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TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast Shamsul Morshed a, Tasnuva Sharmin a, Takashi Ushimaru a, b, * a b
Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8021, Japan Department of Science, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8021, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 November 2019 Accepted 10 November 2019 Available online xxx
Microautophagy is promoted after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) kinase. Invagination of vacuolar membranes by endosomal sorting complex required for transport (ESCRT) is required for microautophagy. Vps27, a subunit of ESCRT-0, is recruited onto vacuolar membranes via dephosphorylation after TORC1 inactivation. Here, we showed that Hse1, another ESCRT0 subunit, is also recruited onto vacuolar membranes after TORC1 inactivation, promoting formation of ESCRT-0 complex on vacuolar membranes. Hse1 recruitment was dependent on Vps27, whereas Vps27 recruitment was independent of Hse1. Not only Vps27 but also Hse1 was required for ESCRT-III recruitment onto vacuolar membranes and microautophagy induction after TORC1 inactivation. This study revealed that ESCRT-0 (Vps27eHse1) complex formation on vacuolar membranes is important for microautophagy inactivation after TORC1 inactivation. © 2019 Elsevier Inc. All rights reserved.
Keywords: ESCRT-0 Vps27 Hse1 Microautophagy TORC1
1. Introduction Microautophagy degrades cargos by direct lysosomal/vacuolar engulfment of the cytoplasmic cargo without the formation of isolation membranes or autophagosomes [1e3]. Microautophagy can selectively degrade various specific cargos, such as the nucleus (micronucleophagy or piecemeal microautophagy of the nucleus), the ER (microERphagy) and lipid droplets (microlipophagy) in budding yeast Saccharomyces cerevisiae [4e6]. Vacuolar membrane proteins together with vacuolar membranes are degraded in the vacuole in the course of microautophagy. Therefore, overall microautophagy flux has been estimated using GFP-tagged vacuolar transmembrane proteins Vph1 and Pho8 [7]. When Vph1-GFP and GFP-Pho8 are incorporated into the vacuole by microautophagy, Vph1 and Pho8, but not the stable GFP moiety, are degraded by vacuolar proteases, producing free GFP, which is
Abbreviations: BiFC, bimolecular fluorescence complementation; ESCRT, endosomal sorting complex required for transport; FYVE, Fab1, YGL023, Vps27, and EEA1; GFP, green fluorescent protein; MVB, multivesicular body; PI3P, phosphatidylinositol 3-phosphate; RFP, red fluorescent protein; TORC1, target of rapamycin complex 1; UIM, ubiquitin-interacting motif; VHS, Vps27, Hrs and STAM. * Corresponding author. Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8021, Japan. E-mail address: [email protected] (T. Ushimaru).
detectable by immunoblotting. Microautophagy including micronucleophagy and microlipophagy is induced after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase [4,6e10]. The endosomal sorting complex required for transport (ESCRT) system, which was originally found as a device for the formation of intraluminal vesicles multivesicular bodies (MVBs) [11,12]. The ESCRT-0 complex first recognizes ubiquitinated cargo and clusters the ESCRT-I and eII complexes, promoting the assembly of ESCRT-III, which promotes the invagination, constriction, and abscission of membranes. ESCRT-0 consists of two subunits, Vps27 and Hse1 (Hrs and STAM1/2 in human), and these subunits interact in a 1:1 ratio. Vps27 binds to phosphatidylinositol 3-phosphate (PI3P) via a Fab1, YGL023, Vps27, and EEA1 (FYVE) domain, and to ubiquitin conjugated with membrane proteins via two ubiquitin-interacting motif (UIM) domains and a Vps27, Hrs and STAM (VHS) domain. In contrast, Hse1 has an UIM domain and a VHS domain, but not a FYVE domain. All of the five ubiquitin-binding sites in ESCRT-0 are required for MVB formation [13]. Vps27 is recruited onto vacuolar membranes after a diauxic shift (carbon starvation) [7]. TORC1 activity is necessary for maintaining Vps27 protein levels [9,14,15]. Vps27, as well as other components of ESCRT-I, eII, and eIII, is required for microautophagy induced by carbon starvation and rapamycin treatment [7,9]. By contrast, the
https://doi.org/10.1016/j.bbrc.2019.11.064 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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properties of Hse1 in microautophagy induction are largely unknown. Here, we show that Hse1 is also required for proper microautophagy induction after TORC1 inactivation. Hse1 recruitment was dependent on Vps27. Furthermore, we found that the ESCRT-0 complex is indeed formed on vacuolar membranes. This study provides basic properties of ESCRT-0 as to TORC1 inactivation-induced microautophagy induction. 2. Results 2.1. TORC1 inactivation promotes recruitment of Vps27 and Hse1 onto vacuolar membranes We wanted to know how TORC1 regulates ESCRT-mediated microautophagy induction. We suspected that a functional Vps27eHse1 ESCRT-0 formation is required for microautophagy induction after TORC1 inactivation. To test this idea, first we examined whether not only Vps27 but also Hse1 is recruited onto the vacuolar membranes after TORC1 inactivation. Faint punctate signals of Vps27 and Hse1 were observed in normal (favorable nutrient) conditions in some cells (Fig. 1A and B). However, strong punctate signals of both proteins appeared on vacuolar membranes after rapamycin treatment (Fig. 1A and B). This indicated that TORC1 inactivation promotes vacuolar membrane recruitment of Hse1, as well as that of Vps27. Thus, Hse1 as well as Vps27 was recruited onto vacuolar membranes after TORC1 inactivation. 2.2. TORC1 inactivation promotes ESCRT-0 complex formation on vacuolar membranes We suspected that both Vps27 and Hse1 are recruited onto vacuolar membranes after rapamycin treatment, forming ESCRT0 complex. To assess this idea, we examined colocalization of Vps27 and Hse1 using cells co-expressing Vps27-GFP and Hse1mCherry. We again found that rapamycin treatment facilitated focus formation of Vps27-GFP and Hse1-mCherry on the vacuolar membranes (Supplementary Figs. S1A and B). We noticed that most of the Vps27 foci on the vacuolar membranes contained Hse1 after rapamycin treatment (>70%) (Fig. 1C). This suggested that ESCRT-0 complex formation on vacuolar membranes is promoted after TORC1 inactivation. Of note, other Vps27 formed foci on vacuolar membranes, where Hse1 was absent (<30%). This indicated that a portion of Vps27 molecules were recruited onto vacuolar membranes without Hse1. In contrast, the vast majority of Hse1 foci on vacuolar membranes after rapamycin contained Vps27 (~95%) (Fig. 1C). In addition, Hse1 foci that did not contain Vps27 were small and faint (Supplementary Fig. S1C). These facts implicated that proper localization of Hse1 on vacuolar membranes after TORC1 inactivation requires Vps27 (see below). To confirm that Vps27 and Hse1 form the ESCRT-0 complex on vacuolar membranes after TORC1 inactivation, we performed bimolecular fluorescence complementation (BiFC) assays, which are useful for studying detection of in vivo proteineprotein interactions [16e18]. We created cells expressing an N-terminal fragment of Venus (VN)-tagged Vps27 (Vps27-VN) and a C-terminal fragment of Venus (VC)-tagged Hse1 (Hse1-VC). There were no obvious signals of the reconstituted Venus in normal conditions (Fig. 1D). However, puncta of the reconstituted Venus signals appeared on vacuolar membranes after rapamycin treatment. This indicated that TORC1 inactivation promotes the formation of Vps27eHse1 (namely, ESCRT-0) complexes on vacuolar membranes.
