Accelerated invagination of vacuoles as a stress response in chronically heat-stressed yeasts

ABSTRACT When exposed to sublethal high temperatures, budding yeast cells can survive for a period of time; however, a sufficient amount of ubiquitin is necessary for this survival. To

understand the nature of the stress, we examined the morphological changes in yeast cells, focusing on the vacuoles. Changes in vacuolar morphology were notable, and ruffled vacuolar

membranes, accelerated invaginations of vacuolar membranes, and vesicle-like formations were observed. These changes occurred in the absence of Atg1, Atg9 or Ivy1 but appeared to require

endosomal sorting proteins, such as Vps23, Vps24 or Pep12. Furthermore, the serial sections of the vacuoles analysed using an electron microscopic analysis revealed that spherical

invaginated structures were linked together in a vacuole. Because degradation of cell surface proteins is induced from heat stress, fusion of endosomal and vacuolar membranes might occur

frequently in heat-stressed cells, and yeast cells might be able to cope with a rapid increase in vacuolar surface area by such invaginations. SIMILAR CONTENT BEING VIEWED BY OTHERS

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September 2020 INTRODUCTION In response to elevated temperatures, organisms initiate a sequence of events that function to cushion these stresses1,2,3,4. In particular, the activities of

protein quality-control systems, such as molecular chaperones or protein degradation machinery, are enhanced. In addition to the canonical heat shock response, the cell wall stress pathway

and oxidative stress response are activated. Moreover, the transport systems, cytoskeletal organization and energy metabolism are also modulated. These heat responses are commonly expected

at elevated temperatures; however, there might be differences in the means of achieving thermotolerance, depending on the temperature and duration of heat. Molecular chaperone heat shock

protein (Hsp)104, which is induced by brief heat stress such as 37 °C along with other heat shock proteins, is necessary for the thermotolerance at acute and lethal high temperatures, such

as 50 °C for 10–20 min (induced thermotolerance)5. In contrast, the polyubiquitin-encoding gene _UBI4_ is not necessary for the induced thermotolerance, but is required for chronic heat

stress of sub-lethal high temperatures such as at 38.5 °C–41 °C (ref.6 and unpublished data)6. Under normal conditions e.g. at 25 °C, _UBI4_ is neither expressed significantly nor required

because the other ubiquitin-encoding genes _UBI1–3_ provide enough of the ubiquitins needed for cell growth. Ubiquitin homeostasis is critical for the maintenance and growth of the cell, and

the fact that _UB14_ is induced under heat stress indicates that many ubiquitins are required for survival under such stress conditions7. Moreover, lysine(K)63-linked ubiquitination but not

K48-linked ubiquitination, is critical for the survival of the heat stress8. Ubiquitination plays a variety roles in the cell; however, in general, K48-linked ubiquitination is utilized

during proteasomal degradation, whereas K63-linked ubiquitination is utilized in events such as endocytosis and endosomal sorting toward vacuoles, suggesting that the latter functions are

critical for survival during chronic heat stress9. The reason why the different proteins are required to mitigate the two different types of heat stress has not been clearly elucidated.

Because Hsp104 has been shown to disaggregate partially unfolded proteins10,11, it is considered that the protein unfolding damage is so severe at the lethal temperatures that disaggregation

of the misfolded proteins, which are caused by the stress, would be the most critical point to rescue the cell. On the other hand, although the reason why cells need many ubiquitins after

sublethal heat stress is not so clear, one can speculate that the protein-folding damage might not be severe enough to cause direct cell death. Rather, by removing unfolded proteins and

using many ubiquitins, the cellular systems might be remodeled or reconstructed to adapt to such heat stress for the long-term survival of the cell. The report by Zhau that the toxic effects

of overexpression of ubiquitin-substrates of cell surface proteins at higher temperatures supports this idea12. Indeed, the cell surface region appears to be remodeled after heat stress.

