The ceramide activated protein phosphatase Sit4 impairs sphingolipid dynamics, 1
mitochondrial function and lifespan in a yeast model of Niemann-Pick type C1 2
3
Rita Vilaça,1,2,3 Ivo Barros,2,3 Nabil Matmati,4 Elísio Silva,2,3 Telma Martins,1,2 Vítor Teixeira,1,2,3 4
Yusuf A. Hannun,4 and Vítor Costa1,2,3 5
6 1
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal. 2Instituto de 7
Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal. 3Departamento de Biologia 8
Molecular, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal. 9
4Stony Brook Cancer Center, Stony Brook University, Health Science Center, Stony Brook, NY, 10
USA 11
12 13
*Corresponding author: Vítor Costa, Instituto de Investigação e Inovação em Saúde, Rua Alfredo 14
Allen, 208, 4200-135 Porto. Portugal. Phone: +351220408800. email: [email protected] 15
Abstract 17
The Niemann-Pick type C is a rare neurodegenerative disease that results from loss-of-function 18
point mutations in NPC1 or NPC2, which affect the homeostasis of sphingolipids and sterols in 19
human cells. We have previously shown that yeast lacking Ncr1, the orthologue of human NPC1 20
protein, display a premature ageing phenotype and higher sensitivity to oxidative stress associated 21
with mitochondrial dysfunctions and accumulation of long chain bases. In this study, a lipidomic 22
analysis revealed specific changes in the levels of ceramide species in ncr1∆ cells, including 23
decreases in dihydroceramides and increases in phytoceramides. Moreover, the activation of Sit4, a 24
ceramide-activated protein phosphatase, increased in ncr1∆ cells. Deletion of SIT4 or CDC55, its 25
regulatory subunit, increased the chronological lifespan and hydrogen peroxide resistance of ncr1∆
26
cells and suppressed its mitochondrial defects. Notably, Sch9 and Pkh1-mediated phosphorylation 27
of Sch9 decreased significantly in ncr1∆sit4∆ cells. These results suggest that phytoceramide 28
accumulation and Sit4-dependent signaling mediate the mitochondrial dysfunction and shortened 29
lifespan in the yeast model of Niemann-Pick type C1, in part through modulation of the Pkh1-Sch9 30 pathway. 31 32 33 Abbreviations: 34
CAPP, ceramide-activated protein phosphatase; CLS, chronological lifespan; COX, cytochrome c 35
oxidase; dhCer, dihydroceramide; DHS, dihydrosphingosine; LCB, long chain sphingoid base; 36
MAPK, mitogen activated protein kinase; NPC, Niemann-Pick type C; PDS, post-diauxic shift; 37
phytoCer, phytoceramide; PHS, phytosphingosine; ROS, reactive oxygen species. 38
39
Keywords: Niemann-Pick type C; ceramide; sphingolipid signaling; mitochondria; Sit4; Sch9 40
41 42
1. Introduction 43
44
Sphingolipids are important structural components of cell membranes, highly conserved among 45
species, and their bioactive metabolites can act as signaling molecules regulating many biological 46
processes. Sphingosine, a long chain sphingoid base (LCB), and ceramide are involved in the 47
regulation of actin cytoskeleton organization, endocytosis, apoptosis, cell senescence and cell cycle 48
arrest whereas sphingosine-1-phosphate plays a key role in proliferation, mitogenesis, cell 49
migration, cell survival and inflammation (reviewed in [1, 2]). The homeostasis of sphingolipids is 50
affected in several age-associated neurological diseases, such as Alzheimer´s [3] and Parkinson´s 51
[4] diseases, in cancer [5], as well as in sphingolipidoses, a group of lysosomal storage diseases 52
that comprise several distinct defects in lysosomal enzymes and lipid transfer proteins [6]. A 53
characteristic feature of the sphingolipidoses is the accumulation of other secondary storage 54