2.3. Hse1 is dispensable for Vps27 recruitment on vacuolar membranes after TORC1 inactivation Vps27 is a tethering factor of ESCRT-0 on MVBs [11]. Vps27 has a FYVE domain that binds to PI3P on lipid membranes. The FYVE domain of Vps27 is essential for the recruitment on vacuolar membranes after a diauxic shift (carbon starvation) [7]. We found that Vps27 formed foci on vacuolar membranes after rapamycin treatment (Fig. 2A). This Vps27 recruitment was not compromised in cells lacking Hse1, indicating that Hse1 is dispensable for Vps27 recruitment. Protein levels of Vps27 were similar in wild-type and hse1D cells in normal conditions (Supplementary Fig. S2A). This suggested that the protein stability of Vps27 is not reduced even if its partner Hse1 is absent in normal conditions. It was reported that Vps27 was degraded by autophagy after rapamycin treatment [9]. Consist with this notion, Vps27 was lost after rapamycin treatment was in wild-type (Supplementary Fig. S2A). Similar degradation of Vps27 after rapamycin treatment was observed in hse1D cells, indicating that autophagic degradation of Vps27 after TORC1 inactivation is not dependent on Hse1. 2.4. Vps27 is essential for Hse1 recruitment onto vacuolar membranes after TORC1 inactivation In contrast, Hse1 recruitment onto vacuolar membranes after rapamycin treatment was largely compromised in vps27D cells (Fig. 2B). This indicated that Vps27 is essential for Hse1 recruitment. Collectively, Vps27, but not Hse1, was a critical tethering factor for the recruitment of ESCRT-0 onto vacuolar membranes after TORC1 inactivation. In addition, these findings showed that Vps27 is recruited onto the vacuolar membranes after TORC1 inactivation in a manner independent of Hse1, but not vice versa. Protein levels of Hse1 were substantially equal in wild-type and vps27D cells in normal conditions (Supplementary Fig. S2B). This suggested that the protein stability of Hse1 is independent of Vps27. In sharp contrast to Vps27, the protein levels of Hse1 were not reduced in wild-type cells after rapamycin treatment. This suggested that Hse1 escapes from microautophagic degradation after TORC1 inactivation. However, Hse1 decreased after rapamycin treatment in vps27D cells. This suggested that Hse1 becomes unstable in the absence of Vps27 after TORC1 inactivation. 2.5. Hse1 is required for proper microautophagy induction after TORC1 inactivation The fact that not only Vps27 but also Hse1 was recruited onto vacuolar membranes after TORC1 inactivation allowed us to suspect that Hse1 is required for microautophagy induction, as well as Vps27. Loss of Vps27 markedly compromised microautophagy induction (monitored using a GFP-Pho8 processing assay) after rapamycin treatment (Fig. 3A), as reported previously [9]. In contrast, microautophagy induction was partially reduced in hse1D cells although to a lesser extent compared with that found in vps27D cells. This indicated that Hse1 is also required for proper microautophagy induction after TORC1 inactivation. 2.6. ESCRT-I to eIII are essential for TORC1 inactivation-induced microautophagy In the case of MVB formation, ESCRT-0 recruits ESCRT-I, eII, and eIII complexes onto endosomes. It has been shown that the ESCRT0 to eIII complexes are all critical for microautophagy induction after a diauxic shift (carbon starvation) [7]. Indeed, loss of Vps28 (ESCRT-I subunit), Vps36 (ESCRT-II subunit), and Vps24 (ESCRT-III subunit) remarkably impaired microautophagy induction after
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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Fig. 1. TORC1 inactivation promotes ESCRT-0 complex formation on vacuolar membranes. (A, B) Exponentially growing cells of strains SCU5926 (VPS27-GFP) and SCU5992 (HSE1-GFP) pretreated with FM4-64 (10 ng/ml) was treated with 200 ng/ml rapamycin for 3 h. Cell and fluorescence images were captured using a microscope. Scale bars, 5 mm. The average (±standard deviation) of percentages of cells with foci of Vps27 (A) or Hse1 (B) on vacuolar membranes was determined from three independent experiments. For statistical analysis, the p-values were calculated using a two-tailed Student’s t-test. ***, p < 0.0001. (C) Cells of strain SCU6116 (VPS27-GFP HSE1-mCherry) were treated with rapamycin for 3 h. Red arrows, Vps27-GFP foci that were not colocalized with Hse1-mCherry. Scale bars, 5 mm. Percentages of colocalization of Vps27-GFP foci colocalized with or without Hse1-mCherry (black bars) and Hse1-mCherry foci colocalized with or without Vps27-GFP (gray bars) on vacuolar membranes after rapamycin treatment for 3 h were determined from three independent experiments. (D) Cells of strain SCU6153 (VPS27-VN HSE1-VC) pretreated with FM4-64 were treated with rapamycin for 3 h. Scale bars, 5 mm. The average (±standard deviation) of percentages of cells with reconstructed Venus signals in BiFC assay on vacuolar membranes was determined from three independent experiments. ***, p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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Fig. 2. Hse1 is dispensable for Vps27 recruitment on vacuolar membranes after TORC1 inactivation, but not vice versa. (A) Cells of strains SCU5926 (VPS27-GFP) and SCU6050 (hse1D VPS27-GFP) pretreated with FM4-64 were treated with rapamycin for 3 h. Cell and fluorescence images were captured using a microscope. Scale bars, 5 mm. The average (±standard deviation) of percentages of cells with Vps27 foci on vacuolar membranes was determined from three independent experiments. (B) Cells of strains SCU5992 (HSE1-GFP) and SCU6070 (vps27D HSE1-GFP) pretreated with FM4-64 were treated with rapamycin for 3 h. Scale bars, 5 mm. The average (±standard deviation) of percentages of cells with Hse1 foci on vacuolar membranes was determined from three independent experiments.
