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MR, JB and KR designed experiments. MR and KR wrote the paper. MR, JB, both MS, MKS, JS, FG, JD, NB, PB, DH and RL performed experiments.
In recent years it turned out that there is not only extensive communication between the nucleus and mitochondria but also between mitochondria and lipid droplets (LDs) as well. We were able to demonstrate that a number of proteins shuttle between LDs and mitochondria and it depends on the metabolic state of the cell on which organelle these proteins are predominantly localized. Responsible for the localization of the particular proteins is a protein domain consisting of two α-helices, which we termed V-domain according to the predicted structure. So far we have detected this domain in the following proteins: mammalian BAX, BCL-XL, TCTP and yeast Mmi1p and Erg6p. According to our experiments there are two functions of this domain: (1) shuttling of proteins to mitochondria in times of stress and apoptosis; (2) clearing the outer mitochondrial membrane from pro- as well as anti-apoptotic proteins by moving them to LDs after the stress ceases. In this way the LDs are used by the cell to modulate stress response.
Mitochondria fulfill a pleiotropy of functions in the cell and are one of the most versatile organelles. Via the electron transport chain and OXPHOS they produce most of the ATP a cell needs. They are involved in fatty acid and amino acid metabolism.1 Furthermore they are a major production site of reactive oxygen species (ROS), which can be used as signaling molecules. ROS are, for example, part of an communication network between mitochondria and the nucleus in homeostasis and stress (reviewed in ref. 2). Although a certain level of ROS can be beneficial for the cell,3 excessive levels have detrimental effects. It has been known for decades that mitochondria and their dysfunction are heavily involved in the aging process as well as age-associated diseases.4,5 Superoxide originating from complexes I and III has the potential to damage mtDNA, proteins and lipids.6 During the aging process, ROS levels increase tremendously, resulting in accumulated mitochondrial damage and creates a vicious cycle.4 As a result the mitochondrial morphology changes from a tubular network to an isolated, blob-shaped appearence.6,7 This change in morphology may represent a way for the cell to separate damaged from undamaged mitochondria.
Systems for counteracting ROS damage include dismutation of superoxide by superoxide dismutases,8 refolding of unfolded proteins by chaperones9 and the degradation of misfolded proteins by the mitochondrial proteases.10 If, however, the accumulated damage exceeds a certain threshold the cell commits to apoptosis leading to specific events: (1) translocation of specific proteins to mitochondria; (2) mitochondrial fission and cristae disorganization; (3) outer membrane permeabilization; (4) calcium influx; (5) release of mitochondrial pro-apoptotic factors such as cytochrome c; and (6) a loss of the mitochondrial membrane potential.11–13 Of crucial importance for apoptosis is the shuttling of pro- and anti-apoptotic proteins to mitochondria.14,15 Prominent examples are MCL1, BMF, BAX, BCL2, BCL-XL, NOXA, Puma, BAD, BAK, BID, BIK and BIM.16,17
Among these BAX is a key protein. After activation it changes its structure, inserts into the outer mitochondrial membrane, and forms a dimer leading to the formation of a higher oligomerized structure. This oligomerization leads to a permeabilization of the outer mitochondrial membrane.18,19 In contrast, BCL-XL is a prominent example for proteins that inhibit this oligomerization.20,21 The cell is able to remove damaged, non-functional mitochondria by mitophagy.22 According to the present article, this is the only mechanism that enables the cell to deal with damaged mitochondria without committing to apoptosis.
In this article, we propose the existence of an alternative mechanism that enables the cell to regulate the translocation of pro- and anti-apoptotic proteins to mitochondria and the initiation of the apoptotic process. We also characterized a special domain in these proteins, responsible for the localization of various proteins to mitochondria as well as their shuttling between mitochondria and lipid droplets (LDs).
