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. 2014 Jun 16;24(12):1314-1322.
doi: 10.1016/j.cub.2014.04.048. Epub 2014 May 29.

Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation

Meiyan Jin  1 , Ding He  1 , Steven K Backues  1 , Mallory A Freeberg  2 , Xu Liu  1 , John K Kim  3 , Daniel J Klionsky  4
Affiliations

Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation

Meiyan Jin et al. Curr Biol. .

Abstract

Background: Autophagy as a conserved lysosomal/vacuolar degradation and recycling pathway is important in normal development and physiology, and defects in this process are linked to many kinds of disease. Because too much or too little autophagy can be detrimental, the process must be tightly regulated both temporally and in magnitude. Two parameters that affect this regulation are the size and the number of autophagosomes; however, although we know that the amount of Atg8 affects the size of autophagosomes, the mechanism for regulating their number has not been elucidated. The transcriptional induction and repression of the autophagy-related (ATG) genes is one crucial aspect of autophagy regulation, but the transcriptional regulators that modulate autophagy are not well characterized.

Results: We detected increased expression levels of ATG genes, and elevated autophagy activity, in cells lacking the transcriptional regulator Pho23. Using transmission electron microscopy, we found that PHO23 null mutant cells contain significantly more autophagosomes than the wild-type. By RNA sequencing transcriptome profiling, we identified ATG9 as one of the key targets of Pho23, and our studies with strains expressing modulated levels of Atg9 show that the amount of this protein directly correlates with the frequency of autophagosome formation and the level of autophagy activity.

Conclusions: Our results identified Pho23 as a master transcriptional repressor for autophagy that regulates the frequency of autophagosome formation through its negative regulation of ATG9.