products because of the lipid nature of the primary storage compound, leading to a traffic jam [7]. 55
Niemann-Pick type C (NPC) is a sphingolipidosis caused by loss-of-function point mutations in 56
NPC1 or NPC2 gene [8, 9] and is clinically characterized by a severe neurodegeneration and 57
accentuated failure of systemic organs such as liver and spleen. The NPC1 protein is a large 58
transmembrane protein that is located in the transient late endosome/lysosome system, while NPC2 59
protein is a soluble glycoprotein with high affinity for cholesterol [10, 11]. Both proteins seem to be 60
involved in intracellular transport of endocytosed cholesterol through the endolysosomal system 61
[12]. In addition to cholesterol accumulation concomitant with sphingomyelin and gangliosides 62
storage [13], there is an increase in the levels of sphingosine that precedes cholesterol entrapment 63
in the lysosome of NPC1 cells [14]. 64
Sphingolipid metabolism is highly conserved from yeast to mammalian and has been extensively 65
studied using this simple model organism (Fig.1) [15]. We have recently reported that yeast cells 66
lacking Ncr1, an orthologue of human NPC1 [16], exhibit high levels of sphingosine and 67
hyperactivation of the Pkh1-Sch9 pathway that are associated with severe mitochondrial 68
dysfunctions, oxidative stress sensitivity and premature ageing [17]. Several studies suggest that 69
sphingolipid homeostasis plays an important role in oxidative stress resistance and longevity in 70
yeast, as also described in mammals [18, 19]. Lag1, a component of ceramide synthase, regulates 71
replicative lifespan [20, 21]. Yeast cells lacking Ydc1, a dihydroceramidase that hydrolyzes 72
dihydroceramide into the LCB dihydrosphingosine, exhibit an increased chronological lifespan 73
(CLS) whereas cells overexpressing YDC1 show mitochondria and vacuolar fragmentation, 74
increased apoptosis and a shortened CLS [22]. The levels of LCBs increase in stationary phase 75
cells due to a decrease in the activity of both ceramide synthase (Lag1) and LCB kinase (Lcb4) 76
[23], and the down-regulation of sphingolipid synthesis extends CLS in part due to a reduction in 77
LCB-mediated activation of Sch9 [24], the yeast homologue of mammalian ribosomal S6K protein 78
kinase also related to Akt/protein kinase B [25, 26]. Moreover, yeast lacking Isc1, an orthologue of 79
mammalian neutral sphingomyelinase-2 (nSMase2) that generates ceramide through the hydrolysis 80
of inositol phosphosphingolipids, display a shortened CLS, oxidative stress sensitivity, mitochondrial 81
dysfunction, iron overload and caspase-dependent apoptosis [27]. 82
Defects in NPC1 function result in the accumulation of ceramide in the liver of NPC1 patients [28]. 83
In addition, the deficiency of a NPC1-related protein in the intracellular parasite Toxoplasma 84
increases the accumulation of cholesteryl esters, sphingomyelin, as well as of ceramide [29]. In 85
mammals, changes in ceramide levels have been implicated in apoptosis through the modulation of 86
signaling proteins [30, 31], including protein kinase C (PKC), cathepsin D, JNK, ceramide-activated 87
protein kinases and ceramide-activated protein phosphatases (CAPPs) [32-35]. The CAPPs are 88
found in all eukaryotes and are composed by one catalytic subunit and two regulatory subunits [36]. 89
The yeast CAPP is constituted by the Tpd3 and Cdc55 regulatory subunits and by the Sit4 catalytic 90
subunit [37, 38]. Sit4 is a serine-threonine protein phosphatase with high homology to human 91
protein phosphatase 6 [39]. It has been implicated in the regulation of the cell cycle [40], 92
mitochondrial function [41-43], carbohydrate metabolism [44, 45], homeostasis of monovalent ion 93
