Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis
2015; Springer Nature; Volume: 34; Issue: 16 Linguagem: Inglês
10.15252/embj.201490315
ISSN1460-2075
AutoresTomer Shpilka, Evelyn Welter, Noam Borovsky, Nira Amar, Muriel Mari, Fulvio Reggiori, Zvulun Elazar,
Tópico(s)Plant responses to water stress
ResumoArticle14 July 2015free access Source Data Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis Tomer Shpilka Tomer Shpilka Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Evelyn Welter Evelyn Welter Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Noam Borovsky Noam Borovsky Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Nira Amar Nira Amar Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Muriel Mari Muriel Mari Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Fulvio Reggiori Fulvio Reggiori Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Zvulun Elazar Corresponding Author Zvulun Elazar Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Tomer Shpilka Tomer Shpilka Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Evelyn Welter Evelyn Welter Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Noam Borovsky Noam Borovsky Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Nira Amar Nira Amar Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Muriel Mari Muriel Mari Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Fulvio Reggiori Fulvio Reggiori Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Zvulun Elazar Corresponding Author Zvulun Elazar Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Author Information Tomer Shpilka1, Evelyn Welter1, Noam Borovsky1, Nira Amar1, Muriel Mari2, Fulvio Reggiori2 and Zvulun Elazar 1 1Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel 2Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands *Corresponding author. Tel: +972 8 9343682; Fax: +972 8 9344112; E-mail: [email protected] The EMBO Journal (2015)34:2117-2131https://doi.org/10.15252/embj.201490315 See also: V Deretic (August 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Autophagy is a major catabolic process responsible for the delivery of proteins and organelles to the lysosome/vacuole for degradation. Malfunction of this pathway has been implicated in numerous pathological conditions. Different organelles have been found to contribute to the formation of autophagosomes, but the exact mechanism mediating this process remains obscure. Here, we show that lipid droplets (LDs) are important for the regulation of starvation-induced autophagy. Deletion of Dga1 and Lro1 enzymes responsible for triacylglycerol (TAG) synthesis, or of Are1 and Are2 enzymes responsible for the synthesis of steryl esters (STE), results in the inhibition of autophagy. Moreover, we identified the STE hydrolase Yeh1 and the TAG lipase Ayr1 as well as the lipase/hydrolase Ldh1 as essential for autophagy. Finally, we provide evidence that the ER-LD contact-site proteins Ice2 and Ldb16 regulate autophagy. Our study thus highlights the importance of lipid droplet dynamics for the autophagic process under nitrogen starvation. Synopsis Under nitrogen starvation, lipid droplet (LD) biogenesis and mobilization enzymes regulate autophagy; the process requires ER-LD contact site proteins. LD biogenesis is pivotal for autophagy under nitrogen starvation in yeast. Both triacylglycerol and steryl esters are essential for autophagy. Mobilization of LDs exerted by lipase Ayr1, lipase/hydrolase Ldh1 and hydrolase Yeh1 is important for efficient autophagy. The ER-LD contact-site proteins Ice2 and Ldb16 regulate autophagy. Introduction Autophagy is an evolutionarily conserved physiological process for the degradation of proteins and organelles in the lysosome/vacuole of the cell, thereby contributing to the maintenance of cell homeostasis (Weidberg et al, 2011). Dysregulation of this catabolic pathway has been implicated in numerous pathological conditions and metabolic diseases (Ravikumar et al, 2010b; Abada & Elazar, 2014). Autophagy starts with formation