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Figures

Figure 1
Figure 1. Pho23 Represses the Transcription of Several ATG Genes When Autophagy Is Suppressed
(A) Protein extracts were generated from wild-type and pho23Δ strains in the indicated backgrounds after growth in YPD to mid-log phase (growing conditions) and then shifted to SD-N medium (nitrogen starvation). Proteins were resolved by SDS-PAGE, then detected by western blot with anti-Atg8 and anti-Pgk1 (loading control) antisera. The Atg8 protein level was increased in growing conditions in pho23Δ cells relative to the wildtype in all three strain backgrounds. (B) The ratio of pho23Δ to wild-type mRNA levels of the indicated ATG genes was measured by qRT-PCR. RNA extracts were prepared from wild-type (SEY6210) and pho23Δ (JMY047) cells after growth in YPD to mid-log phase. The error bars represent the SEM of at least three independent experiments. Two-tailed t test was used for statistical significance; *p < 0.05, **p < 0.01. (C) Protein extracts were prepared as in (A) from wild-type and pho23Δ strains in growing conditions. The indicated proteins were detected by western blot using antisera to the endogenous proteins or an antibody that detects the protein A (PA) tag. Pgk1 was used as a loading control. See also Figure S1 and Table S1.
Figure 2
Figure 2. Pho23 Negatively Regulates Autophagy Activity
(A) Wild-type (BY4742) and pho23Δ (JMY018) cells with a centromeric plasmid expressing CUP1 promoter-driven GFP-ATG8 were grown to mid-log phase inSMD-Ura and shifted to SD-N for the indicated times. Autophagy activity was measured by the GFP-Atg8 processing assay. (B) Wild-type (WLY176) and pho23Δ (JMY048) cells were grown to mid-log phase in YPD and shifted to SD-N for the indicated times of nitrogen starvation, and autophagy activity was monitored by the Pho8Δ60 assay. Pho8Δ60 activity was normalized to the wild-type strain (set to 100%) after 3 hr of nitrogen starvation. The graph shows the average activity from three different experiments. Error bars represent the SEM. Two-tailed paired t test was used for statistical significance; *p < 0.05, **p < 0.01. (C) Wild-type (SEY6210) and pho23Δ (JMY047) cells were grown overnight, diluted to 0.1 optical density 600 (OD600), grown to mid-log phase (0.6 OD600) in YPD, and shifted to SD-N for 30 min of nitrogen starvation. The precursor (pr) and mature (m) forms of Ape1 were separated by SDS-PAGE and detected with anti-Ape1 antiserum by western blotting. Pgk1 was detected with anti-Pgk1 antiserum as a loading control. (D) Precursor Ape1 processing in wild-type (vac8Δ; CWY230) and pho23Δ (pho23Δ vac8Δ; JMY146) cells at 0, 30, and 60 min after nitrogen starvation was detected by western blotting as in (C).
Figure 3
Figure 3. pho23Δ Cells Have an Increased Frequency of Autophagosome Formation
(A) Representative TEM images of wild-type (pep4Δ vps4Δ; FRY143) and pho23Δ (pep4Δ pho23Δ vps4Δ; JMY050) cells after 3 hr of nitrogen starvation. More autophagic bodies accumulated in the vacuole of pho23Δ cells. Scale bar, 500 nm. (B) Estimated average number of autophagic body numbers per cell in wild-type and pho23Δ strains after 3 hr of nitrogen starvation. Estimation was based on the number of autophagic body cross sections observed by TEM in two independent experimental sets of more than 100 cells each for each strain. Two-tailed t test was used for statistical significance; *p < 0.05. (C) Representative images of GFP-Atg8 in wildtype (MZY089) and pho23Δ (SKB233) cells. Brackets indicate the lifetime of each punctum. Scale bar, 2 μm. (D) Distribution of GFP-Atg8 puncta lifetimes. Cells were imaged for 45 min beginning 40 min after a shift to nitrogen starvation. The lifetime of each individual punctum was determined as the time from when the punctum first appeared and began brightening to the time when it either disappeared or began a second round of brightening (indicating a second round of autophagosome formation from the same phagophore assembly site). N > 300 puncta from >65 cells per condition. Mean lifetime numbers show the SEM. See also Table S2.
Figure 4
Figure 4. pho23Δ Cells Maintain Higher ATG9 Expression Levels Relative to the Wild-Type after Autophagy Is Activated
(A and B) Gene RPKM (reads per kilobase per million mapped reads) values under growing (A) or 2 hr nitrogen starvation (B) conditions are shown for the wild-type (SEY6210) versus the pho23Δ (JMY047) strains. Genes with expression changes more than 2-fold in pho23Δ cells are highlighted in red, while ATG genes are indicated by gray-outlined yellow circles and ATG9 is indicated by a blue triangle. (C) The ratio of the gene RPKM of the pho23Δ strain to the wild-type is calculated for the top hits among the ATG genes. (D) Atg1 and Atg9 protein levels in wild-type (TVY1) and pho23Δ (JMY020) cells in the pep4Δ background after 0, 0.5, 1, and 2 hr nitrogen starvation were analyzed by western blot. Pgk1 was used as a loading control. See also Tables S3 and S4.
Figure 5
Figure 5. The Atg9 Protein Level Correlates with Autophagosome Formation Frequency and Autophagy Activity
(A) Four strains expressing different levels of Atg9-GFP (A9Δ, the null control strain; A9Lo, expression controlled by the ATG23 promoter and thus lower than WT; A9WT, controlled by the endogenous promoter; and A9OE, expression controlled by the ATG8 promoter in addition to the endogenous copy of ATG9 and thus higher than WT) were grown in YPD to mid-log phase and shifted to SD-N for 3 hr. Proteins were resolved by SDS-PAGE and detected by western blot with anti-Atg9 and anti-Pgk1 (loading control) antisera. Atg9 intensity was measured in ImageJ and represents an average of three independent experiments. (B) Cell lysates were generated as in (A) and autophagy activity was monitored by the Pho8Δ60 assay. Pho8Δ60 activity was normalized to the A9WT strain (set to 100%) after 3 hr starvation. The graph shows the average activity from at least four different experiments. Error bars represent the SEM. (C) Representative TEM images of the four A9 strains after 3 hr starvation. Scale bar, 500 nm. (D) Estimated average number of autophagic bodies per cell in the four A9 strains after 3 hr of nitrogen starvation. Estimation was based on the number of autophagic body cross sections observed by TEM in two independent experimental sets of more than 190 cells each for each strain. (E) Atg9 protein level detected by western blot, average autophagic body number per cell estimated by TEM, and starvation-induced Pho8Δ60 activity (the average Pho8Δ60 activity in growing condition was subtracted) of the four different A9 strains after 3 hr starvation were plotted on one graph for comparison. See also Table S5.
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