rapamycin treatment (Fig. 3B). Thus, TORC1 negatively regulates microautophagy induction, which was mediated by ESCRT-0 to ESCRT-III. 2.7. Vps27 and Hse1 promote ESCRT-III recruitment onto vacuolar membranes after TORC1 inactivation Because Hse1, as well as Vps27, was required for proper microautophagy induction after TORC1 inactivation, we suspected that Hse1 is also a key factor for the recruitment of the following ESCRT complexes onto vacuolar membranes after TORC1 inactivation. To test this idea, we observed localization of ESCRT-III, a key factor for membrane deformation, after rapamycin treatment. We found clear promotion of recruitment of ESCRT-III (monitored using Vps24-GFP) onto vacuolar membranes in wild-type cells after
rapamycin treatment (Fig. 4). The recruitment of ESCRT-III was dramatically hindered in vps27D cells, whereas it was moderately reduced in hse1D cells. This was consistent with the fact that loss of Vps27 more remarkably compromised microautophagy induction after rapamycin treatment than loss of Hse1 (Fig. 3A). Protein levels of Vps24 decreased after rapamycin treatment (Supplementary Fig. S3). We found similar protein levels of Vps24 in vps27D and hse1D cells before and after rapamycin treatment. This indicated that the defect recruitment of Vps24 in cells lacking ESCRT-0 is not due to alterations in protein levels in Vps24. 3. Discussion In this study, we obtained several important findings on ESCRT0 formation after TORC1 inactivation: (1) TORC1 inactivation
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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Fig. 3. ESCRT is required for proper microautophagy induction after TORC1 inactivation. (A) Cells of strains SCU5466 (wild-type), SCU5457 (vps27D), and SCU6048 (hse1D) harboring plasmid pSCU2366 (pGFP-PHO8) [8] were treated with rapamycin for 6 h. Whole cell extracts were subjected to western blotting using the anti-GFP antibody. Free GFP processed from GFP-Pho8 after rapamycin treatment was measured using ImageJ software, and quantified by calculating the ratio of cleaved free GFP versus sum of uncleaved GFP-Pho8 and free GFP. The average (±standard deviation) was determined from three independent experiments, and relative values were normalized against the value in control cells are shown. The p-values were calculated using a two-tailed Student’s t-test. **, p < 0.001; ***, p < 0.0001. (B) Cells of strains SCU2684 (wild-type), SCU6187 (vps28D), SCU6188 (vps36D), and SCU4337 (vps24D) harboring plasmid pSCU2366 (pGFP-PHO8) were treated with rapamycin for 6 h. Whole cell extracts were subjected to western blotting. The ratio of cleaved free GFP versus sum of uncleaved GFP-Pho8 and free GFP was determined. ***, p < 0.0001.
promoted ESCRT-0 formation on the vacuolar membranes; (2) Vps27 was recruited on vacuolar membranes after TORC1 inactivation in a manner independent of Hse1, but not vice versa; and (3) Vps27 was critical for ESCRT-III recruitment onto the vacuolar membranes and microautophagy induction after TORC1 inactivation, whereas Hse1 was also required for the ESCRT-III recruitment and microautophagy induction after TORC1 inactivation. Both Vps27 and Hse1 have ubiquitin-binding UIMs and VHS domains and these domains is required for the function of ESCRT0 on endosomal membranes [13]. We showed that Hse1 could not be recruited onto vacuolar membranes after TORC1 inactivation
if Vps27 was absent. This indicated that the interaction between ubiquitin and UIM/VHS of Hse1 is insufficient for the localization of Hse1 onto vacuolar membranes after TORC1 inactivation. This further suggested that the interaction of the FYVE domain of Vps27 with PI3P on vacuolar membranes after TORC1 inactivation is critical for Vps27 recruitment onto vacuolar membranes (Supplementary Fig. S4). This is consistent with the finding that the FYVE domain of Vps27 is essential for the recruitment on vacuolar membranes after a diauxic shift (carbon starvation) [7]. Here, we utilized the BiFC assay for assessment for ESCRT0 (Vps27eHse1) formation. We found that the ESCRT-0 complex
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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Fig. 4. Vps27 and Hse1 promote ESCRT-III recruitment onto vacuolar membranes after TORC1 inactivation. Cells of strains SCU6092 (VPS24-GFP), SCU6094 (vps27D VPS24-GFP), and SCU6096 (hse1D VPS24-GFP) pretreated with FM4-64 were treated with rapamycin for 3 h. Cell and fluorescence images were captured using a microscope. Scale bars, 5 mm. The average (±standard deviation) of percentages of cells with Vps27 foci on vacuolar membranes was determined from three independent experiments.
was massively formed on vacuolar membranes after TORC1 inactivation. In contrast, there were no obvious BiFC signals in normal conditions using our BiFC system (Vps27-VN and Hse1-VC), although Vps27 and Hse1 form the ESCRT-0 complex on endosomes, and both proteins are indispensable for MVB formation and vacuolar protein sorting [19]. The BiFC assay is sensitive to the structure of complex of two proteins with interaction [16]. The structure of ESCRT-0 complexes on vacuolar membranes might be different from that on endosomes. TORC1 phosphorylates multiple residues of Vps27, probably causing structural changes in Vps27 [9]. Changes in the phosphorylation status of Vps27 might affect the structure of ESCRT-0 complexes. This study showed that the formation of the ESCRT-0 consisting of Vps27 and Hse1 on vacuolar membranes after TORC1 inactivation promoted the subsequential recruitment of ESCRT-III onto vacuolar membranes and microautophagy induction. Microautophagy is thought to be involved in various human diseases, such as neurodegenerative diseases [20]. However, in sharp contrast to macroautophagy, microautophagy is largely unknown with regard to its molecular mechanisms in not only yeast but also human cells. This study provides valuable insights into microautophagy-related diseases in human. 4. Materials and methods 4.1. Strains and media The S. cerevisiae strains used are listed in Supplementary Table S1. Glucose-containing YPAD (YPD containing 0.01% adenine) and synthetic minimal medium (SD) complemented with the appropriate nutrients for plasmid maintenance were prepared
