A sterol-enriched vacuolar microdomain mediates stationary phase lipophagy in budding yeast - PubMed (original) (raw)
A sterol-enriched vacuolar microdomain mediates stationary phase lipophagy in budding yeast
Chao-Wen Wang et al. J Cell Biol. 2014.
Abstract
Stationary phase (stat-phase) is a poorly understood physiological state under which cells arrest proliferation and acquire resistance to multiple stresses. Lipid droplets (LDs), organelles specialized for cellular lipid homeostasis, increase in size and number at the onset of stat-phase. However, little is known about the dynamics of LDs under this condition. In this paper, we reveal the passage of LDs from perinuclear endoplasmic reticulum association to entry into vacuoles during the transition to stat-phase. We show that the process requires the core autophagy machinery and a subset of autophagy-related (Atg) proteins involved in selective autophagy. Notably, the process that we term stat-phase lipophagy is mediated through a sterol-enriched vacuolar microdomain whose formation and integrity directly affect LD translocation. Intriguingly, cells defective in stat-phase lipophagy showed disrupted vacuolar microdomains, implying that LD contents, likely sterol esters, contribute to the maintenance of vacuolar microdomains. Together, we propose a feed-forward loop in which lipophagy stimulates vacuolar microdomain formation, which in turn promotes lipophagy during stat-phase.
© 2014 Wang et al.
Figures
Figure 1.
The dynamics of LDs during transition to stat-phase. (A) Cells expressing the indicated proteins grown in SC medium to log phase, diauxic shift (DS), or the indicated days after log phase (D1–D7) were subjected to fluorescence microscopy. DIC, differential interference contrast. Bar, 5 µm. (B) Quantification of data in A for the indicated localization patterns from three independent experiments was plotted as mean ± SD. (C) The thin-sectioned EM pictures of wild-type cells grown in SC medium to D4. Va, vacuole. Asterisks show LDs. Bars, 0.5 µm. Yellow arrows indicate the LDs in the vacuole lumen. The LDs (n = 96) inside the vacuole lumen with or without outer membranes as depicted were quantified, and the percentage is shown.
Figure 2.
Translocation of LDs into the vacuole lumen is mediated by a microautophagy mechanism. (A) Representative images of LDs and vacuoles in the indicated strains grown to D5. Quantification of localization based on the indicated patterns. Three independent data were plotted as mean ± SD. (B) Summary of A based on their roles in autophagy. (C) Wild type (WT) and various strains as indicated expressing Faa4-GFP were grown in SC medium to the indicated growth conditions. Cells were lysed, and the lysates were analyzed by immunoblotting with the anti-GFP antibody. (D) The comparison of LD diameter in the indicated strains grown to D1 and D5. The data shown are from one experiment (n = 100 for each cell type). *, P < 0.01. (E) Cells expressing the indicated proteins were grown in SC medium to D5 and imaged by fluorescence microscopy. DIC, differential interference contrast. (F) Quantification of data in E for the indicated localization patterns from three independent experiments, which were plotted as mean ± SD. (G) Wild-type cells stained with FM4-64 (vacuole) at DS and BODIPY (LDs) on D3 were subjected to time-lapse fluorescence microscopy. Images were taken every 1 min. Arrowheads denote an LD during its translocation into the vacuole lumen. Bars, 5 µm.
Figure 3.