and pH [46], the Pkc1-MAPK pathway [47] and traffic from the ER to the Golgi complex [48]. 94
Moreover, Sit4 is down regulated by the Target of Rapamycin Complex 1 (TORC1) [49, 50]. 95
Interestingly, a recent study implicated NPC1 in mTORC1 regulation in response to lysosomal 96
cholesterol. Castellano et al showed that lysosomal cholesterol drives mTORC1 recruitment to 97
lysosomes and activation. Moreover, NPC1 mediates mTORC1 inhibition upon cholesterol 98
depletion and mTORC1 is constitutively active in NPC1 deleted cells [51]. 99
Our laboratory has shown that SIT4 deletion increases CLS and H2O2 resistance through 100
modulation of mitochondrial function [52]. Moreover, we reported that Hog1 MAPK is a downstream 101
effector of Sit4 that regulates mitochondrial function, being activated in response to ceramide by a 102
Sch9-dependent mechanism [53, 54]. Both Sch9 and Sit4 are also modulators of sphingolipid 103
metabolism [55, 56]. These results support a functional cross talk between Sit4 and Sch9 in 104
response to ceramide changes. 105
In this report, we show that the activation of Sit4/CAPP associated with an increase in the levels of 106
long chain phytoceramide species impairs oxidative stress resistance, mitochondrial function and 107
chronological lifespan in ncr1∆ cells. Notably, the suppression of ncr1∆ phenotypes by SIT4 108
deletion is associated with downregulation of the Pkh1-Sch9 pathway. 109
110
2. Materials and Methods 111
112
2.1. Yeast strains and growth conditions 113
The Saccharomyces cerevisiae strains used in this work are listed in table 1. The growth media 114
used were YPD [1 % (w/v) yeast extract, 2 % (w/v) bactopeptone, 2 % (w/v) glucose], YPG [1 % 115
(w/v) yeast extract, 2 % (w/v) bactopeptone, 2 % (v/v) glycerol], synthetic complete (SC) drop-out 116
medium containing 2 % (w/v) glucose 0.67 % yeast nitrogen base without amino acids or minimal 117
medium containing 2 % (w/v) glucose 0.67 % yeast nitrogen base without amino acids 118
supplemented with appropriate amino acids [0.008 % (w/v) histidine, 0.04 % (w/v) leucine, 0.008 % 119
(w/v) tryptophan)] and 0.008 % (w/v) uracil. Yeast cells were grown aerobically at 26 ºC in an orbital 120
shaker (at 140 rpm), with a ratio of flask volume:medium volume of 5:1, to early exponential phase 121
(OD600nm=0.6) or to post-diauxic shift phase (PDS; OD600nm=7-9). 122
Deletion strains were generated using PCR-derived deletion cassettes containing URA3, HIS3 or 123
KanMX4 with flanking regions of each gene. Yeast cells were transformed using the lithium acetate/ 124
single-stranded DNA/ polyethylene glycol protocol [57]. 125
To evaluate the levels of Lag1 and Ypc1 expression, ncr1∆ LAG1-9MYC and ncr1∆ YPC1-9MYC 126
cells were generated by disruption of the NCR1 gene in BY4741 cells with LAG1 and YPC1 127
genomically tagged with 9Myc, respectively [55] All the disruptions were confirmed by standard 128
PCR procedures. 129
Plasmids used in this work are detailed in table 2. To study Sit4-Gln3-dependent MEP2 expression, 130
yeast cells were transformed with the YCpMEP2-lacZ plasmid [58] and selected in minimal medium 131
lacking uracil. To assess mitochondrial morphology, cells were transformed with pYX222-mtDsRed 132
plasmid [59] and selected in minimal medium lacking histidine. To evaluate the expression of 133
ceramide synthases and ceramidases, cells were transformed with plasmids expressing lacZ 134
reporter fusions of the promoters of LAG1, LAC1, YDC1 and YPC1 genes [55] and selected in 135
minimal medium lacking uracil. 136
137
2.2 Sphingolipid analysis by HPLC-MS/MS 138
Yeast cells were grown in SC-glucose medium and 1.9x109 cells were collected at exponential and 139
