Figure 7.
Complementation of an atg4 mutant strain with WT or mutant forms of Atg4. (A) atg4 mutant strain was transformed with empty vector (−) or a plasmid encoding Atg4 WT, Atg4C147S, Atg4C338S, or Atg4C394S, to express a HA-tagged version of the corresponding protein under the control of the ATG4 promoter. The wild-type strain (SEY6210) was used as a positive control. Thirty micrograms of total extracts from stationary phase cells grown in SD were resolved by 12% SDS-PAGE followed by western blotting with anti-Ape1, anti-Pgk1, anti-HA and anti-GFP. The precursor (prApe1) and mature (mApe1) forms of Ape1, Pgk1, HA-Atg4, GFP-Atg8 fusion or free GFP proteins are marked with arrowheads. (B) Cells growing exponentially in SD were collected and processed for fluorescence microscopy analysis. The signal corresponds to GFP-Atg8. (C) The percentage of cells from (B) displaying GFP-Atg8 puncta were quantified. The data are represented as mean ± standard deviation from 3 independent experiments. “*,” Differences were significant at P < 0.05 according to the Student t test between atg4 (WT) and atg4 (C338S) or atg4 (C394S). Number of cells quantified: n = 620 for atg4 (−), n = 700 for atg4 (WT), n = 600 for atg4 (C147S), n = 1275 for atg4 (C338S) and n = 1465 for atg4 (C394S).