Besides some of the more well-known pro- and anti-apoptotic proteins there are others, whose involvement in apoptosis is more complex. One of these proteins is TCTP (or its yeast homolog Mmi1p), a highly conserved protein assumed to participate in a wide variety of cellular processes such as apoptosis, stress response, microtubule organization and ion homeostasis.23,24 This protein roughly resembles chaperones.25 A flexible loop region and a helical domain are the structural characteristics of TCTP.26 The flexible loop region harbors a calcium-binding motif,27 whereas it is assumed that the helical domain is responsible for tubulin binding.28 Besides its many intracellular functions, TCTP is also secreted and can be detected in the extracellular space.29 A re-import of this protein is made possible by a special protein-transduction domain that penetrates all biological membranes with high efficiency.30 In a previous work, we were able to show that stress is able to initiate a shift of Mmi1p from the cytosol to the outer mitochondrial membrane.31 This finding was later on confirmed in mice.32 In 2013, we could demonstrate that the binding of Mmi1p to mitochondria is mediated by an α-helical domain (V-domain) consisting of two helices.33 This was shown by cloning the V-domain of MMI1 via PCR into the yeast vector pUG35 and expressing it as a C-terminal GFP fusion protein. Surprisingly, this domain was found in vesicular structures after yeast cells depleted all nutrients and the culture reached stationary phase (Figure 1a). In a follow-up approach, we tried to identify other proteins containing a V-domain. The structure of the Mmi1p V-domain was predicted with Swiss-Model (http://swissmodel.expasy.org) (Figure 1b) and was then submitted to the ‘DaliLite v. 3’ server (http://ekhidna.biocenter.helsinki.fi/dali_server/start). Among others, BAX, the master regulator of apoptosis, showed a V-domain too. BAX contains nine α-helices (reviewed in ref. 34) and α5 and α6, two amphipathic helices that together form a hairpin show an overlap with Mmi1p (Figure 1c). It was demonstrated that mutations in this two helices prevent mitochondrial localization of BAX.35,36 Therefore, we cloned the GFP-tagged V-domain of BAX into the yeast expression vector pUG35. During the early exponential phase, when nutrients are plenty, a mitochondrial localization of this BAX domain was observed (Figure 1e). In mid-log phase, when the concentrations of nutrients drop considerably, yeast cells begin to store energy in form of lipids. Interestingly, during this phase, the V-domain starts to detach from mitochondria and accumulates in vesicular structures (Figure 1f). These vesicles become more prominent during cell growth and reach a maximum in size during stationary phase.
By co-localizing the V-domain of BAX with a RFP-tagged Erg6p (Delta(24)-sterol C-methyltransferase) and a RFP-tagged Loa1p (lysophosphatidic acid acyltransferase), we demonstrated that these vesicles are in fact LDs (Figures 2a and b). Of special interest is Erg6p, because this protein localizes to LDs,37 as well as to the outer mitochondrial membrane.38 For Erg6p, a potential 3D structure based on homology modeling (http://swissmodel.expasy.org/) was predicted (Figures 2c and d) as no NMR or X-ray defraction structure data are available. A domain resembling the V-domain was GFP-tagged by cloning it into pUG35. This V-domain peptide is localized either to mitochondria (early log phase) or to LDs (late exponential and stationary phase) in yeast cells. Therefore, we hypothesize that we have identified a domain responsible for communication between mitochondria and LDs. Compared to Mmi1p the V-domain of BAX and Erg6p seems to have a higher affinity for LDs than for the outer mitochondrial membrane.
The localization of this Mito-LD-shuttle domain was also tested in mammalian cells. Preferred cells for these studies were hepatocytes, because they have a very high content of LDs. In an initial step the V-domain of murine, BAX was cloned into the mammalian shuttle vector pEGFP-N3 and transfected into primary rat hepatocytes. Fluorescence microscopic visualization revealed both a tubular network and vesicular structures (Figure 3). To prove mitochondrial as well as LD localization, the bi-cistronic vector pIRES2 was used for the following constructs: pIRES2-V-domain (BAX)/TOMM20-RFP, pIRES2-V-domain (BAX)/PLIN2-RFP and pIRES2-V-domain (BAX)/PLIN3-RFP. In higher eukaryotes, different types of LDs can be distinguished: smaller, more peripheral LDs and larger, more centrally localized LDs. Plin3 is a marker protein for peripheral LDs, while Plin2 is a marker protein for larger ones.39,40 TOMM20 is a translocase of the outer mitochondrial membrane.41 Transfection of the human liver cancer cell line HepG2 revealed a co-localization of the V-domain from the murine BAX protein with TOMM20-RFP (Figure 4b) and PLIN2-RFP (Figure 4a) and only a partial co-localization with PLIN3-RFP (Supplementary Figure 1). This experiment demonstrates the evolutionary conserved function of the V-domain as a Mito-LD-shuttle domain. This indicates that the V-domain can only attach to large LDs. According to DaliLite, BCL-XL also harbors a V-domain. A GFP-tagged version showed a clear LD localization in HepG2 cells (Figure 4c) and yeast cells (Figure 4d).