using standard methods. For assessment of microautophagy after rapamycin treatment, when cells harbored plasmids, cells were precultured in SD medium with the appropriate nutrients, and then cultured in YPAD, followed by rapamycin treatment. 4.2. Western blotting analysis Exponentially growing cells were used for these experiments. Proteins were extracted using a post-alkaline extraction method in accordance with a previous report [10]. Briefly, cells (10 ml culture, OD600 ¼ 0.2e0.8) were treated with 200 ml of 0.1 M NaOH for 5 min and then the pellet was collected by centrifugation. The pellet was resuspended in sample buffer (60 mM Tris-HCl [pH 6.8], 5% glycerol, 2% SDS, 4% 2-mercaptoethanol, and 0.0025% bromophenol blue) at 95 C for 5 min. Crude extracts were cleared by centrifugation and the supernatant was used for western blotting analysis. We used the following antibodies: anti-GFP mouse monoclonal antibody (Santa Cruz, Cat#sc-9996; RRID: AB_627695), HRPconjugated anti-GFP goat polyclonal antibody (Rockland, #600103-215; RRID: AB_218193), and anti-Pgk1 mouse monoclonal antibody (Abcam, Cat# ab113687; RRID: AB_10861977). Chemiluminescence signals from Western BLoT Quant HRP Substrate (Takara, Cat #DS-T7102) for horseradish peroxidase (HRP) and Immuno Shot (Cosmo Bio, Cat #IS-012-250) as an immunoreaction enhancer solution were detected using an image analyser (Fuji LAS4000mini). All western blotting experiments were performed independently at least three times to confirm the reproducibility of the results. Relative protein amounts were measured using ImageJ software. The average and standard deviation were determined for each sample from three independent experiments, and relative values normalized against the value in control cells are shown. For
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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statistical analysis, the p-values were calculated using a two-tailed Student’s t-test. 4.3. Microscope observations Exponentially growing cells were used for experiments. Cell, GFP, Venus, and RFP (mCherry) images were captured using a Carl Zeiss Axio Imager M1 microscope with a cooled CCD camera (Carl Zeiss AxioCam MRm). All microscope observations were performed independently at least three times to confirm the reproducibility of the results. 4.4. BiFC assay Strain expressing Vps27-VN and Hse1-VC was constructed according to the previous study [16]. We confirmed expression of Vps27-VN and Hse1-VC by microscopic observation and western blotting analysis using the anti-GFP polyclonal or monoclonal antibodies. All microscope observations were performed independently at least three times to confirm the reproducibility of the results. Funding This work was supported in part by the Japan Society for the Promotion of Science (grant No. 18K06212). Author contributions TU designed the experiments. SM conducted most of the experiments. TS performed some experiments. TU mainly wrote the paper. SM helped to write the paper. Declaration of competing interest The authors declare no conflict of interests. Acknowledgments We thank Won-Ki Huh and Koji Okamoto for generous gifts of materials, and Ayumu Yamamoto, Yoko Kimura and laboratory members of TU for helpful discussions. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.064.
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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.064.
References [1] O. Muller, T. Sattler, M. Flotenmeyer, H. Schwarz, H. Plattner, A. Mayer, Autophagic tubes: vacuolar invaginations involved in lateral membrane sorting and inverse vesicle budding, J. Cell Biol. 151 (2000) 519e528. [2] T. Sattler, A. Mayer, Cell-free reconstitution of microautophagic vacuole invagination and vesicle formation, J. Cell Biol. 151 (2000) 529e538. [3] J.B. Kunz, H. Schwarz, A. Mayer, Determination of four sequential stages during microautophagy in vitro, J. Biol. Chem. 279 (2004) 9987e9996. [4] P. Roberts, S. Moshitch-Moshkovitz, E. Kvam, E. O’Toole, M. Winey, D.S. Goldfarb, Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae, Mol. Biol. Cell 14 (2003) 129e141. [5] S. Schuck, C.M. Gallagher, P. Walter, ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery, J. Cell Sci. 127 (2014) 4078e4088. [6] T. van Zutphen, V. Todde, R. de Boer, M. Kreim, H. Hofbauer, H. Wolinski, M. Veenhuis, I.J. van der Klei, S.D. Kohlwein, Lipid droplet autophagy in the yeast Saccharomyces cerevisiae, Mol. Biol. Cell (2014) 290e301. [7] M. Oku, Y. Maeda, Y. Kagohashi, T. Kondo, M. Yamada, T. Fujimoto, Y. Sakai, Evidence for ESCRT- and clathrin-dependent microautophagy, J. Cell Biol. 216 (2017) 3263e3274. [8] M.A. Rahman, M. Terasawa, M.G. Mostofa, T. Ushimaru, The TORC1-Nem1/ Spo7-Pah1/lipin axis regulates microautophagy induction in budding yeast, Biochem. Biophys. Res. Commun. 504 (2018) 505e512. [9] R. Hatakeyama, M.P. Peli-Gulli, Z. Hu, M. Jaquenoud, G.M. Garcia Osuna, A. Sardu, J. Dengjel, C. De Virgilio, Spatially distinct pools of TORC1 balance protein homeostasis, Mol. Cell 73 (2019) 325e338, e8. [10] M.G. Mostofa, M.A. Rahman, N. Koike, A.M. Yeasmin, N. Islam, T.M. Waliullah, S. Hosoyamada, M. Shimobayashi, T. Kobayashi, M.N. Hall, T. Ushimaru, CLIP and cohibin separate rDNA from nucleolar proteins destined for degradation by nucleophagy, J. Cell Biol. 217 (2018) 2675e2690. [11] R.L. Williams, S. Urbe, The emerging shape of the ESCRT machinery, Nat. Rev. Mol. Cell Biol. 8 (2007) 355e368. [12] W.M. Henne, N.J. Buchkovich, S.D. Emr, The ESCRT pathway, Dev. Cell 21 (2011) 77e91. [13] X. Ren, J.H. Hurley, VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo, EMBO J. 29 (2010) 1045e1054. [14] N. Dobzinski, S.G. Chuartzman, R. Kama, M. Schuldiner, J.E. Gerst, Starvationdependent regulation of golgi quality control links the TOR signaling and vacuolar protein sorting pathways, Cell Rep. 12 (2015) 1876e1886. [15] K. Dauner, W. Eid, R. Raghupathy, J.F. Presley, X. Zha, mTOR complex 1 activity is required to maintain the canonical endocytic recycling pathway against lysosomal delivery, J. Biol. Chem. 292 (2017) 5737e5747. [16] M.K. Sung, W.K. Huh, Bimolecular fluorescence complementation analysis system for in vivo detection of protein-protein interaction in Saccharomyces cerevisiae, Yeast 24 (2007) 767e775. [17] Y. Kodama, C.D. Hu, Bimolecular fluorescence complementation (BiFC): a 5year update and future perspectives, Biotechniques 53 (2012) 285e298. [18] K.E. Miller, Y. Kim, W.K. Huh, H.O. Park, Bimolecular fluorescence complementation (BiFC) analysis: advances and recent applications for genome-wide interaction studies, J. Mol. Biol. 427 (2015) 2039e2055. [19] P.S. Bilodeau, J.L. Urbanowski, S.C. Winistorfer, R.C. Piper, The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting, Nat. Cell Biol. 4 (2002) 534e539. [20] W.W. Li, J. Li, J.K. Bao, Microautophagy: lesser-known self-eating, Cell. Mol. Life Sci. 69 (2012) 1125e1136.