of the phagophore, a cup-shaped vesicle that elongates and enwraps parts of the cytoplasm including organelles, and seals itself to form a unique double-membrane structure termed the autophagosome (Weidberg et al, 2011). Several organelles including the endoplasmic reticulum (ER) (Axe et al, 2008; Hayashi-Nishino et al, 2009), mitochondria (Hailey et al, 2010), and Golgi apparatus (Young et al, 2006; Mari et al, 2010; Nair et al, 2011), as well as the plasma membrane (Ravikumar et al, 2010a), were recently reported to contribute to formation of the autophagosome (Rubinsztein et al, 2012; Abada & Elazar, 2014). Numerous autophagy-related proteins (Atgs) are essential for autophagosome biogenesis. In yeast, the site of autophagosome biogenesis is the pre-autophagosomal structure (PAS). Upon induction of autophagy, Atgs are hierarchically recruited to the PAS (Suzuki et al, 2007). The Atg1 kinase complex, the class III phosphoinositide 3-kinase (PI3K) complex, and Atg9 are required at early stages of phagophore formation, whereas the Atg12-Atg5-Atg16 complex and Atg8 are recruited at later stages. Atg8 is a key player in autophagosome formation and is regulated by several essential autophagy factors that enable it to conjugate to phosphatidylethanolamine (PE) on the autophagic membrane (Kirisako et al, 2000; Suzuki et al, 2001; Hanada et al, 2007; Shpilka et al, 2012). Conjugation of Atg8 to PE is a hallmark event in autophagy and is widely utilized to assess autophagic activity (Klionsky et al, 2007; Shpilka et al, 2012). Lipid droplets (LDs) are organelles that store neutral lipids. They are found in most organisms and cell types (Walther & Farese, 2012) and are most probably formed in the ER (Fujimoto et al, 2008; Jacquier et al, 2011). They are comprised of a neutral lipid core that predominantly contains triacylglycerols (TAGs) and steryl esters (STEs) and is surrounded by a phospholipid monolayer and a specific set of proteins (Rajakumari et al, 2008; Farese & Walther, 2009; Walther & Farese, 2012). In yeasts, STE synthesis requires the activity of two acyl-CoA:sterol acyltransferases, Are1 and Are2 (Jensen-Pergakes et al, 2001), while the enzymes mainly responsible for the synthesis of TAG are the diacylglycerol acyltransferases Dga1 and Lro1 (Sorger & Daum, 2002). Yeast strains devoid of all four enzymes lack LDs (Sandager et al, 2002). LDs serve important functions in the cell by providing lipids and energy as well as by storing free fatty acids that may otherwise become cytotoxic (Beller et al, 2010). Recent studies point to a complex interplay between autophagy and LDs. On the one hand, LDs are degraded by autophagy via lipophagy (Singh et al, 2009; Wang et al, 2014b; van Zutphen et al, 2014), while, on the other hand, LDs have been implicated in regulation of the autophagic process in mammals (Dupont et al, 2014). In the present study, we utilized the yeast system to investigate the roles of LDs in autophagy. We show that LDs and their component STEs and TAGs are important for autophagosome biogenesis. Deletion of biosynthetic enzymes of STEs and of TAGs has opposite effects on the lipidation state of Atg8, suggesting novel and complementary roles for these neutral lipids in regulation of the autophagic process. Moreover, we identified the TAG and the STE lipases that participate in the autophagic process and found that the ER-LD contact-site proteins Ldb16 and Ice2, which couple LD lipolysis to phospholipid formation in the ER, are also needed. Our results point to a novel role for neutral lipids in regulation of the autophagic process. Results Autophagy is inhibited upon depletion of free fatty acids We first set out to characterize the role of fatty acid synthesis in autophagosome biogenesis. To this end, we utilized the antifungal antibiotic cerulenin that binds to and inhibits the activity of fatty acid synthase (FAS), the enzyme responsible for the entire synthesis of C16 and C18 fatty acids (Vance et al, 1972; Wakil et al, 