LDs associate with the sterol-enriched vacuolar microdomain Lo. (A) Wild-type cells stained with FM4-64 (vacuole) and BODIPY (LDs) were imaged by fluorescence microscopy. Images were processed by deconvolution followed by maximal projection. (B) Same as A, except that the images were acquired by a time interval of every 1 min. The yellow and white arrowheads and blue arrows define three independent LDs. (C) Cells expressing Vph1-mCherry were imaged and processed as in A. Representative images of various Vph1-mCherry patterns are shown. The plots show quantification of these patterns under various growth conditions. (left) The data shown are from a single representative experiment (n > 200 for each indicated condition) out of three repeats. (right) The data shown are from three independent experiments and plotted as mean ± SD. (D) Cells expressing Erg6-mCherry (LDs) and microdomain (Ld or Lo) markers as indicated were imaged by fluorescence microscopy on D1. LDs and microdomain patterns as indicated were quantified and plotted. The data shown are from a single representative experiment (n > 60 for each cell type) out of three repeats. (E) Cells expressing Erg6-mCherry and Vph1-GFP were imaged for periphery and center on D3. The localization of types 3 and 4 of Vph1-GFP patterns were compared. The LDs in type 3 images were further quantified based on the two indicated patterns from three independent experiments. Error bars show mean ± SEM. (F) Cells expressing proteins as indicated were imaged by fluorescence microscopy. DIC, differential interference contrast. Bars, 5 µm.
Figure 4.
Vacuolar microdomain organization is crucial for stat-phase lipophagy. (A) Cells as indicated expressing Vph1-mCherry were imaged by fluorescence microscopy, and data were quantified based on the three indicated patterns. The data shown are from a single representative experiment (n > 200 for each condition) out of three repeats. (B) Cells as indicated stained with FM4-64 (vacuole) at DS and BODIPY (LDs) on D3 were imaged by fluorescence microscopy. Data were quantified based on the indicated patterns and plotted as mean ± SD from three independent experiments. WT, wild type. (C) Cells as indicated grown in SC medium were shifted to SD-N for 3 h. Cells were lysed, and the lysates were analyzed by immunoblotting with the anti-Ape1 antibody. pr, precursor; m, mature. (D) Cells expressing GFP-Pho8Δ60 under growth conditions as indicated were lysed, and the lysates were analyzed by immunoblotting with the anti-GFP antibody. (E) Same as B, except that wild type and pep4Δ were compared. (F) Same as A, except that wild type and pep4Δ were compared. Error bars show mean ± SEM from three experiments. (G) Cells expressing Vph1-GFP and Erg6-mCherry were imaged by fluorescence microscopy on D4. The indicated patterns of Vph1-GFP were quantified in cells with (+) and without (−) Erg6-mCherry inside vacuole lumen (lipophagy). The data shown are from a single representative experiment (n > 80 for each condition) out of three repeats. DIC, differential interference contrast. Bars, 5 µm.
Figure 5.
Stat-phase lipophagy is needed for vacuolar microdomain maintenance. (A) Cells as indicated expressing Vph1-mCherry were imaged by fluorescence microscopy on D1 and D3. (B, left) A table lists selective autophagy defects for the indicated strains. Cvt, cytoplasm-to-vacuole targeting. (right) Same as A. (C) Cells as indicated were quantified based on the Vph1-mCherry patterns and culture conditions as indicated. Data from three independent experiments were plotted as mean ± SD. (D, left) Lipids in strains as indicated were analyzed by TLC. SE, sterol ester; TAG, triacylglycerol; ERG, ergosterol. (right) Wild-type and are1Δ are2Δ cells expressing Vph1-mCherry were imaged by fluorescence microscopy, and three independent data were quantified. Error bars show mean ± SEM from three experiments. (E, top) Cells as indicated expressing GFP-Pho8Δ60 were grown in SC medium to various growth conditions. Cells were lysed, and the lysates were analyzed by immunoblotting with the anti-GFP antibody. (bottom) Same as top, except that cells expressing either GFP-Pho8Δ60 or GFP-Atg8 were grown in SC medium to log phase (SCD) or shifted to SD-N for 3 h from log phase. (F) Working model for stat-phase lipophagy. N, nucleus; V, vacuole. During DS, vacuolar proteins start to sort into either one of the vacuolar microdomains, Ld or Lo. Several proteins, Fab1, Vps4, Nem1, Atg6, and Atg8, are needed for microdomain formation at the stage. The pattern of quasisymmetrical microdomains appears later in the stationary phase. During this stage, LDs are engulfed by the Lo microdomain and feed vacuoles with sterols to maintain lipid phase partitioning, which in turn stimulates stat-phase lipophagy. Bars, 5 µm.
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