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TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast Shamsul Morshed a, Tasnuva Sharmin a, Takashi Ushimaru a, b, * a b
Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8021, Japan Department of Science, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8021, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 November 2019 Accepted 10 November 2019 Available online xxx
Microautophagy is promoted after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) kinase. Invagination of vacuolar membranes by endosomal sorting complex required for transport (ESCRT) is required for microautophagy. Vps27, a subunit of ESCRT-0, is recruited onto vacuolar membranes via dephosphorylation after TORC1 inactivation. Here, we showed that Hse1, another ESCRT0 subunit, is also recruited onto vacuolar membranes after TORC1 inactivation, promoting formation of ESCRT-0 complex on vacuolar membranes. Hse1 recruitment was dependent on Vps27, whereas Vps27 recruitment was independent of Hse1. Not only Vps27 but also Hse1 was required for ESCRT-III recruitment onto vacuolar membranes and microautophagy induction after TORC1 inactivation. This study revealed that ESCRT-0 (Vps27eHse1) complex formation on vacuolar membranes is important for microautophagy inactivation after TORC1 inactivation. © 2019 Elsevier Inc. All rights reserved.
Keywords: ESCRT-0 Vps27 Hse1 Microautophagy TORC1
1. Introduction Microautophagy degrades cargos by direct lysosomal/vacuolar engulfment of the cytoplasmic cargo without the formation of isolation membranes or autophagosomes [1e3]. Microautophagy can selectively degrade various specific cargos, such as the nucleus (micronucleophagy or piecemeal microautophagy of the nucleus), the ER (microERphagy) and lipid droplets (microlipophagy) in budding yeast Saccharomyces cerevisiae [4e6]. Vacuolar membrane proteins together with vacuolar membranes are degraded in the vacuole in the course of microautophagy. Therefore, overall microautophagy flux has been estimated using GFP-tagged vacuolar transmembrane proteins Vph1 and Pho8 [7]. When Vph1-GFP and GFP-Pho8 are incorporated into the vacuole by microautophagy, Vph1 and Pho8, but not the stable GFP moiety, are degraded by vacuolar proteases, producing free GFP, which is
Abbreviations: BiFC, bimolecular fluorescence complementation; ESCRT, endosomal sorting complex required for transport; FYVE, Fab1, YGL023, Vps27, and EEA1; GFP, green fluorescent protein; MVB, multivesicular body; PI3P, phosphatidylinositol 3-phosphate; RFP, red fluorescent protein; TORC1, target of rapamycin complex 1; UIM, ubiquitin-interacting motif; VHS, Vps27, Hrs and STAM. * Corresponding author. Graduate School of Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, 422-8021, Japan. E-mail address: [email protected] (T. Ushimaru).
detectable by immunoblotting. Microautophagy including micronucleophagy and microlipophagy is induced after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase [4,6e10]. The endosomal sorting complex required for transport (ESCRT) system, which was originally found as a device for the formation of intraluminal vesicles multivesicular bodies (MVBs) [11,12]. The ESCRT-0 complex first recognizes ubiquitinated cargo and clusters the ESCRT-I and eII complexes, promoting the assembly of ESCRT-III, which promotes the invagination, constriction, and abscission of membranes. ESCRT-0 consists of two subunits, Vps27 and Hse1 (Hrs and STAM1/2 in human), and these subunits interact in a 1:1 ratio. Vps27 binds to phosphatidylinositol 3-phosphate (PI3P) via a Fab1, YGL023, Vps27, and EEA1 (FYVE) domain, and to ubiquitin conjugated with membrane proteins via two ubiquitin-interacting motif (UIM) domains and a Vps27, Hrs and STAM (VHS) domain. In contrast, Hse1 has an UIM domain and a VHS domain, but not a FYVE domain. All of the five ubiquitin-binding sites in ESCRT-0 are required for MVB formation [13]. Vps27 is recruited onto vacuolar membranes after a diauxic shift (carbon starvation) [7]. TORC1 activity is necessary for maintaining Vps27 protein levels [9,14,15]. Vps27, as well as other components of ESCRT-I, eII, and eIII, is required for microautophagy induced by carbon starvation and rapamycin treatment [7,9]. By contrast, the
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Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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properties of Hse1 in microautophagy induction are largely unknown. Here, we show that Hse1 is also required for proper microautophagy induction after TORC1 inactivation. Hse1 recruitment was dependent on Vps27. Furthermore, we found that the ESCRT-0 complex is indeed formed on vacuolar membranes. This study provides basic properties of ESCRT-0 as to TORC1 inactivation-induced microautophagy induction. 2. Results 2.1. TORC1 inactivation promotes recruitment of Vps27 and Hse1 onto vacuolar membranes We wanted to know how TORC1 regulates ESCRT-mediated microautophagy induction. We suspected that a functional Vps27eHse1 ESCRT-0 formation is required for microautophagy induction after TORC1 inactivation. To test this idea, first we examined whether not only Vps27 but also Hse1 is recruited onto the vacuolar membranes after TORC1 inactivation. Faint punctate signals of Vps27 and Hse1 were observed in normal (favorable nutrient) conditions in some cells (Fig. 1A and B). However, strong punctate signals of both proteins appeared on vacuolar membranes after rapamycin treatment (Fig. 1A and B). This indicated that TORC1 inactivation promotes vacuolar membrane recruitment of Hse1, as well as that of Vps27. Thus, Hse1 as well as Vps27 was recruited onto vacuolar membranes after TORC1 inactivation. 2.2. TORC1 inactivation promotes ESCRT-0 complex formation on vacuolar membranes We suspected that both Vps27 and Hse1 are recruited onto vacuolar membranes after rapamycin treatment, forming ESCRT0 complex. To assess this idea, we examined colocalization of Vps27 and Hse1 using cells co-expressing Vps27-GFP and Hse1mCherry. We again found that rapamycin treatment facilitated focus formation of Vps27-GFP and Hse1-mCherry on the vacuolar membranes (Supplementary Figs. S1A and B). We noticed that most of the Vps27 foci on the vacuolar membranes contained Hse1 after rapamycin treatment (>70%) (Fig. 1C). This suggested that ESCRT-0 complex formation on vacuolar membranes is promoted after TORC1 inactivation. Of note, other Vps27 formed foci on vacuolar membranes, where Hse1 was absent (<30%). This indicated that a portion of Vps27 molecules were recruited onto vacuolar membranes without Hse1. In contrast, the vast majority of Hse1 foci on vacuolar membranes after rapamycin contained Vps27 (~95%) (Fig. 1C). In addition, Hse1 foci that did not contain Vps27 were small and faint (Supplementary Fig. S1C). These facts implicated that proper localization of Hse1 on vacuolar membranes after TORC1 inactivation requires Vps27 (see below). To confirm that Vps27 and Hse1 form the ESCRT-0 complex on vacuolar membranes after TORC1 inactivation, we performed bimolecular fluorescence complementation (BiFC) assays, which are useful for studying detection of in vivo proteineprotein interactions [16e18]. We created cells expressing an N-terminal fragment of Venus (VN)-tagged Vps27 (Vps27-VN) and a C-terminal fragment of Venus (VC)-tagged Hse1 (Hse1-VC). There were no obvious signals of the reconstituted Venus in normal conditions (Fig. 1D). However, puncta of the reconstituted Venus signals appeared on vacuolar membranes after rapamycin treatment. This indicated that TORC1 inactivation promotes the formation of Vps27eHse1 (namely, ESCRT-0) complexes on vacuolar membranes.