1983). Cells were grown to mid-log phase in complete medium (YPD; 4) and prior to nitrogen starvation were mock-treated or treated for 30 min with cerulenin. Delivery of green fluorescent protein (GFP)-tagged Atg8 to vacuoles was monitored by the GFP-cleavage assay (Shintani & Klionsky, 2004) and fluorescence microscopy. Upon delivery of GFP-tagged proteins to the vacuole, the proteins are degraded while GFP remains relatively stable, enabling delivery of the tagged protein to the vacuole to be assessed (Shintani & Klionsky, 2004). Under nitrogen starvation, GFP-Atg8 was readily delivered to the vacuoles in mock-treated cells, but its delivery was blocked in cells treated with cerulenin (Fig 1A). Addition of exogenous fatty acid to the cerulenin-treated cells reversed the autophagic defect, supporting the need for fatty acids in the autophagic process (Fig 1A). Fluorescence imaging of cerulenin-treated cells demonstrated the failure of Atg8 to reach the vacuole and its accumulation in puncta (Fig 1B). This phenotype could be rescued by the addition of fatty acids (Fig 1B). Inhibition of autophagy by cerulenin was further tested with the autophagy kinase Atg1. In line with a previous report (Nakatogawa et al, 2012b), under nitrogen starvation, GFP-Atg1 localized to the vacuole and to a single punctum (presumably the PAS). In contrast, treatment of the cells with cerulenin led to accumulation of Atg1 in the cytosol in multiple puncta (Fig 1C). Cerulenin treatment also inhibited the delivery of the ER protein Scs2 for degradation, suggesting fatty acid synthesis is also needed for ERphagy (Fig EV1A and B). Figure 1. Autophagy is inhibited upon depletion of free fatty acids A, B. Wild-type (WT) (BY4741) cells expressing GFP-Atg8 were grown to mid-log phase in YPD and were then preincubated with 50 μM cerulenin or 50 μM cerulenin + 0.1 mM palmitic/stearic/myristic acids or with DMSO (−) in the rich medium for 30 min. Cells were washed and shifted to nitrogen starvation medium (SD-N) for 4 h in the presence of 50 μM cerulenin or 50 μM cerulenin + 0.1 mM palmitic/stearic/myristic acids or DMSO. Cells were then lysed and subjected to SDS–PAGE, followed by Western blot analysis using anti-GFP and anti-Pgk1 antibodies (A) or were visualized by fluorescence microscopy (B). Scale bar, 5 μm; **, non-specific band. C. Cells (TOS038) expressing Atg1-GFP were grown to mid-log phase and were then preincubated in YPD with 50 μM cerulenin or with DMSO for 30 min. Cells were washed, shifted to SD-N for 4 h, and then visualized by fluorescence microscopy. Scale bar, 5 μm. D. WT (BY4741) and pep4∆ (TOS015) strains were grown to mid-log phase and preincubated in YPD with cerulenin or DMSO for 30 min. Cells were pulse-labeled for 10 min with [35S] methionine and cysteine and chased for the indicated time periods. Cell lysates were subjected to immunoprecipitation with anti-Ape1 antibodies followed by SDS–PAGE and X-ray film to detect radioactive signals. prApe1, premature Ape1; mApe1, mature Ape1. E. Cells of the fas1∆ (TOS029) strain expressing GFP-Atg8 were grown to mid-log phase in YPD + 0.1 mM palmitic/stearic/myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min. Cells were then shifted to SD-N for the indicated times. Cell lysates were subjected to SDS–PAGE, followed by Western blot analysis using anti-GFP antibodies. F. WT (BY4741) cells were grown to mid-log phase and then preincubated in YPD with 25 μM or 50 μM cerulenin (25, 50) or with DMSO (−) in rich medium for 30 min. Cells were washed and shifted to SD-N in the presence of cerulenin (25, 50) or DMSO (−). Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using anti-GFP and anti-Pgk1 antibodies. Data information: cer, cerulenin; DIC, differential interference contrast; FA, fatty acids; SD-N, nitrogen starvation medium; WT, wild type; YPD, complete medium. Source data are available online for this figure. Source Data for Figure 1 [embj201490315-sup-0003-SDataFig1.tif] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Autophagy is inhibited upon depletion of free fatty acids WT (BY4741), atg1∆ (TOS001), and atg7∆ (TOS005) cells expressing GFP-Scs2 were grown to mid-log phase in YPD and shifted to SD-N for 12 h in the presence (SD-N cer) or absence (SD-N) of 50 μM cerulenin and visualized by fluorescence microscopy. Scale bar, 5 μm. WT (BY4741) and atg1∆ (TOS001) cells were grown as in (A). Lysates were subjected to SDS–PAGE, followed by Western blot analysis using anti-GFP antibodies. **, non-specific band. fas2∆ (TOS030) cells expressing GFP-Atg8 were grown to mid-log phase in YPD + 0.1 mM palmitic/stearic/myristic acids and shifted either to the same medium or to YPD without fatty acids for 30 min. The cells were then shifted to SD-N for the indicated time periods. Cell lysates were subjected to SDS–PAGE, followed by Western blot analysis using anti-GFP antibodies. Data information: cer, cerulenin; SD-N, nitrogen starvation medium; WT, wild type; YPD, complete medium. Download figure Download PowerPoint Cytoplasm-to-vacuole targeting (CVT) is a selective pathway in which the autophagic machinery is utilized to deliver, under vegetative growth conditions, enzymes such as aminopeptidase 1 (Ape1) to the vacuole (Lynch-Day & Klionsky, 2010). Using pulse–chase analysis of radiolabeled Ape1, we examined whether the CVT pathway is also blocked by cerulenin. As a control, we used pep4∆ strains that are incapable of maturing Ape1 (Fig 1D) (Lynch-Day & Klionsky, 2010). In untreated wild-type (WT) cells, Ape1 was readily processed and matured, whereas it failed to mature upon cerulenin treatment, suggesting that FAS activity is essential for CVT (Fig 1D). Fatty acid synthase is an essential enzymatic complex composed of two subunits, Fas1 and Fas2 (Henry, 1973; Lomakin et al, 2007) To further characterize the effect of FAS inhibition on autophagy, we utilized deletion strains of FAS, which are able to grow only in rich medium supplemented with fatty acids (Schweizer & Bolling, 1970; Henry, 1973). Using the GFP-cleavage assay, we monitored autophagic activity in fas1∆ and fas2∆ strains expressing GFP-Atg8 under the endogenous promoter. Depletion of fatty acids from these cells by allowing them to grow for 30 min in the absence of exogenous fatty acids prior to nitrogen starvation prevented the degradation of GFP-Atg8 (Figs 1E and EV1C). Depletion of fatty acids prior to nitrogen starvation was essential for the inhibition of autophagy, as shown by the finding that fas1∆ and fas2∆ cells subjected directly to nitrogen starvation (without preincubation in rich medium lacking fatty acids) exhibited normal autophagic activity (Figs 1E and EV1). Cerulenin was also able to block the autophagy process only in cells that were exposed to such preincubation prior to nitrogen starvation (Fig 1F). Lipid droplets are essential for efficient autophagy To better characterize the role of fatty acids in the autophagic process we measured autophagic activity by the Pho8∆60 assay (Noda & Klionsky, 2008) in the presence or absence of cerulenin. Pho8∆60, a genetically engineered version of the resident vacuolar enzyme Pho8, lacks the N-terminal transmembrane domain that enables translocation of this enzyme into the ER. It therefore accumulates in the cytosol and can be delivered to the vacuole only by autophagy (Noda & Klionsky, 2008). Cells were grown in complete medium (YPD; see 4) and were either shifted directly to nitrogen starvation medium or were first preincubated with cerulenin for different times in rich medium to deplete the cells of their free fatty acids prior to nitrogen starvation. We observed that Pho8∆60 activity gradually decreased with increasing preincubation time (Fig 2A). This finding further supported the notion that depletion of fatty acids from the cells blocks autophagy. Figure 2. Lipid droplets are important for autophagy Cells (TN124 strain) were grown to mid-log phase and preincubated in YPD with 50 μM cerulenin for the indicated time periods (Cer