For example, the degradation of several cell surface proteins, such as transporters, proton pumps or pheromone receptors, after various heat stresses, and the increase in chitin content,

which is most likely activated by the cell wall stress pathway, have been reported12,13,14,15; however, the physiological changes or consequences that occur in the cell after chronic heat

stress and that change the cell into a heat-tolerant state have not been fully investigated. The vacuole is a degradative and dynamic organelle whose morphology changes in response to

various stimuli or stresses16,17. Vacuoles fuse or fission depending on the stimuli: vacuoles fuse during stationary phase, starvation or under hypotonic conditions, and fission under

hypertonic conditions. The autophagic responses are induced in response to nutrient starvation18,19. After heat stress, negative curvature formations of vacuolar membranes have been

reported20. In addition, the vacuole is the place where several cellular surface proteins are degraded after heat stress. To investigate the nature of the reactions to chronic sub-lethal

heat stress, we started to look for the differences between wild type and _ubi4_Δ cells, and examine changes in cellular morphology in the yeast _Saccharomyces cerevisiae_. In this study, we

focused on the morphological changes of the vacuoles after the stress, and further investigated any relevance to Δ_ubi4_ and other mutations. RESULTS PHYSIOLOGICAL PROPERTIES UNDER THE

CHRONIC HEAT STRESS To better understand the effects of chronic sublethal heat stress, we first evaluated the viability at 40.5 °C, a sublethal temperature for yeast. Cell growth was

retarded in wild-type cells after the shift to 40.5 °C, increasing by only 2.0-fold after 6 h compared with 13.4-fold and 7.9-fold at 25 °C and at 37 °C, respectively; however, staining the

dead cells with Phloxine B revealed that 95% of the cells were alive after 6 h at 40.5 °C (Fig. 1a,b), but that this percentage decreased after 9 h, and reached 57% after 24 h. In the

Δ_ubi4_ mutant cells, the number of Phloxine B-stained cells sharply increased after 9 h, and nearly all cells were stained after 24 h. After heat stress, several plasma membrane proteins,

including Dip5, Ste3, Pma1, Can1, Hxt3, Mup1, Gap1, and Lyp1, have been reported to be ubiquitinated, transported and degraded in the vacuoles12,13,14. Moreover, overexpression of plasma

membrane proteins have been shown to cause toxicity at elevated temperatures12. In accordance with these findings, Hxt3-3HA and Lyp1-3HA levels decreased in wild-type cells at 40.5 °C (Fig. 

1c,d). However, in _ubi4_Δ mutant cells, Hxt3-3HA levels decreased, whereas Lyp1-3HA levels increased after an initial decrease, suggesting that the accumulation of the proteins that should

be removed might be one of the reasons for the loss of viability in the _ubi4_Δ cells. MORPHOLOGICAL CHANGES IN VACUOLES AFTER CHRONIC HEAT STRESS After a temperature shift to 40.5 °C, cells

became larger and rounder (Fig. 2a). We decided to carefully examine the vacuolar structures in the cell after exposure to chronic heat stress because the protein transport toward the

vacuoles involves the K63-linked ubiquitination, and the ruffling of vacuolar membranes by heat stress has been reported20. FM4-64 staining revealed that compared with the round and

single-lobed structures of vacuoles at 25 °C, wild-type cells exhibited various vacuolar morphologies along with the fissions of vacuoles at 40.5 °C (Fig. 2a, Supplementary Fig. 1a). At 25 

°C, 92% of cells contained only one vacuole and the rest contained more than two vacuoles, whereas after 6 h at 40.5 °C only 25% of cells contained only one vacuole. Vacuoles were highly

distorted in some cells, such as negative membrane curvatures. In addition, we observed small vesicles in the vacuoles of some cells; some of which appeared to be anchored to vacuolar

membranes, whereas others were mobile. Using a different strain, BY20695, we observed similar vesicles at 40.5 °C, but vacuole fission was not enhanced after heat stress. In this strain, 53%

and 63% of the cells contained only one vacuole at 25 °C and 40.5 °C, respectively. The cells with vesicles were Phloxine B negative, indicating that they were viable (Supplementary Fig. 