2.3. Hse1 is dispensable for Vps27 recruitment on vacuolar membranes after TORC1 inactivation Vps27 is a tethering factor of ESCRT-0 on MVBs [11]. Vps27 has a FYVE domain that binds to PI3P on lipid membranes. The FYVE domain of Vps27 is essential for the recruitment on vacuolar membranes after a diauxic shift (carbon starvation) [7]. We found that Vps27 formed foci on vacuolar membranes after rapamycin treatment (Fig. 2A). This Vps27 recruitment was not compromised in cells lacking Hse1, indicating that Hse1 is dispensable for Vps27 recruitment. Protein levels of Vps27 were similar in wild-type and hse1D cells in normal conditions (Supplementary Fig. S2A). This suggested that the protein stability of Vps27 is not reduced even if its partner Hse1 is absent in normal conditions. It was reported that Vps27 was degraded by autophagy after rapamycin treatment [9]. Consist with this notion, Vps27 was lost after rapamycin treatment was in wild-type (Supplementary Fig. S2A). Similar degradation of Vps27 after rapamycin treatment was observed in hse1D cells, indicating that autophagic degradation of Vps27 after TORC1 inactivation is not dependent on Hse1. 2.4. Vps27 is essential for Hse1 recruitment onto vacuolar membranes after TORC1 inactivation In contrast, Hse1 recruitment onto vacuolar membranes after rapamycin treatment was largely compromised in vps27D cells (Fig. 2B). This indicated that Vps27 is essential for Hse1 recruitment. Collectively, Vps27, but not Hse1, was a critical tethering factor for the recruitment of ESCRT-0 onto vacuolar membranes after TORC1 inactivation. In addition, these findings showed that Vps27 is recruited onto the vacuolar membranes after TORC1 inactivation in a manner independent of Hse1, but not vice versa. Protein levels of Hse1 were substantially equal in wild-type and vps27D cells in normal conditions (Supplementary Fig. S2B). This suggested that the protein stability of Hse1 is independent of Vps27. In sharp contrast to Vps27, the protein levels of Hse1 were not reduced in wild-type cells after rapamycin treatment. This suggested that Hse1 escapes from microautophagic degradation after TORC1 inactivation. However, Hse1 decreased after rapamycin treatment in vps27D cells. This suggested that Hse1 becomes unstable in the absence of Vps27 after TORC1 inactivation. 2.5. Hse1 is required for proper microautophagy induction after TORC1 inactivation The fact that not only Vps27 but also Hse1 was recruited onto vacuolar membranes after TORC1 inactivation allowed us to suspect that Hse1 is required for microautophagy induction, as well as Vps27. Loss of Vps27 markedly compromised microautophagy induction (monitored using a GFP-Pho8 processing assay) after rapamycin treatment (Fig. 3A), as reported previously [9]. In contrast, microautophagy induction was partially reduced in hse1D cells although to a lesser extent compared with that found in vps27D cells. This indicated that Hse1 is also required for proper microautophagy induction after TORC1 inactivation. 2.6. ESCRT-I to eIII are essential for TORC1 inactivation-induced microautophagy In the case of MVB formation, ESCRT-0 recruits ESCRT-I, eII, and eIII complexes onto endosomes. It has been shown that the ESCRT0 to eIII complexes are all critical for microautophagy induction after a diauxic shift (carbon starvation) [7]. Indeed, loss of Vps28 (ESCRT-I subunit), Vps36 (ESCRT-II subunit), and Vps24 (ESCRT-III subunit) remarkably impaired microautophagy induction after
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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Fig. 1. TORC1 inactivation promotes ESCRT-0 complex formation on vacuolar membranes. (A, B) Exponentially growing cells of strains SCU5926 (VPS27-GFP) and SCU5992 (HSE1-GFP) pretreated with FM4-64 (10 ng/ml) was treated with 200 ng/ml rapamycin for 3 h. Cell and fluorescence images were captured using a microscope. Scale bars, 5 mm. The average (±standard deviation) of percentages of cells with foci of Vps27 (A) or Hse1 (B) on vacuolar membranes was determined from three independent experiments. For statistical analysis, the p-values were calculated using a two-tailed Student’s t-test. ***, p < 0.0001. (C) Cells of strain SCU6116 (VPS27-GFP HSE1-mCherry) were treated with rapamycin for 3 h. Red arrows, Vps27-GFP foci that were not colocalized with Hse1-mCherry. Scale bars, 5 mm. Percentages of colocalization of Vps27-GFP foci colocalized with or without Hse1-mCherry (black bars) and Hse1-mCherry foci colocalized with or without Vps27-GFP (gray bars) on vacuolar membranes after rapamycin treatment for 3 h were determined from three independent experiments. (D) Cells of strain SCU6153 (VPS27-VN HSE1-VC) pretreated with FM4-64 were treated with rapamycin for 3 h. Scale bars, 5 mm. The average (±standard deviation) of percentages of cells with reconstructed Venus signals in BiFC assay on vacuolar membranes was determined from three independent experiments. ***, p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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Fig. 2. Hse1 is dispensable for Vps27 recruitment on vacuolar membranes after TORC1 inactivation, but not vice versa. (A) Cells of strains SCU5926 (VPS27-GFP) and SCU6050 (hse1D VPS27-GFP) pretreated with FM4-64 were treated with rapamycin for 3 h. Cell and fluorescence images were captured using a microscope. Scale bars, 5 mm. The average (±standard deviation) of percentages of cells with Vps27 foci on vacuolar membranes was determined from three independent experiments. (B) Cells of strains SCU5992 (HSE1-GFP) and SCU6070 (vps27D HSE1-GFP) pretreated with FM4-64 were treated with rapamycin for 3 h. Scale bars, 5 mm. The average (±standard deviation) of percentages of cells with Hse1 foci on vacuolar membranes was determined from three independent experiments.
