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. 2011 Apr 1;7(4):375–385. doi: 10.4161/auto.7.4.14369

Figure 4.

Figure 4

Overexpression of Atg35 inhibits MIPA formation. (A and B) Overexpression of Atg35, but not of Atg17, specifically inhibits micropexophagy. The cells from WT (PPY12 h) and WT overexpressing Atg35-eYFP (STN274), eCFP-Atg17 (STN350), or both Atg35-eYFP and eCFP-Atg17 (STN322), were grown overnight in SM and transferred to (A) SD or (B) SE. At the indicated time-points, culture samples were collected and processed for immunoblotting for AOX. (C) Overexpression of Atg35 specifically affects MIPA formation. The WT strain overexpressing Atg35-eYFP and expressing the endogenous levels of mCherry-Atg8 (STN338) was grown overnight in SM and transferred to SD or SE for 1 h. VSM, MIPA and pexophagosomes were labeled with mCherry-Atg8. Arrows point to pexophagosomes. Bar, 5 µm. (D and E) Overexpression of Atg35 does not affect general autophagy, but delays the Cvt pathway under vegetative, but not starvation, conditions. (D) GFP-Atg8 processing and (E) prApe1 maturation assays. The WT (STN70), WT overexpressing Atg35-eYFP (SJCF1376) and atg1Δ (STN66) cells, all expressing the endogenous levels of GFP-Atg8, were grown overnight in SD and transferred to SD-N. At the indicated time-points, culture samples were collected and processed for immunoblotting for (D) GFP and (E) Ape1. *, nonspecific band; red box, maturation of prApe1 under vegetative conditions.