1c). It should be noted that the overall FM4-64 fluorescence on the vacuolar membrane was much brighter in cells at 40.5 °C than at 25 °C (Fig. 2a; Supplementary Fig. 1d,e) although the

fluorescence was not always evenly distributed along the membrane at 40.5 °C (Fig. 2e(ii)), suggesting that FM4-64 uptake was accelerated at 40.5 °C. To evaluate invaginations, we followed

vacuolar morphological changes using yeasts that expressed Rpl24A-green fluorescent protein (GFP), a cytoplasmic ribosome subunit fused with GFP, following a temperature shift to 40.5 °C.

Vesicle-like structures started to appear 1 h after the shift, although rarely, and increased by 6 h at 40.5 °C (Fig. 2b,c). We found that the majority of the vesicle-like structures

exhibited GFP fluorescence surrounded by a FM4-64 fluorescent contour, and a portion of the vesicle-like structures was surrounded by both FM 4-64 and GFP fluorescence (Fig. 2d,

Supplementary Fig. 1f). Ruffled vacuole membranes were increased after 3 h at 40.5 °C, and at a similar level after 6 h (Fig. 2e). On examination of vacuolar structures in _ubi4_Δ cells, we

observed that many vacuoles were swollen and remained single-lobed after exposure to heat stress (Fig. 2a, Supplementary Fig. 1a). At 25 °C, 89% of the cells contained only one vacuole, and

at 40.5 °C, 77% of cells still contained one vacuole. Vacuole membranes were not significantly ruffled compared with those of the wild-type. Although vesicles were also detected within the

vacuoles, these tended to be located near the vacuolar membranes, and extended vacuolar invaginations were not frequently observed compared with those in wild-type cells. Another method by

which to follow the vacuolar membrane changes is to observe the localization of Vph1, a subunit of a vacuolar ATPase on the vacuolar membrane21. It has been reported that Vph1-GFP is rather

homogeneously distribution on vacuolar membranes at normal temperatures but exhibits dotted localization after 3 h at 37 °C22. Consistent with that report, Vph1-GFP showed homogeneous

distribution at 25 °C and dot-like localization after 6 h at 40.5 °C in wild-type cells (Fig. 3a). With Vph1-GFP fluorescence, vesicle-like structures were also observed. Interestingly,

Vph1-GFP was uniformly distributed on vacuolar membranes at 40.5 °C in _ubi4_Δ cells. Because it has been suggested that the region that lacks Vph1-GFPis enriched with sterols in

stationary-phase cells, there is a possibility that domains with different lipid contents are generated after the heat stress in wild-type cells, but not in _ubi4_Δ cells. This observation

of _ubi4_Δ cells was similar to the results of a previous report that some vesicular transport mutants exhibited a uniform distribution of Vph1-GFP at the stationary phase22; therefore, we

considered Vph1-GFP fluorescence in the _vps23_Δ cells in which endosomal sorting was impaired. After heat stress at 40.5 °C, the mutant showed homogeneously distributed GFP fluorescence on

the vacuolar membranes along with a foci structure, most likely the class E compartment, an aberrant late endosomal structure23. In addition, its vacuolar membrane was observed to be smooth,

and budding-like structures of vacuolar membranes were not detected (Fig. 3a). On the basis of these observations, we further examined the vacuole morphology of vesicular transport mutants

with FM4-64 staining after chronic heat stress (Fig. 3b). We observed that _vps24_Δ, _vps23_Δ and _pep12_Δ mutants exhibited similar phenotypic vacuolar morphology; their vacuolar membranes

did not show significant invaginated structures and remained smooth after chronic heat stress. In these cells, highly mobile FM4-64 fluorescent materials, which did not look like vesicle

structures, were often observed in their vacuoles. These mutants were reported to be heat-sensitive24,25. Indeed, some of the _vps23_Δ and _vps24_Δ cells appeared to be dying after 6 h at