To demonstrate that full-length proteins also have the ability to shuttle between mitochondria and LDs, an yeast strain was constructed expressing a GFP-tagged version of full-length Mmi1p. LDs were isolated from stressed (10min 46°C) and unstressed yeast cultures. Only LDs from the stressed cultures showed a fluorescence signal (Figure 5a), a fact also confirmed by western blot analysis. In times of stress, a strong band at ~50kDa (predicted MW for Mmi1-GFP: 47kDa) appeared, whereas blots from LDs from unstressed cultures revealed only faint signals (Figure 5b). To exclude an influence from the GFP-tag, a FLAG-tagged version of Mmi1p was created and analyzed in the same way. Mmi1p-FLAG (predicted MW: 20.58kDa) was accumulated in LDs of stressed cells, whereas only low amounts were found in LDs from unstressed cells (Figure 5c). Finally, a mass spectral analysis was performed with LDs from stressed/apoptotic and unstressed yeast cultures. Mmi1p could only be detected in LDs from apoptotic yeast cells.
Working with full-length murine BAX in yeast cells turned out to be difficult, because murine BAX induces apoptosis in yeast.42,43 There is no known BAX homolog in yeast cells, although a possible candidate is under investigation.44 However, there are also no anti-apoptotic counterparts, resulting in an upregulated cytotoxic effect of BAX. Cloning BAX into the vector p416GPD-RFP and expression of the resulting BAX-RFP-fusion in the Saccharomyces cerevisiae strain BY4741, which contains already a pESC-HIS-ERG6-GFP plasmid resulted in very few and slow-growing yeast cells. Co-localization of the full-length BAX with Erg6p was observed, but mobile LDs (Supplementary Video 1) were predominantly found in the vacuole (Figure 5d). In some cases, faint BAX-GFP signals co-localizing with LDs in the cytosol were detected (Figure 5e). This BAX-localization underlines its cytotoxic function and suggests a detoxifying role for LDs in yeast, which led to the following hypothesis: mobile LDs (Supplementary Video 2) attach to mitochondria after stress and V-domain containing proteins are shuttled from mitochondria to LDs. This offers the possibility to reset ‘mitochondrial clock’ after stress ends. Protein-loaded LDs ultimately detach from mitochondria and are degraded in the vacuole. As a positive side effect the degradation of LDs results in a release of neutral lipids. These lipids form a pool for the recovery of membranes and provide energy, a prerequisite for starting growth after a period of stress.
In mammalian cells, TCTP translocates to mitochondria during apoptosis and interacts with two anti-apoptotic proteins: Mcl-1 and BCL-XL. The result is a decrease of apoptotic cell death (reviewed in ref. 45). The role of the yeast Mmi1p protein at mitochondria is much less clear. Therefore, we tested if this protein has an anti-apoptotic/anti-BAX function. The murine BAX gene and the yeast MMI1 gene were cloned into the yeast expression vector pESC-HIS, both under control of an inducible, bidirectional GAL1/10 promoter. Addition of galactose leads to the expression of both Mmi1p and mBAX. Expression of mBAX alone causes a clear increase in cell death (survival rate of 49%), whereas a combination leads to a statistically significant increase in cell survival (survival rate 59%, P-value=0.0046) (Figure 6a). This indicates that Mmi1p has anti-apoptotic properties too.