rapamycin treatment (Fig. 3B). Thus, TORC1 negatively regulates microautophagy induction, which was mediated by ESCRT-0 to ESCRT-III. 2.7. Vps27 and Hse1 promote ESCRT-III recruitment onto vacuolar membranes after TORC1 inactivation Because Hse1, as well as Vps27, was required for proper microautophagy induction after TORC1 inactivation, we suspected that Hse1 is also a key factor for the recruitment of the following ESCRT complexes onto vacuolar membranes after TORC1 inactivation. To test this idea, we observed localization of ESCRT-III, a key factor for membrane deformation, after rapamycin treatment. We found clear promotion of recruitment of ESCRT-III (monitored using Vps24-GFP) onto vacuolar membranes in wild-type cells after
rapamycin treatment (Fig. 4). The recruitment of ESCRT-III was dramatically hindered in vps27D cells, whereas it was moderately reduced in hse1D cells. This was consistent with the fact that loss of Vps27 more remarkably compromised microautophagy induction after rapamycin treatment than loss of Hse1 (Fig. 3A). Protein levels of Vps24 decreased after rapamycin treatment (Supplementary Fig. S3). We found similar protein levels of Vps24 in vps27D and hse1D cells before and after rapamycin treatment. This indicated that the defect recruitment of Vps24 in cells lacking ESCRT-0 is not due to alterations in protein levels in Vps24. 3. Discussion In this study, we obtained several important findings on ESCRT0 formation after TORC1 inactivation: (1) TORC1 inactivation
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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Fig. 3. ESCRT is required for proper microautophagy induction after TORC1 inactivation. (A) Cells of strains SCU5466 (wild-type), SCU5457 (vps27D), and SCU6048 (hse1D) harboring plasmid pSCU2366 (pGFP-PHO8) [8] were treated with rapamycin for 6 h. Whole cell extracts were subjected to western blotting using the anti-GFP antibody. Free GFP processed from GFP-Pho8 after rapamycin treatment was measured using ImageJ software, and quantified by calculating the ratio of cleaved free GFP versus sum of uncleaved GFP-Pho8 and free GFP. The average (±standard deviation) was determined from three independent experiments, and relative values were normalized against the value in control cells are shown. The p-values were calculated using a two-tailed Student’s t-test. **, p < 0.001; ***, p < 0.0001. (B) Cells of strains SCU2684 (wild-type), SCU6187 (vps28D), SCU6188 (vps36D), and SCU4337 (vps24D) harboring plasmid pSCU2366 (pGFP-PHO8) were treated with rapamycin for 6 h. Whole cell extracts were subjected to western blotting. The ratio of cleaved free GFP versus sum of uncleaved GFP-Pho8 and free GFP was determined. ***, p < 0.0001.
promoted ESCRT-0 formation on the vacuolar membranes; (2) Vps27 was recruited on vacuolar membranes after TORC1 inactivation in a manner independent of Hse1, but not vice versa; and (3) Vps27 was critical for ESCRT-III recruitment onto the vacuolar membranes and microautophagy induction after TORC1 inactivation, whereas Hse1 was also required for the ESCRT-III recruitment and microautophagy induction after TORC1 inactivation. Both Vps27 and Hse1 have ubiquitin-binding UIMs and VHS domains and these domains is required for the function of ESCRT0 on endosomal membranes [13]. We showed that Hse1 could not be recruited onto vacuolar membranes after TORC1 inactivation
if Vps27 was absent. This indicated that the interaction between ubiquitin and UIM/VHS of Hse1 is insufficient for the localization of Hse1 onto vacuolar membranes after TORC1 inactivation. This further suggested that the interaction of the FYVE domain of Vps27 with PI3P on vacuolar membranes after TORC1 inactivation is critical for Vps27 recruitment onto vacuolar membranes (Supplementary Fig. S4). This is consistent with the finding that the FYVE domain of Vps27 is essential for the recruitment on vacuolar membranes after a diauxic shift (carbon starvation) [7]. Here, we utilized the BiFC assay for assessment for ESCRT0 (Vps27eHse1) formation. We found that the ESCRT-0 complex
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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Fig. 4. Vps27 and Hse1 promote ESCRT-III recruitment onto vacuolar membranes after TORC1 inactivation. Cells of strains SCU6092 (VPS24-GFP), SCU6094 (vps27D VPS24-GFP), and SCU6096 (hse1D VPS24-GFP) pretreated with FM4-64 were treated with rapamycin for 3 h. Cell and fluorescence images were captured using a microscope. Scale bars, 5 mm. The average (±standard deviation) of percentages of cells with Vps27 foci on vacuolar membranes was determined from three independent experiments.
was massively formed on vacuolar membranes after TORC1 inactivation. In contrast, there were no obvious BiFC signals in normal conditions using our BiFC system (Vps27-VN and Hse1-VC), although Vps27 and Hse1 form the ESCRT-0 complex on endosomes, and both proteins are indispensable for MVB formation and vacuolar protein sorting [19]. The BiFC assay is sensitive to the structure of complex of two proteins with interaction [16]. The structure of ESCRT-0 complexes on vacuolar membranes might be different from that on endosomes. TORC1 phosphorylates multiple residues of Vps27, probably causing structural changes in Vps27 [9]. Changes in the phosphorylation status of Vps27 might affect the structure of ESCRT-0 complexes. This study showed that the formation of the ESCRT-0 consisting of Vps27 and Hse1 on vacuolar membranes after TORC1 inactivation promoted the subsequential recruitment of ESCRT-III onto vacuolar membranes and microautophagy induction. Microautophagy is thought to be involved in various human diseases, such as neurodegenerative diseases [20]. However, in sharp contrast to macroautophagy, microautophagy is largely unknown with regard to its molecular mechanisms in not only yeast but also human cells. This study provides valuable insights into microautophagy-related diseases in human. 4. Materials and methods 4.1. Strains and media The S. cerevisiae strains used are listed in Supplementary Table S1. Glucose-containing YPAD (YPD containing 0.01% adenine) and synthetic minimal medium (SD) complemented with the appropriate nutrients for plasmid maintenance were prepared