40.5 °C (data not shown), and they showed loss of viability after the chronic heat stress (Supplementary Fig. 3). To determine whether the vesicle-like structures were generated from

autophagic events, we examined the effects of _atg1_Δ and _atg9_Δ mutations (Fig. 3b). Both Atg1 and Atg9 are required for macroautophagy26. The vacuolar morphologies of these mutants after

6 h at 40.5 °C were very similar to those of the wild type. Next, we examined _ivy1_Δ mutations after chronic heat stress, because Ivy1 was reported to be localized at the invaginated

vacuolar membranes and implicated in microautophagy, an inward budding of the cytoplasm from the vacuolar membrane20,27. Consistent with previous reports on invaginations after heat stress

in the _ivy1_Δ mutant20, we observed both the invaginations and the vesicle-like formations in the mutant after chronic heat stress (Fig. 3b). These results suggest that the formation of

vesicle-like structures after chronic heat stress does not involve either the macroautophagy process or Ivy1. In addition, increases in vesicle-like structures were not observed in the

_pep4_Δ mutant after 6 h at 40.5 °C (Fig. 3c), which suggested that these structures were apparently not degraded by Pep4. We then examined the fine structures using electron microscopy

(EM). The vacuoles exhibited smooth membranes in wild-type cells at 25 °C (Fig. 4a). In contrast, vacuoles of cells at 40.5 °C exhibited various shapes (Fig. 4b–e, Supplementary Fig. 4).

Vacuolar membranes were angular or not smooth in about half of the cells. At a frequency similar to the light microscopic analysis, the invagination of the cytoplasm and vesicle-like

structures were observed in EM. Multiple vesicular bodies (MVBs) were occasionally observed in bud-like invaginations (Fig. 4c). Invaginated structures with a bumpy morphology were also

occasionally observed (Fig. 4d). In addition, doughnut-like structures in which a belt of cytoplasm surrounded the vacuolar lumen were occasionally observed (Fig. 4b,e). These might be the

vesicle-like structures surrounded by GFP and FM4-64 fluorescence in cells expressing Rpl24a-GFP as shown in Fig. 2d and Supplementary Fig. 1f; however, how these structures were formed

remains unclear. During EM analysis, we unexpectedly noticed more lipid droplets in cells after 6 h at 40.5 °C than at 25 °C (Fig. 4b,e,f). In addition, these were often observed at the

invaginated vacuolar membrane or in contact with vacuolar membranes. In _ubi4_Δ cells, we also observed bud-like invaginations after 6 h at 40.5 °C, although these were tended to be located

near the vacuolar membranes, and vacuolar membranes were smoother than those in wild-type cells (Fig. 5a, Supplementary Fig. 5), which was consistent with the images seen in FM4-64 staining.

Vacuolar membranes in _vps23_Δ cells were much smoother, and vesicle-like cytosolic structures were not observed after 6 h at 40.5 °C (Fig. 5b, Supplementary Fig. 6). Instead, ~40% of the

_vps23_Δ cells contained unidentified constituents within the vacuoles; these might be the FM4-64 fluorescent highly mobile constituents shown in the vacuole in Fig. 3b. Finally,

three-dimensional observations (3D) of serial sections of vacuoles were conducted to analyse the vesicle-like structures (Fig. 6; Supplementary Fig. 7; Supplementary Videos 1,2,3,4,5 and 6).

Surprisingly, vesicle-like structures did not exist independently; instead, several vesicle-like structures were either linked together in a vacuole or connected to the cytoplasm. The

average diameter of the vesicle-like structures was calculated to be ~0.6 μm using the images of the serial sections. Sometimes, we observed lipid droplets within the linked structures.