To test our hypothesis that LDs have an important role in modulating stress response by clearing the OMM, we checked the amount of LDs after stress induction. According to our theory, the number of LDs should increase. In yeast cells, stress was induced in two different ways. Either by expression of mBAX or by heat shock at 46°C. LDs were stained with Nile Red, a fluorescent hydrophobic dye that selectively stains neutral lipids.46 Response to heat stress is very fast and after only 4min, an ~10% increase in LD content (Figure 6b) could be observed. LD numbers further increase until a maximum is reached after 12min (~60% increase; P=0.0038). After this point, the number of LDs decreases and starts to rise again after 16min. A possible explanation for this cyclic appearance/disappearance of LDs could be: after 4min of stress the content of LDs starts to increase removing potentially harmful proteins from the OMM. The LDs with their protein cargo are then moved to the vacuole for degradation (after 12min of stress) and a new cycle is started (after 16min of stress). Besides heat-shock treatment, an increase in LDs was confirmed after expression of mBAX under control of the tetracycline inducible promoter tetO7 (yeast transformed with plasmids pCM666 or pCM666-mBAX). Doxycycline addition triggers the expression of mBAX, leading to severe stress and the induction of apoptotic cell death. Sixteen hours after addition of doxycycline a 269.34% (P-value<0.0001) increase in LD numbers was observed. These LD ‘waves’ resemble the cyclic release of cytochrome c in mammalian cells.47 A correlation between stress and LD numbers was also tested in HepG2 cultures. Cells were treated with 50μM hydrogen peroxide for 16h and were stained with Sudan III (ref. 48) after fixation. As can be seen in Figure 6c, the size as well as number of LDs increases significantly. In another approach, HepG2 cells transfected with the vector pEGP-N3-V-domain-BAX-GFP were treated with staurosporine, a kinase inhibitor that induces apoptosis. Confirming our results obtained with hydrogen peroxide, the LD content increases enormously in apoptotic hepatic cells (Supplementary Figure 2).
In follow-up experiments, the role of LDs in stress response was tested. In yeast cells, the number of LDs was boosted with the overexpression of LRO1. This gene encodes a phospholipid:diacylglycerol acyltransferase, which forms triacylglycerols out of diacylglycerols. The resulting surplus of triacylglycerols in yeast cells (transformed with the vector p416GPD-LRO1) led to an increased number of LDs visualized by a co-expression of the V-domain of mBAX (vector Yeplac181-MET25-V-domain-BAX-GFP) (Supplementary Figure 3). In yeast cells transformed with the plasmid pCM666-mBAX, expression and shuttling of BAX to mitochondria was induced by the addition of doxycycline (50mg/l). After 16h, a clear induction of apoptosis was observed and cell survival dropped to 37% (P<0.0001). Interestingly, overproduction of Lro1p induced a partial redirection of mBAX to LDs and cell survival increased to 49% (P=0.0003) (Figure 6d). We also tested other stressors (hydrogen peroxide and acetic acid) and LD number was further increased by co-expressing Lro1p and Dga1p at the same time. Dga1, a diacylglycerol acyltransferase, also stimulates triacylglycerol synthesis. It turned out that yeast cells (BY4741 p416GPD-LRO1 pESC-DGA1) overexpressing both, Lro1p and Dga1p, were resistant to high concentrations of acetic acid as well as hydrogen peroxide as shown by spot test experiments (Figure 6e). In addition, we created a strain that was deleted for ARE1, ARE2, DGA1 and LRO1 genes at the same time. It has been published that this quadruple mutant is devoid of LDs.49 Minor amounts of LDs were still visible (visualized by expressing the GFP-tagged V-domain of mBAX), but their number and size were dramatically decreased compared to the wild-type. This strain showed a reduced resistance to hydrogen peroxide and acetic acid (Supplementary Figure 4). However, this effect is less pronounced than the increased resistance to acetic acid upon LD stimulation. A correlation between LDs and mitochondria could also be demonstrated in another way: we show here that deficiency of LDs in the quadruple mutant results in the progressive fragmentation of the tubular mitochondrial network in yeast cells (Figure 6g). According to our hypothesis, harmful proteins accumulate in the outer mitochondrial membarne in the quadruple mutant causing to the observed phenotype.