preincubation) or incubated with DMSO without preincubation (−). Cells were then shifted to SD-N for 3 h with the addition of DMSO or 50 μM cerulenin (cer). Autophagic activity was measured by alkaline phosphatase assay. Error bars represent the s.e.m. of three independent experiments. *P < 0.05, ***P < 0.001 (Student's t-test). Cells were grown as in (A), stained with BODIPY, and visualized by fluorescence microscopy. Scale bar, 5 μm. WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 3 h. Cells were stained with Nile red and visualized by fluorescence microscopy. Scale bar, 5 μm. WT (SCY62) and tag∆ste∆ (H1246) cells expressing GFP-Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cell lysates were subjected to SDS–PAGE, followed by Western blot analysis using anti-GFP, anti-Ape1 (prApe1, premature Ape1; mApe1, mature Ape1), and anti-Fas1 and anti-Pgk1 antibodies. WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 2 h. GFP-Atg8 was visualized by fluorescence microscopy. Scale bar, 5 μm. Data information: cer, cerulenin; DIC, differential interference contrast; FA, fatty acids; SD-N, nitrogen starvation medium; WT, wild type; YPD, complete medium. Source data are available online for this figure. Source Data for Figure 2 [embj201490315-sup-0004-SDataFig2.zip] Download figure Download PowerPoint Inhibition of fatty acid synthesis under growing conditions leads to the utilization of LD pools in order to supply cells with the necessary lipids (Kurat et al, 2009). We therefore examined the amount and distribution of LDs in cerulenin-treated cells, using the LD dye BODIPY and the protein Erg6, an LD marker (Figs 2B and EV2A) (Greenspan et al, 1985; Jacquier et al, 2011). We found that the decrease in autophagic activity upon cerulenin treatment correlated with a reduction in the amount of LDs (Figs 2B and EV2A). Under conditions of nitrogen starvation, both BODIPY and Erg6−red fluorescent protein (Erg6−RFP) localized to LDs, whereas their localization shifted to the ER and the number of LDs decreased at a rate proportional to the period of preincubation with cerulenin (Figs 2B and EV2A). Addition of cerulenin directly to the starvation medium also inhibited the accumulation of LDs (Fig EV2B). These results led us to hypothesize that LDs are essential for the autophagic process. Click here to expand this figure. Figure EV2. Lipid droplets are important for autophagy RFP-Erg6 (TOS039)-expressing cells were grown to mid-log phase in YPD and preincubated with 50 μM cerulenin for the indicated time periods (Cer preincubation) or with DMSO (0). Cells were shifted to SD-N with 50 μM cerulenin for 3 h and then visualized by fluorescence microscopy. Scale bar, 5 μm. WT cells were grown to mid-log phase in YPD and shifted to SD-N in the presence or absence of 50 μM cerulenin for 16 h. Cells were stained with BODIPY and visualized by fluorescence microscopy. Scale bar, 5 μm. WT (SCY62) and tag∆ste∆ (H1246) cells were grown to mid-log phase in YPD and shifted to SD-N for 2 h. Cells were lysed and the lysate was subjected to subcellular fractionation as described in 4. An equal volume of each fraction was subjected to immunoblotting with anti-Atg8, anti-Kar2, anti-Ape1, and anti-Atg3 antibodies. Data information: cer, cerulenin; SD-N, nitrogen starvation medium; WT, wild type; YPD, complete medium. Source data are available online for this figure. Download figure Download PowerPoint To directly test whether LDs participate in autophagy, we utilized a quadruple deletion strain unable to synthesize LDs (H1246) (Sandager et al, 2002). This strain lacks the enzymes Lro1 and Dga1, which are responsible for the synthesis of TAGs (Sandager et al, 2002), as well as the Are1 and Are2 enzymes responsible for the synthesis of STEs (Sandager et al, 2002). Nile red and BODIPY fluorescence staining confirmed that this strain lacks LDs (Figs 2C and 4A). Deficiency of LDs led to the inhibition of autophagy, as indicated by the inability of this strain to degrade GFP-Atg8 and