Among the six series of serial sections observed, we did not find any free spherical vesicles. Although the possibility of microautophagy for the vesicle-like structures remained, it was

suggested that many vesicle-like structures observed in the vacuoles did not form a free single sphere in chronically heat-stressed cells. DISCUSSION To understand the nature of chronic

sublethal heat stress on yeast, we examined the physiological changes in the cells after the stress, and focused on the specific changes in vacuolar morphology in this study. We observed

vesicle-like structures that formed in vacuoles when subjected to the chronic heat stress, and observations of serial sections of the vacuoles using EM revealed that these vesicle-like

structures were linked. In addition, our results suggest that endosomal sorting proteins Vps23, Vps24 and Pep12 are necessary for invagination of vacuolar membranes and vesicle-like

formation after heat stress. We suggest that linked vesicle-like structures are a new type of vacuolar invaginated structures observed in chronically heat-stressed cells. Together with our

results that showed these endosomal sorting proteins are needed, we propose a model that explains the formation of these structures (Fig. 6e). After heat stress, several endosomes fuse with

vacuolar membranes, and bud-like invaginations are formed because of the rapid increase in these membranes. It could be speculated that the number of endosomes that fuse with the vacuoles

might increase after heat stress because more cell surface proteins were reported to be lost as the temperature increased12. The endosomes travel to these invaginations, forming new

invaginations from existing ones. The subsequent endosomes are formed in the same manner, and result in chains of vesicle-like structures. If our speculation is correct, the cells might be

able to cope with massive increases in vacuolar membranes by forming such invaginated structures without an increase in vacuolar volume; however, to test this model and to determine the

process by which the linked vesicle-like structures are formed, time-lapse analysis is needed to capture the onset of invagination. In addition, additional studies are needed to determine

whether the formation of these linked structures is a reversible process. A preliminary study demonstrated that when the temperature was returned to 25 °C following exposure to a temperature

of 40.5 °C for 4 h, a decrease of vesicle-like structures occurred in the vacuole after 2 h; however, when the temperature was returned to 25 °C after 40.5 °C for 6 h, this significant

decrease was not evident after 2 h (data not shown). Thus, we speculate that invaginations are easily reversible in the early stages, and that excessive invaginations are irreversible or

take time to revert. Vesicle-like structures in vacuoles have been reported previously27,28. For example, vesicle-like structures in the vacuole were reported by the addition of glycerol28.

We are not certain whether these are the same vesicle-like structures; such structures could be formed not only because of chronic heat stress but also because of other environmental

changes. In addition, vesicle-like structures might be intralumenal fragments, which are produced during vacuolar fusions29. However, in the W303 strains used in this study, vacuoles were

rather frequently fragmented by heat stress, therefore, we do not think this is likely. Moreover, from the results of Vph1-GFP distribution, we suggest that the raft-like domains having

different sterol contents, which could be formed in the vacuolar membranes under heat stress, might affect the invagination region and frequency in forming vesicle-like structures. In our

study, the _ubi4_Δ mutant exhibited less clear phenotypes in terms of vacuolar structures. The vesicle-like structures in the vacuole were observed in the mutant like as wild-type, but the

vacuolar membranes of the _ubi4_Δ mutant were smoother than those of the wild type, but less smooth than those of the _vps23_Δ mutant. Regarding the Vph1-GFP fluorescence, both _ubi4_Δ and

_vps23_Δ mutants exhibited homogeneous distribution, whereas wild-type cells exhibited dotted localizations on the vacuolar membrane at 40.5 °C. We speculate that the impairment of

ubiquitination in _ubi4_Δ cells might result in decreased transport of endosomes to vacuoles and fewer fusion events of vacuolar membranes and endosomes, resulting in the observed vacuolar

structures and membranes. The invagination of the vacuoles and the vesicle-like formations in the vacuoles occurred in the absence of either Atg1 or Atg9, which are essential for

macroautophagy, and of Ivy1, which has been implicated in microautophagy; therefore, the phenomena of macroautophagy and microautophagy, which are dependent on these Atg proteins and Ivy1,

are not involved in this process. Although the increase in vesicle-like structures containing Rpl24A-GFP was not detected in _pep4_Δ cells after 6 h at 40.5 °C, whether microautophagy occurs

after chronic heat stress has yet to be investigated. Microautophagy that was dependent on endosomal sorting proteins but independent of core Atg protein(s) was recently reported to occur