Similar experiments were carried out with HepG2 cells where oleate was added to cell cultures. Oleate has been demonstrated to increase the number of LDs. Apoptosis was induced by addition of 0.05μM staurosporine. After 24h, HepG2 cells were fixed, nuclei were stained with DAPI and the morphology of DAPI stained nuclei was used to identify viable, apoptotic and necrotic cells. Addition of staurosporine leads to a clear increase in apoptotic cell death (~5%). The number of apoptotic cells increases more than threefold when combining staurosporine and oleate (~17%; Figure 6f). This result was surprising because an anti-apotpotic effect was expected. Therefore mitochondria were isolated from HepG2 cells that were either treated with 0.05μM staurosporine or a combination of staurosporine and oleate. Western blot analysis confirmed that addition of staurosporine induced a translocation of BAX and BCL-XL to mitochondria (Figures 7a and b). After addition of oleate both proteins completely disappeared from the outer mitochondrial membrane. In case of oleate-treated cells BCL-XS, a pro-apoptotic splice variant of BCL2L1,50 translocated to mitochondria (Figure 7b) and this protein seems to be responsible for the increased cell death in oleate-treated HepG2 cells. Compared with BCL-XL, BCL-XS cannot be removed because the V-domain in this splice variant is missing (Figures 7c–e).
LDs are often named the forgotten organelle. They have been underestimated for quite a while, but finally shifted into the focus of research in the past decade.51,52 LDs are originating from the ER53 and consist of a neutral lipid core surrounded by a protein-decorated phospholipid monolayer. The hydrophobic core is mainly composed of triacylglycerols (TGs) and cholesterylesters.40,51 To preserve their structural integrity, LDs have a unique protein composition, allowing them to interact with other organelles and release or synthesize triacyglycerols.54 Besides their birthplace, the ER, LDs are also found in contact with other organelles such as mitochondria and peroxisomes.55 Inside the cell LDs are highly mobile and can shuttle between organelles (Supplementary Video 2). The life of a LD ends in the vacuole/lysosome. It was demonstrated that this is actually a type of autophagy termed lipophagy.56 LDs are storage organelles for neutral lipids which meet the cell’s requirement for energy, membranes and lipid-derived molecules. There is evidence that LDs also have a role in protein modifications, histone storage and antioxidant defense.57–59 The direct interaction with most, if not all organelles enables a direct lipid transfer and could be a tool to prevent lipotoxicity.60 Of special interest is the mitochondria-LD-junction mediated via PLIN5.61 It is assumed that this interaction enables a flux of fatty acyl-CoAs for oxidation that is essential for energy production. We demonstrate that LDs have an important role in clearing the mitochondrial outer membrane from unwanted proteins and reset the ‘mitochondrial clock’. This is probably a way to rescue mitochondria from being removed via mitophagy. Our hypothesis is briefly summarized in Figure 8: an apoptotic stimulus leads to a conformational change in the pro-apoptotic protein BAX, which re-localizes from the cytosol to mitochondria. After integration into the outer mitochondrial membrane via the V-domain BAX oligomerizes with BAX and BAK and forms a mitochondrial-apoptosis-induced channel (MAC). Formation of MAC marks the point-of-no-return, since it leads to the release of cytochrome c and subsequently to apoptotic cell death. Apoptotic death can be prevented by TCTP or BCL-XL. Both proteins contain a structurally similar V-domain and localize to mitochondria after external stress. TCTP/Mmi1 prevents oligomerization of BAX and formation of MACs and in this way exerts its anti-apoptotic activity. In this work, we show that Mmi1p, similar to human TCTP, can prevent BAX-induced apoptosis. The effect is not as distinct as in mammals, but cell survival is clearly improved from 49 to 59%. TCTP and BAX are not part of healthy mitochondria and have to be removed. LDs are in close contact with mitochondria and TCTP, as well as BAX, can be transferred from mitochondria to LDs via the V-domain. As a direct consequence the number of LDs increases in times of stress. We have shown this for BAX expression, hydrogen peroxide and heat stress in yeast as well as staurosporine and hydrogen peroxide-induced apoptosis in hepatocytes. Our experiments also indicate that BAX decorated LDs are highly toxic and have to be moved to the vacuole for degradation. In the vacuole, LDs are lysed, providing energy for the restart of the cell cycle. Besides Mmi1p/TCTP and BAX we have been able to demonstrate that other proteins, such as BCL-XL and yeast Erg6p, harbor a V-domain. Our data clearly indicate that this domain is a shuttle domain between mitochondria and LDs. In current literature there is growing evidence that LDs are involved in stress response. In murine lymphoma cells, for example, etoposide, an anticancer drug of the topoisomerase inhibitor class, induces an increase in LD numbers.62 Emerging data also demonstrate that several tumors utilize LDs for survival. In colon cancer and human breast cancer cells, increased LD numbers were detected.63,64 Our results indicate that these tumors use LDs to redirect BAX and other pro-apoptotic proteins from mitochondria to LDs. This way the progression of the apoptotic program is prevented which represents a key event in reaching malignancy.
