Figure 1. Proteasomes are rapidly degraded upon nitrogen but not carbon starvation.

(A, B and C) Measurement of proteaphagy upon nitrogen and/or carbon starvation by monitoring the release of free GFP from the CP and RP proteasome subunit reporters Pre10-GFP and Rpn5-GFP, respectively. Cells expressing PRE10-GFP or RPN5-GFP, and also containing the Δatg7 or Δatg13 mutations (panel B only), were switched from nutrient-rich medium (+N + C) to medium lacking either nitrogen (–N), carbon (–C), or both (–N –C). Total protein extracts from cells collected at the indicated times were assayed for GFP release by immunoblot analysis with anti-GFP antibodies. Open and closed arrowheads locate the GFP fusions and free GFP, respectively. The full gels are shown for the Pre10-GFP reporter, whereas only the regions of the gels containing the GFP fusion and free GFP are shown for the Rpn5-GFP reporter. Immunodetection of histone H3 was used to confirm near equal protein loading. (D) Proteasomes rapidly coalesce into PSG-type puncta soon after carbon starvation. PRE10-GFP or RPN5-GFP cells were examined by confocal fluorescence microscopy immediately before and 1 hr after switching from +N +C medium to –C medium. Scale bar, 2 µm. (E) Proteasomes are deposited into vacuoles upon nitrogen starvation, but form cytoplasmic PSG-type puncta in response to carbon starvation. PRE10-GFP or RPN5-GFP cells were grown on +N +C medium and then switched to +N +C, –N, –C, or –N –C media for 24 hr before imaging by confocal fluorescence microscopy. Scale bar, 2 µm. (F) Quantification of the cellular distribution of proteasomes when grown in +N +C, –N, –C, or –N –C media. Cells were treated and imaged as in panel (E). Each bar represents analysis of at least 200 cells. (G) Aggregation of proteasomes into IPODs, but not PSGs, requires the Hsp42 chaperone. PRE10-GFP cells with or without the Δerg6 and/or Δhsp42 mutations were switched from +N +C medium to either –C medium or +N +C medium containing 80 µM MG132 (+MG132) for 24 hr before imaging as in panel (E). Scale bar, 2 µm. (H) PSGs formed upon carbon starvation are distinct from IPOD puncta. PRE10-GFP cells also expressing the IPOD marker RNQ1-mCherry were switched from +N +C medium to –C medium for 24 hr before imaging as in panel E. Shown are the GFP, mCherry and merged fluorescence images. Scale bar, 1 µm. In panels D, E, G, and H: N, nucleus; V, vacuole; P, PSG; I, IPOD.

Figure 1—figure supplement 1. Carbon starvation activates both bulk and selective autophagy.

(A) Both carbon and nitrogen starvation, and growth at high or low pH, strongly attenuates yeast cell growth. Cells were grown in nutrient-rich (+N +C) medium at pH 6.0 and then switched to either medium lacking nitrogen (–N), carbon (–C), or both (–N –C; left panel), or to medium buffered to pH 3.0, 6.0 or 9.0 and containing 100 μM CCCP (right panel). Cell growth at the indicated times was monitored by measuring culture density at OD₆₀₀. (B) Bulk autophagy is induced upon nitrogen and carbon starvation. Cells expressing Pho8Δ60 were grown for either 0, 4, 8 or 20 hr after a switch from +N +C medium to –N, –C or –N –C media. Cells were assayed for bulk autophagy using the phosphatase activity generated upon vacuolar activation of the Pho8Δ60 reporter. Values were normalized to those obtained at 0 hr. Each bar represents the mean (±SD) of three biological replicates, each comprised of three technical replicates. Asterisks indicate data points that are statistically significantly different to the 0 hr time point (p<0.05). (C) Both carbon and nitrogen starvation induce Atg8-mediated autophagy, as judged by release of free GFP from the GFP-Atg8 reporter. GFP-ATG8 cells were switched from +N +C medium to –N, –C or –N –C media. Total protein extracts from cells collected at the indicated times were assayed for GFP release by immunoblot analysis with anti-GFP antibodies. Open and closed arrowheads locate the GFP-Atg8 fusion and free GFP, respectively. Immunodetection of histone H3 antibodies was used to confirm near equal protein loading. (D) Both carbon and nitrogen starvation activate multiple selective autophagic routes. Cells expressing the GFP-Ape1 (CVT), Om45-GFP (mitophagy), Pex14-GFP (pexophagy), or Rpl25-GFP (ribophagy) reporters were switched from +N +C medium to –N, –C or –N –C media. Total protein extracts from cells collected at the indicated times were assayed for GFP release by immunoblot analysis with anti-GFP antibodies, as in panel (C). Open and closed arrowheads highlight the different GFP fusions and free GFP, respectively.

Figure 1—figure supplement 2. Formation of PSGs occurs rapidly in response to carbon starvation, is independent of the pre-autophagosomal structure (PAS), and is reversible.

(A) Proteasomes rapidly coalesce into PSG-type puncta soon after carbon starvation. PRE10-GFP or RPN5-GFP cells were grown on nutrient-rich (+N +C) medium and then switched to medium lacking carbon (–C) for the indicated periods of time before imaging by confocal fluorescence microscopy. Scale bar, 2 µm. (B) Time course for the changes in the cellular distribution of proteasomes when switched to growth in –C medium. The intracellular distribution of proteasomes was quantified from cells treated and imaged as in panel (A). Each bar represents analysis of at least 200 cells. (C) PSGs form upon carbon starvation even in mutants that cannot scaffold the PAS. PRE10-GFP cells with or without the Δatg1, Δatg11, Δatg13 or Δatg17 mutations were grown on nutrient-rich (+N +C) medium and then switched to –C medium for 6 hr before imaging as in panel (A). Scale bar, 2 µm. (D) PSG formation upon carbon starvation is rapidly reversible. PRE10-GFP cells were grown on +N +C medium, switched to –C medium for 6 hr, and then returned to +C medium for 30 min before imaging as in panel (A). Scale bar, 2 µm. (E) PSG formation upon treatment with 2-deoxyglucose (2-DG) is rapidly reversible. PRE10-GFP cells were grown on +N +C medium, switched to +C medium containing 5 mM 2-DG and 2 mM NaN₃ medium for 6 hr, and then returned to +C medium lacking 2-DG and NaN₃ for 1 hr before imaging as in panel (A). Scale bar, 2 µm. In panels A, C, D and E: N, nucleus; V, vacuole, P, PSG.