using standard methods. For assessment of microautophagy after rapamycin treatment, when cells harbored plasmids, cells were precultured in SD medium with the appropriate nutrients, and then cultured in YPAD, followed by rapamycin treatment. 4.2. Western blotting analysis Exponentially growing cells were used for these experiments. Proteins were extracted using a post-alkaline extraction method in accordance with a previous report [10]. Briefly, cells (10 ml culture, OD600 ¼ 0.2e0.8) were treated with 200 ml of 0.1 M NaOH for 5 min and then the pellet was collected by centrifugation. The pellet was resuspended in sample buffer (60 mM Tris-HCl [pH 6.8], 5% glycerol, 2% SDS, 4% 2-mercaptoethanol, and 0.0025% bromophenol blue) at 95 C for 5 min. Crude extracts were cleared by centrifugation and the supernatant was used for western blotting analysis. We used the following antibodies: anti-GFP mouse monoclonal antibody (Santa Cruz, Cat#sc-9996; RRID: AB_627695), HRPconjugated anti-GFP goat polyclonal antibody (Rockland, #600103-215; RRID: AB_218193), and anti-Pgk1 mouse monoclonal antibody (Abcam, Cat# ab113687; RRID: AB_10861977). Chemiluminescence signals from Western BLoT Quant HRP Substrate (Takara, Cat #DS-T7102) for horseradish peroxidase (HRP) and Immuno Shot (Cosmo Bio, Cat #IS-012-250) as an immunoreaction enhancer solution were detected using an image analyser (Fuji LAS4000mini). All western blotting experiments were performed independently at least three times to confirm the reproducibility of the results. Relative protein amounts were measured using ImageJ software. The average and standard deviation were determined for each sample from three independent experiments, and relative values normalized against the value in control cells are shown. For
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064
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statistical analysis, the p-values were calculated using a two-tailed Student’s t-test. 4.3. Microscope observations Exponentially growing cells were used for experiments. Cell, GFP, Venus, and RFP (mCherry) images were captured using a Carl Zeiss Axio Imager M1 microscope with a cooled CCD camera (Carl Zeiss AxioCam MRm). All microscope observations were performed independently at least three times to confirm the reproducibility of the results. 4.4. BiFC assay Strain expressing Vps27-VN and Hse1-VC was constructed according to the previous study [16]. We confirmed expression of Vps27-VN and Hse1-VC by microscopic observation and western blotting analysis using the anti-GFP polyclonal or monoclonal antibodies. All microscope observations were performed independently at least three times to confirm the reproducibility of the results. Funding This work was supported in part by the Japan Society for the Promotion of Science (grant No. 18K06212). Author contributions TU designed the experiments. SM conducted most of the experiments. TS performed some experiments. TU mainly wrote the paper. SM helped to write the paper. Declaration of competing interest The authors declare no conflict of interests. Acknowledgments We thank Won-Ki Huh and Koji Okamoto for generous gifts of materials, and Ayumu Yamamoto, Yoko Kimura and laboratory members of TU for helpful discussions. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.064.
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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.064.
References [1] O. Muller, T. Sattler, M. Flotenmeyer, H. Schwarz, H. Plattner, A. Mayer, Autophagic tubes: vacuolar invaginations involved in lateral membrane sorting and inverse vesicle budding, J. Cell Biol. 151 (2000) 519e528. [2] T. Sattler, A. Mayer, Cell-free reconstitution of microautophagic vacuole invagination and vesicle formation, J. Cell Biol. 151 (2000) 529e538. [3] J.B. Kunz, H. Schwarz, A. Mayer, Determination of four sequential stages during microautophagy in vitro, J. Biol. Chem. 279 (2004) 9987e9996. [4] P. Roberts, S. Moshitch-Moshkovitz, E. Kvam, E. O’Toole, M. Winey, D.S. Goldfarb, Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae, Mol. Biol. Cell 14 (2003) 129e141. [5] S. Schuck, C.M. Gallagher, P. Walter, ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery, J. Cell Sci. 127 (2014) 4078e4088. [6] T. van Zutphen, V. Todde, R. de Boer, M. Kreim, H. Hofbauer, H. Wolinski, M. Veenhuis, I.J. van der Klei, S.D. Kohlwein, Lipid droplet autophagy in the yeast Saccharomyces cerevisiae, Mol. Biol. Cell (2014) 290e301. [7] M. Oku, Y. Maeda, Y. Kagohashi, T. Kondo, M. Yamada, T. Fujimoto, Y. Sakai, Evidence for ESCRT- and clathrin-dependent microautophagy, J. Cell Biol. 216 (2017) 3263e3274. [8] M.A. Rahman, M. Terasawa, M.G. Mostofa, T. Ushimaru, The TORC1-Nem1/ Spo7-Pah1/lipin axis regulates microautophagy induction in budding yeast, Biochem. Biophys. Res. Commun. 504 (2018) 505e512. [9] R. Hatakeyama, M.P. Peli-Gulli, Z. Hu, M. Jaquenoud, G.M. Garcia Osuna, A. Sardu, J. Dengjel, C. De Virgilio, Spatially distinct pools of TORC1 balance protein homeostasis, Mol. Cell 73 (2019) 325e338, e8. [10] M.G. Mostofa, M.A. Rahman, N. Koike, A.M. Yeasmin, N. Islam, T.M. Waliullah, S. Hosoyamada, M. Shimobayashi, T. Kobayashi, M.N. Hall, T. Ushimaru, CLIP and cohibin separate rDNA from nucleolar proteins destined for degradation by nucleophagy, J. Cell Biol. 217 (2018) 2675e2690. [11] R.L. Williams, S. Urbe, The emerging shape of the ESCRT machinery, Nat. Rev. Mol. Cell Biol. 8 (2007) 355e368. [12] W.M. Henne, N.J. Buchkovich, S.D. Emr, The ESCRT pathway, Dev. Cell 21 (2011) 77e91. [13] X. Ren, J.H. Hurley, VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo, EMBO J. 29 (2010) 1045e1054. [14] N. Dobzinski, S.G. Chuartzman, R. Kama, M. Schuldiner, J.E. Gerst, Starvationdependent regulation of golgi quality control links the TOR signaling and vacuolar protein sorting pathways, Cell Rep. 12 (2015) 1876e1886. [15] K. Dauner, W. Eid, R. Raghupathy, J.F. Presley, X. Zha, mTOR complex 1 activity is required to maintain the canonical endocytic recycling pathway against lysosomal delivery, J. Biol. Chem. 292 (2017) 5737e5747. [16] M.K. Sung, W.K. Huh, Bimolecular fluorescence complementation analysis system for in vivo detection of protein-protein interaction in Saccharomyces cerevisiae, Yeast 24 (2007) 767e775. [17] Y. Kodama, C.D. Hu, Bimolecular fluorescence complementation (BiFC): a 5year update and future perspectives, Biotechniques 53 (2012) 285e298. [18] K.E. Miller, Y. Kim, W.K. Huh, H.O. Park, Bimolecular fluorescence complementation (BiFC) analysis: advances and recent applications for genome-wide interaction studies, J. Mol. Biol. 427 (2015) 2039e2055. [19] P.S. Bilodeau, J.L. Urbanowski, S.C. Winistorfer, R.C. Piper, The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting, Nat. Cell Biol. 4 (2002) 534e539. [20] W.W. Li, J. Li, J.K. Bao, Microautophagy: lesser-known self-eating, Cell. Mol. Life Sci. 69 (2012) 1125e1136.
Please cite this article as: S. Morshed et al., TORC1 regulates ESCRT-0 complex formation on the vacuolar membrane and microautophagy induction in yeast, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.064