Taken together LDs are able to collect pro- as well as anti-apoptotic proteins from mitochondria. This either attenuates or increases apoptosis, an effect that obviously depends on the cell type and the metabolic state. In yeast, a higher number of LDs provide the cell with a higher resistance against apoptosis. In HepG2 cells more LDs cause a higher sensitivity towards induction of apoptosis. HepG2 cells redirect both BAX and BCL-XL from mitochondria to LDs, but induce a translocation of BCL-XS to mitochondria that finally kills them. Similar effects have been observed by other groups as oleate induces apoptosis in 3T3-L1 adipocytes65 but rescues INS-1E β-cells from apoptosis.66
All strains used in this study are based on the S. cerevisiae strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and the null mutants of the same genetic background were obtained from the EUROSCARF deletion collection. The strains were grown at 28°C in either complex medium (YPD) (1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) d-glucose) or synthetic complete glucose medium (SC-glucose) (2% (w/v) d-glucose, 0.17% (w/v) yeast nitrogen base without amino acids, 0.5% ammonium sulphate and 10ml of complete dropout mixture (0.2% Arg, 0.1% His, 0.6% Ile, 0.6% Leu, 0.4% Lys, 0.1% Met, 0.6% Phe, 0.5% Thr, 0.4% Trp, 0.1% Ade, 0.4% Ura, 0.5% Tyr) per liter). The quadruple mutant (Δare1, Δare2, Δdga1, Δlro1) was created by mating of the appropriate parent strains from the EUROSCARF collection, diploid selection, sporulation and spore dissection after zymolyase digestion using a micromanipulator (Singer, MSM System series 200, Roadwater, UK). All yeast strains used and created for this study are summarized in Supplementary Table 1.
For PCR amplifications the NEB Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA, USA) was used. All restriction enzymes were provided by either Promega (Mannheim, Germany), NEB or Thermo Scientific (Waltham, MA, USA). For Gibson Assembly the NEB Gibson Assembly Master Mix was used according to the manufacturer’s protocol. The cloning strategies, sequence of primers (obtained from Sigma-Aldrich, Darmstadt, Germany) enzymes used for cloning and the target vectors are summarized in Supplementary Table 2. All constructs were sequenced by Eurofins-MWG-OPERON (Ebersberg, Germany).
For chromosomal integration C-terminal RFP fusion proteins were created. Integration cassettes containing RFP as well as kanMX4 were amplified via PCR from the template plasmid pRFP. An aliquot of 1μg of the PCR product was transformed into yeast cells, chromosomal integration was selected by plating the cells on YPD plates containing 200μg/ml G418 (Geneticin, Sigma-Aldrich). Integration of the cassette in the correct chromosomal region, creating an RFP fusion, was controlled by PCR.
The distribution of various fusion proteins (or RFP fusions) was analyzed with a ×100 PlanApochromat objective (NA=1.4) either using an Olympus IX-81 inverted microscope equipped with a Hamamatsu Orca/ER digital camera (Hamamatsu Photonics, Hamamatsu City, Japan) and an Olympus (Tokyo, Japan) CellR detection and analyzing system (GFP filter block U-MGFPHQ, exc. max. 488, em. max. 507; RFP filter block U-MWIY2, exc. max. 545–580, em. max. 610; DAPI filter block U-MNUA2, exc. max. 360–370, em. max. 420–460), a Leica TCS SP5 AOBS confocal laser scanning system (Leica Microsystems, Wetzlar, Germany) coupled to a Leica DMI 6000 Cs inverted microscope, a Carl Zeiss AG Axioscope (Oberkochen, Germany) or Nikon (Tokyo, Japan) Eclipse Ni-U equipped with a DS-Fi2 digital camera.