the autophagic substrate FAS1 upon nitrogen starvation (Fig 2D). Ape1 was in its mature form in vegetative growing cells, suggesting that the CVT pathway is not dependent on LDs. Upon nitrogen starvation, however, the Ape1 that accumulated in the LD-deficient strain was premature compared with that in the WT, further supporting the inhibition of autophagy (Fig 2D). Fluorescence microscopy revealed no apparent differences in GFP-Atg8 localization in the WT and the LD-deficient strains under vegetative growth conditions (Fig 2E). Shifting of the cells to the nitrogen starvation medium (SD-N), however, resulted in GFP-Atg8 accumulation in the vacuoles of WT cells, whereas the LD-deficient strain exhibited multiple puncta and failed to reach the vacuole efficiently (Fig 2E). Subcellular fractionation indicated that Atg8 accumulated mainly in the ER fraction (Fig EV2B). The need for LDs in autophagy was further emphasized by the use of an LD-deficient strain harboring the DGA1 and ARE2 genes under a galactose-inducible promoter (are1∆lro1∆ pGAL-DGA1 pGAL-ARE2). We found that it was only when this strain was grown on a galactose medium that LDs were formed (Fig 3A). Autophagy was inhibited when these cells were grown on glucose, as indicated by the GFP-Atg8 processing assay and by the localization of GFP-Atg8 to multiple puncta under nitrogen starvation (Fig 3B and C). Upon galactose induction, however, GFP-Atg8 was readily processed and accumulated inside the vacuoles (Fig 3B and C). We also observed that the core autophagy protein and substrate Atg1 failed to reach the vacuole under nitrogen starvation when grown on glucose-containing medium but was readily delivered to the vacuole when grown on galactose (Fig 3D). Together, these results strongly indicate that LDs are key regulators of autophagy. Figure 3. Induction of STE and TAG enzymes rescues autophagy A. GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118 strain) was grown on SC (synthetic minimal medium without dextrose) + raffinose overnight, diluted to OD 0.4, and grown to mid-log phase in either SC + glucose or SC + galactose medium. Cells were shifted to SD-N for 2 h, stained with BODIPY, and visualized by fluorescence microscopy. Scale bar, 5 μm. B. WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP-Atg8 were grown as in (A). Cells were lysed at the indicated times and subjected to SDS–PAGE, followed by Western blot analysis using anti-GFP antibodies. C, D. WT (BY4741) and GAL-DGA1 GAL-ARE2 lro1∆ are1∆ (FYS118) cells expressing GFP-Atg8 (C) or GFP-Atg1 (D) were grown as in (A) and visualized by fluorescence microscopy after 2 h in SD-N. Scale bar, 5 μm. Data information: DIC, differential interference contrast; SD-N, nitrogen starvation medium; WT, wild type. Source data are available online for this figure. Source Data for Figure 3 [embj201490315-sup-0005-SDataFig3.tif] Download figure Download PowerPoint Exogenous fatty acids cannot rescue lipid droplet deficiency Under nitrogen starvation, fatty acids are stored in the form of LDs (Fig 4A). Both STE- and TAG-containing LDs accumulated under these conditions (Fig 4A). We speculated that in the absence of LDs, the cells might encounter a shortage of fatty acids that would result in autophagy blockage. To test this hypothesis, we supplied exogenous fatty acids to WT and LD-deficient strains. Addition of fatty acids to the LD-deficient strain failed to rescue autophagy, as indicated by the GFP-Atg8 cleavage assay (Fig 4B) and also failed to overcome the accumulation of GFP-Atg8 puncta upon nitrogen starvation (Fig 4C). In agreement with the inhibition of the autophagic process and the inability of fatty acids to overcome autophagic defects, we observed that FAS activity remained high and relatively constant in the LD-deficient strain but showed a marked decrease upon nitrogen starvation in the WT strain presumably owing to its degradation by autophagy (Fig 4D). In the absence of LDs, therefore, cells may need to rely mostly on FAS