after a diauxic shift or a chronic phospholipid deficient state30,31. Similarly, the degradation of an ubiquitinated vacuolar membrane protein in the vacuoles has shown to require endosomal

sorting proteins32. Therefore, it is reasonable to think that similar events might occur after chronic heat stress. During the course of EM study, we unexpectedly found that more lipid

droplets were produced after a shift to 40.5 °C for 6 h. We found that lipid droplets were present in the invaginations, and, from EM images of serial sections of the vacuoles, that some

lipid droplets were linked to cytoplasmic vesicle-like structures. The physiological implication of lipid droplet localization around the vacuoles has not been investigated, but it is

possible that lipid droplets are sequestered in the invaginated area of the vacuoles during heat stress. The lipid droplets produced under several different conditions were reported to be

digested, possibly through microautophagy31,33,34,35,36. Thus, we are currently investigating whether microlipophagy occurs under chronic heat stress. Finally, due to the progress of global

warming, it is likely that more and more organisms are expected to have a chance to be exposed to sub-lethal temperatures in the future37. We believe that our study will contribute to the

understanding how the cellular activities are influenced by these temperatures. METHODS MEDIA AND YEAST STRAINS Yeast strains were grown in YPAD medium [1% yeast extract, 2% Bacto–Peptone or

Hipolypepton (Nihon Seiyaku), 2% glucose, and 0.002% adenine]. A list of yeast strains is provided in Supplementary Table 1. W303 strains were used if not indicated.

Vph1-GFP-expressing-yeasts and _pep12_Δ mutants were SEY6210 and BY4741 strains, respectively. DETERMINATION OF CELL VIABILITY For yeast viability assays, yeast cells at the early log phase

grown at 25 °C were transferred to 40.5 °C, and incubated with a moderately slow shaking speed (70 turns/min) in a water temperature-chamber. Staining with Phloxine B was performed using the

modified method of Noda38. Briefly, cells were pelleted by centrifugation at 300 × g for 3 min and washed twice with 1x PBS. This was followed by resuspension in 1x PBS containing Phloxine

B at a final concentration of 5 μg/ml. Cells were observed under a fluorescence microscope using a red filter. In total, approximately 200 cells were observed after each staining, and the

ratio of Phloxine B-negative cells to the total number of cells was calculated. For the colony formation assay, 10 μl of culture was diluted in 10 ml of 1x PBS, plated onto YPAD medium in

duplicates, and incubated at 25 °C for 2 days. The culture was directly plated onto the YPAD medium when the cell viability was very low. IMMUNOBLOTTING Preparation of whole-cell extracts

and immunoblot analysis were performed essentially as described previously39. Cells (1–3 × 107) were washed with water and suspended in 200 μl of cold ethanol containing 2 mM PMSF. Cells

were broken by agitation with 200 μl of glass beads for 10 min, and chilled at −20 °C. Cells were dried, suspended in sample buffer and heated at 96 °C for 5 min. In western blotting, the

blots were incubated with mouse anti-GFP monoclonal antibody (Roche), anti-HA antibody (TANA2, MBL), anti-ubiquitin antibody (P4D1, Santa Cruz), or anti-yeast phosphoglycerate kinase (PGK)

antibody (Molecular Probes, Eugene, OR), followed by horseradish peroxidase (HRP)-conjugated anti-mouse IgG (#NA931V, GE Healthcare), and visualized using a chemiluminescent reagent.

MICROSCOPY FM4-64 staining was performed as described previously40, and the cells were treated with FM 4–64 just before the temperature shift. For the treatment of FM4-64, 2 ml culture of

cells grown at 25 °C in YPAD medium were centrifuged at 5000 rpm for 1 min, and suspended in 49 μl of YPAD. To the cells, 1 μl of 2 mM FM4-64 (Molecular Probes, Inc.) was added at a final

concentration of 40 μM, and incubated for 20 min at room temperature. Then cells were washed with 1x PBS and suspended in 2 ml of YPAD, followed by the heat treatment. After heat treatment,

cells were collected by centrifugation and imaged at room temperature using a confocal microscope (LSM700; Carl Zeiss) equipped with a 100x oil or 40x water objective lens. Microscopic

observation started 10–15 min after the heat treatment. Images were processed using the LSM image browser, and the brightness and contrast were adjusted using Zen. ELECTRON MICROSCOPY