Equal amounts of LDs (normalized to OD600) were mixed with sample buffer, were loaded to and separated in 15% SDS-PAGE gels and then blotted on protran BA85 nitrocellulose membranes (Schleicher & Schuell BioScience GmbH, Dassel, Germany) (250mA, 90min at RT). After blocking the membranes with MTBS-T (25mM TRIS pH 7.6, 137mM NaCl, 0.1% TWEEN 20, 5% nonfat milk powder) for 90min at RT and washing for 30min with TBS-T the primary-antibodies diluted in MTBS-T were added and incubated overnight at 4°C under constant shaking. The antibodies were diluted as follows: GFP-antibody (B-2) HRP (sc-9996 HRP; Santa Cruz; 1:1000); FLAG M2 antibody (F3165; Sigma-Aldrich; 1:500); BAX-antibody (D2E11) (5023; Cell Signal Technologies; 1:1000); BCL-XL-antibody (54H6) (2764; Cell Signal Technologies; 1:1000). After three washing steps with TBS-T (3×10min at RT) the membranes were incubated with the secondary-antibody, diluted in MTBS-T (5% w/v milk powder in TBS-T) (Polyclonal rabbit anti-mouse immunoglobulins/HRP; P0161; Dako; 1:25000). Detection was carried out with the Pierce (Thermo Fisher Scientific, Waltham, MA, USA) ECL western blotting substrate according the manufacturerś instructions.
Primary liver cells were grown on glass slides in 24-well plates and lipofected 24h after perfusion. In case of human HepG2 cells, cells were grown to 80–90% confluency on glass slides. Lipofection took place in six-well plates according to the manufacturer' protocol. Glass slides were then taken out of the 24/6-well plates and were washed with PBS buffer and fixed for 10min at 37°C in 4% para-formaldehyd (in PBS). The cells were washed three times with PBS and enclosed in a Mowiol-DABCO solution.
Starting with an OD600 of 0.1 yeast cell were grown for 24h in YPD medium. After centrifugation, the cells were washed and resuspended in 20ml of isolaton buffer 1 (0.1M Tris-HCl, 10mM DTT pH9.4) and incubated for 15min at 28°C. After centrifugation and washing, the cells were resuspended in 25ml isolation buffer 2 (1.2M Sorbitol, 20mM Tripotassium phosphate pH 7.4) supplemented with 0.5mg/ml zymolyase and incubated for 1h at 28°C. Yeast cells were then pelleted and resuspended in 15ml of a 200mM Tris pH 7.4 buffer. After cell wall digestion the cells were lysed with 10 strokes in a potter homogenizer and centrifugated for 5min at 4000×g. The supernatant was then centrifuged at 50000×g for 10min. The next centrifugation step was carried out in an ultracentrifuge at 100000×g for 30min. Special self-made suction devices were used to collect LDs floating on top. To cleanse the samples of cytosolic contaminations they were mixed with sucrose and sodium carbonate (end concentrations of 25% and 10mM, respectively) and layered on top of a 60% sucrose cushion. The sample was then overlaid with 10mM sodium carbonate followed by the 200mM Tris pH 7.4 buffer. After ultracentrifugation at 29000r.p.m. (100000×g) for 30min the purified LDs were collected.
LDs were mixed with the same volume of diethyl ether and vortexed thoroughly. After a centrifugation step of 20000×g for 15min the layer containing the ether was removed and the ether was evaporated. The proteins in the liquid layer were then precipitated using one-tenth of its volume of 100% TCA. Samples were kept on ice for 30min before centrifugation (30min at 14000r.p.m.). The supernatant was removed and the pellet was washed thoroughly using aceton. Proteins were reduced, alkylated and digested with the ProteoExtract All-in-One Trypsin Digestion Kit (Merck Millipore, Billerica, MA, USA). Peptides were loaded onto the trap column (PepSwift Monolithic Trap Column, Dionex, Thermo Fisher Scientific, Waltham, MA, USA) and desalted with 0.1% (v/v) heptafluorobutyric acid at a flow rate of 10μl/min. After 5min, trap and separation column (PepSwift Monolithic Nano Column, 100μm×25cm, Dionex) were coupled with a switching valve and the peptides were eluted with an acetonitrile gradient (Solvent A: 0.1% (v/v) FA/0.01% (v/v) TFA/5% (v/v) ACN; solvent B: 0.1% (v/v) FA/0.01% (v/v) TFA/90% (v/v) ACN; 5–45% B in 60min) at flow rate of 1μl/min at 55° C. The HPLC was directly coupled via nano electrospray to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Capillary voltage was 2kV. A top 12 method was used, the normalized fragmentation energy was 28%. Survey and fragment spectra were analyzed with Proteome Discoverer version 1.4 (Thermo Fisher Scientific) or Peaks Studio 7.5 (Bioinformatics Solutions, Waterloo, ON, Canada), respectively with the human sequence database from UniProt.
