures represent 20 μm.