for their fatty acid supply. Inhibition of FAS by cerulenin under nitrogen starvation led to a rapid and complete loss of viability in the LD-deficient strain, whereas its effect on WT cells was not significant (Fig 4E). These results showed that LDs and not fatty acids are needed for regulation of the autophagic process. Figure 4. Fatty acids cannot rescue autophagy in LD-deficient strains WT (SCY62), tag∆ ste∆ (H1246), tag∆ (H1226), and ste∆ (H1112) cells expressing GFP-Atg8 were grown to mid-log phase and shifted to SD-N for 6 h. Cells were stained with BODIPY and visualized by fluorescence microscopy. Scale bar, 5 μm. WT (SCY62) and tag∆ ste∆ (H1246) cells expressing GFP-Atg8 were grown to mid-log phase in YPD + 0.1 mM palmitic/stearic/myristic acids (FA) and shifted to SD-N + 0.1 mM palmitic/stearic/myristic acids for the indicated time periods. Cell lysates were subjected to SDS–PAGE followed by Western blot analysis using anti-GFP antibodies. Fluorescence visualization of GFP-Atg8 in tag∆ ste∆ (H1246) cells grown in the presence or absence of 0.1 mM palmitic/stearic/myristic acids (FA) and shifted to SD-N for 2 h. Scale bar, 5 μm. WT (SCY62) and tag∆ ste∆ (H1246) cells expressing GFP-Atg8 were grown to mid-log phase and shifted to SD-N for 4 h. FAS activity was determined at 0 h and after 4 h in SD-N. Error bars represent the s.e.m. of three independent experiments. ***P < 0.001 (Student's t-test). WT (SCY62) and tag∆ ste∆ (H1246) cells were grown to mid-log phase and shifted to SD-N with DMSO or 50 μM cerulenin for the indicated time periods. Cell viability was determined at the indicated times using phloxine B. Error bars represent the s.e.m. of three independent experiments. ***P < 0.001 (Student's t-test). Data information: Cer, cerulenin; DIC, differential interference contrast; FA, fatty acids; FAS, fatty acid synthase; SD-N, nitrogen starvation medium; WT, wild type; YPD, complete medium. Source data are available online for this figure. Source Data for Figure 4 [embj201490315-sup-0006-SDataFig4.zip] Download figure Download PowerPoint Triacylglycerols and steryl esters are both essential for efficient autophagy Lipid droplets are composed of TAGs and STEs surrounded by a phospholipid monolayer. To gain a deeper understanding of the role of LDs in autophagy, we analyzed the effect of LDs deprived of either tag∆ (dga1∆ lro1∆) or ste∆ (are1∆ are2∆). Using the GFP-Atg8 processing assay, we observed that GFP cleavage in both tag∆ and ste∆ cells is defective compared to that in WT cells (Fig 5A and B). In addition, fluorescence microscopy of GFP-Atg8 revealed that GFP-Atg8 fails to efficiently localize to the vacuole in these strains (Fig 5C). Notably, Atg8 in both tag∆ste∆ and ste∆ strains accumulated in puncta-like structures, whereas no such accumulation was observed in the tag∆ strain (Fig 5C). Similar accumulation in puncta was observed when we visualized GFP-Atg1 (Fig EV3A), but were not seen with the GFP-tagged housekeeping gene phosphoglycerokinase 1 GFP-Pgk1 (Fig EV3B), suggesting that the puncta are autophagy-related structures. In agreement with these results, we found that upon nitrogen starvation, Atg8 accumulated in its unlipidated form in the tag∆ strain, whereas lipidated Atg8 accumulated in the ste∆ and the ste∆tag∆ strains (Figs 5D and EV3C). In further support of autophagy inhibition, the starvation-induced degradation of long-lived proteins was significantly inhibited (Fig EV3D). These results indicated that both TAGs and STEs are important for autophagy. Notably, we could not detect the autophagy proteins Atg8 or Atg3 on LDs, suggesting that Atg8 lipidation does not occur on LDs (Fig EV3E). Figure 5. TAG and STE are both essential for efficient autophagy WT (SCY62), tag∆ste∆ (H1246), tag∆ (H1226), and ste∆ (H1112) cells expressing GFP-Atg8 were grown to mid-log phase in YPD and shifted to SD-N for the indicated time periods. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using anti-GFP antibodies. Qua
Referência(s)