SC-galactose plates containing H2O2 (in a concentration range from 1–5mM) and acetic acid (3–50mM) were prepared to test for the sensitivity of yeast strains that are either overexpressing or are deleted for genes essential for the formation of LDs. These cells were then grown to stationary phase in liquid SC, were washed twice with water and serially diluted to OD600 values of 3.0; 1.0; 0.3; 0.1. 10μl aliquots were then spotted onto the plates prepared as described above. Sensitivity was determined by comparison of growth after 3 days at 28°C.
HepG2 cells were grown to ~80% confluency in 75cm2 cell culture flasks. Medium was aspirated and cells were washed 2 times with STE buffer (5mM Tris pH7.4, 250mM sucrose, 2mM EGTA) before scraping them off an collecting them. Cells were resuspended in 5ml of cold STE buffer and transferred to a 50ml centrifuge tube. They were then centrifuged at 2000r.p.m. for 10min and 4°C before removing the supernatant. Cells were resuspended in 2.5ml cold STE buffer containing protease inhibitor and 0.5% BSA before transferring them to a potter homogenizer. They were then homogenized with 20–30 strokes and transferred to a 50ml centrifuge tube. The homogenate was spinned down at 3000r.p.m. for 3min and 4°C. The supernatant was then spinned down at 10000r.p.m. for 11min at 4°C, afterwards the supernatant was removed and the mitochondrial pellet was resuspended in ice cold STE before transferring it to a 15ml centrifuge tube and topping it up with STE buffer. It was then spinned down at 11600×g for 10min and pellet was resuspended in 100μl of STE buffer.
Yeast strains were grown in YPD to mid-exponential phase and were then washed and resuspended in PBS. After exposure to heat shock (42°C for 2, 4, 6, 8, 10, 12, 14 and 16min) cells were fixed with 3.7% formaldehyde. In addition, yeast cells harboring either the plasmid pCM666-mBAX (murine BAX under control of a tet-inducible promoter) or pCM666 were incubated for 16h in the presence of 10μg/ml doxycycline hyclate at 28°C in SC-leu medium. All cells were then stained with 1mg/ml Nile Red (Thermo Fisher Scientific, N-1142) and incubated in the dark for 20min before measuring relative fluorescence with the multi-plate reader Anthos Zenyth 3100 (Anthos Labtec Instruments GmbH, Wals-Siezenheim, Austria).
Two deletion strains from the EUROSCARF library with different mating types were mated and selected on SD +his +leu +ura plates. Cells were then sporulated on SPO plates. The sporulated cells were incubated for 5min at 37°C with a 0.5mg/ml zymolyase solution (Seikagaku Biobusiness, 20T; Seikagaku Corporation, Tokyo, Japan). The spores were separated using a micromanipulator (Singer, MSM System series 200) on YPD plates. The strains carrying a double deletion were selected on YPD plates containing kanamycin. They were further analyzed by PCR with the respective primer pairs provided by EUROSCARF (Scientific Research and Development GmbH, Oberusel, Germany). The two resulting double mutants were then crossed with each other in exactly the same way resulting in a quadruple mutant strain.
Data are reported as arithmetic means±S.E.M. Data were tested using unpaired two-tailed Student’s t-test, and results with P<0.05 were considered statistically significant.
We express our gratitude for project 7AMB14016 to MR and JH, project CSF 16-05497S to JH, project GAUK309315 to RL and to the OeAD for the project CZ 10/2014 to MR. We are grateful to JM Shaw (University of Utah, UT, USA) for providing pYX142-mtRFPm plasmid.
Supplementary Information accompanies the paper on the Cell Death and Discovery website (http://www.nature.com/cddiscovery)
Edited by N Barlev
The authors declare no conflict of interest.