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. Author manuscript; available in PMC: 2016 Jun 11.
Published in final edited form as: Mol Cell. 2015 May 21;58(6):1053–1066. doi: 10.1016/j.molcel.2015.04.023

Figure 2. 26S Proteasomes Are Delivered to the Vacuole upon Nitrogen Starvation.

Figure 2

(A) Delivery of vesicles containing PAG1-GFP and RPN5a-GFP to the vacuole is blocked in strong autophagy mutants. WT, atg7-2, atg10-1, and nbr1-2 seedlings were switched to N-free medium containing 1 μM ConA for 16 hr. Root cells were examined for autophagic bodies by confocal microscopy. Scale bars, 10 μm.

(B) Time course of free GFP release from PAG1-GFP and RPN5a-GFP during N starvation. WT and atg7-2 seedlings expressing each reporter were starved for the indicated times, and total protein extracts were immunoblotted with anti-GFP antibodies. The GFP fusions and free GFP are indicated by closed and open arrowheads, respectively.

(C) Effect of autophagy mutants on the cleavage of PAG1-GFP and RPN5a-GFP during N starvation. WT and mutant seedlings were either kept on N-rich medium or switched to N-free medium for 16 hr. Immunoblotting was performed as in (B).

(D) Quantification of the free GFP/PAG1-GFP ratio during N starvation by densitometric scans of the immunoblots shown in (B). Bars represent the mean (± SD) of three biological replicates.

(E) N starvation induces the loss of many 26S proteasome components. WT and atg7-2 seedlings were N-starved for the indicated times. Total protein extracts were probed with antibodies against various proteasome subunits.