Electron microscopy was performed by Tokai-EMA, Inc. Rapid freezing and freeze-fixation methods were employed. After collection of cells by centrifugation, the small number of cells were

sandwiched between copper disks, and frozen in liquid propane at −175 °C. They were freeze-substituted with 2% glutaraldehyde and 0.5% tannic acid in acetone, followed by 2% distilled water

at −80 °C for 2 days. The cells were fixed with 2% osmium tetroxide in acetone. The cells were then dehydrated, infiltrated with propylene oxide, embedded in Quetol-651 and observed using a

transmission electron microscope (JEM-1400Plus; JEOL Ltd.). Serial sections were sliced at approximately 80 nm. Images were adjusted using GIMP. 3-D reconstructions of EM images were

performed by using Image J software. Flip EM images were assembled using GIMP. DATA AVAILABILITY No datasets were generated or analyzed during the current study. REFERENCES * Parsell, D. a.

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Integrated Imaging Research Support for electron microscopic analysis. We also thank M Ohsumi, Y Ohsumi, T Ushimaru, A Kawamata, and M Welte for discussions, Y Ohsumi, A Kawamata, T

Ushimaru, H Yashiroda, N Matsuda, and Y Yoshida for materials, and H Akabayashi for statistical analysis. Strain BY20695 was provided by the National Bio-Resource Project of the MEXT, Japan.

This work was supported by a Grant-in aid for Scientific Research (C) No. JP2644007. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Graduate School of Integrated Science and Technology,

Shizuoka University, Shizuoka, 422-8529, Japan Ayane Ishii, Masahito Kawai, Haruka Noda, Kohei Takeda, Tomohiro Sasanami & Yoko Kimura * Department of Agriculture, Shizuoka University,

Shizuoka, 422-8529, Japan Hiroyuki Kato, Kotomi Asakawa, Yoshinobu Ichikawa & Yoko Kimura * Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science,

Setagaya-ku, Tokyo, 113-8613, Japan Keiji Tanaka Authors * Ayane Ishii View author publications You can also search for this author inPubMed Google Scholar * Masahito Kawai View author

publications You can also search for this author inPubMed Google Scholar * Haruka Noda View author publications You can also search for this author inPubMed Google Scholar * Hiroyuki Kato

View author publications You can also search for this author inPubMed Google Scholar * Kohei Takeda View author publications You can also search for this author inPubMed Google Scholar *

Kotomi Asakawa View author publications You can also search for this author inPubMed Google Scholar * Yoshinobu Ichikawa View author publications You can also search for this author inPubMed

 Google Scholar * Tomohiro Sasanami View author publications You can also search for this author inPubMed Google Scholar * Keiji Tanaka View author publications You can also search for this

author inPubMed Google Scholar * Yoko Kimura View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS A.I. performed microscopic analysis, western

blotting and made figures. M.K. did microscopic studies and made figures. H.N. did western blotting and made figures. H.K. performed viability assays. Ko.T. did microscopic analysis. Y.I.

did microscopic analysis. T.S. did microscopic analysis. K.A. made strains. Ke.T. designed the initial experiments and made an input for writing manuscript, and Y.K. designed the

experiments, wrote the manuscript and supervised all the experiments. CORRESPONDING AUTHOR Correspondence to Yoko Kimura. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

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ARTICLE Ishii, A., Kawai, M., Noda, H. _et al._ Accelerated invagination of vacuoles as a stress response in chronically heat-stressed yeasts. _Sci Rep_ 8, 2644 (2018).

https://doi.org/10.1038/s41598-018-20781-8 Download citation * Received: 09 May 2017 * Accepted: 24 January 2018 * Published: 08 February 2018 * DOI:

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