PDS phase. Cell pellets were re-suspended in 1mL of lipid extraction solvent: 50 % (v/v) 140
isopropanol, 10 % (v/v) diethyl ether, 2 % (v/v) pyridine, 25 % (v/v) ammonia. A 200 µL volume of 141
glass beads were added into a 2 ml screw cup plastic tubes. Tubes were shaken in a bead beater 5 142
times, 3 min on, 1 min off at 4 ºC. The content of the tubes was poured into a 13x100 mm glass 143
tubes. An additional 1 mL solvent was used to wash the plastic tubes and was added into the glass 144
tubes. The tubes containing 2 mL solvent with cells and glass beads were dried in an analytical 145
nitrogen evaporator (N-EVAP). The dried samples were sent to the Lipidomic Core at the Medical 146
University of South Carolina for lipid analysis. Levels of dihydroceramides, phytoceramides, α -147
hydroxylated phytoceramides, long-chain sphingoid bases and their phosphorylated forms were 148
measured by the high-performance liquid chromatography/mass spectrometry (LC-MS/MS) 149
methodology as previously described [60]. Analytical results of lipids were expressed as pmol 150
sphingolipid/total cell number. 151
2.3. Oxidative stress resistance and chronological lifespan 153
For analysis of oxidative stress resistance, yeast cells were grown to exponential phase and treated 154
with 1.5 mM H2O2 for 1 h. CLS was assayed as previously described [61]. Briefly, overnight cultures 155
were diluted to OD600nm=0.6 and grown for 24 h (PDS phase) or 48 h (stationary phase; considered 156
t0 in the lifespan assay) and kept in culture media at 26 ºC. Cell viability was determined by 157
standard dilution plate counts on YPD medium containing 1.5 % (w/v) agar. Colonies were counted 158
after growth for 3 days at 26 ºC. Viability was expressed as a percentage of colony-forming units in 159
relation to time 0 or to untreated cells, as indicated. 160
161
2.4. Enzymatic activities 162
All the procedures were carried out at 4 ºC. Yeast cells were grown to respiratory phase and 163
harvested by centrifugation. Cytochrome c oxidase (COX) activity was determined as previously 164
described [62], by measuring cytochrome c oxidation. For the β-galactosidase assay, yeast cells 165
were grown in selective SC-glucose medium to PDS phase, and the activity was measured as 166
previously described [52], with some modifications: a cellular extract was prepared in 100 mM Tris-167
HCl, 1 mM DTT, 10 % (v/v) glycerol, and 150 µg of total protein was used in the assay. 168
169
2.5. Oxygen consumption and growth in glycerol 170
Oxygen consumption rate was measured for 3x108 cells at 26 ºC in phosphate buffer using an 171
oxygen electrode (Oxygraph, Hansatech). Data were analyzed using the Oxyg32 v2.25 software. 172
For analysis of respiratory capacity, yeast cells were grown to exponential phase, diluted to an 173
OD600nm=0.1 and five-fold serial dilutions were plated in SC media containing glucose or glycerol 174
(non-fermentable carbon source) supplemented with 1.5 % (w/v) agar. 175
176
2.6. Mitochondrial morphology 177
Mitochondrial morphology was analyzed in cells transformed with a plasmid expressing 178
mitochondrial DsRed (pYX222-mtDsRed) [59]. Cells were grown in SC-glucose medium lacking 179
histidine to PDS phase. The mitochondrial network was observed in live cells mounted on agarose 180
coated slides by fluorescence microscopy (AxioImager Z1, Carl Zeiss). Data image stacks were 181
deconvolved by QMLE algorithm of Huygens Professional v3.0.2p1 (Scientific Volume Imaging 182
B.V.). Maximum intensity projection was used to output final images using ImageJ 1.51n software. 183
Final scientific figures were designed using Figure J tool. 184
185
2.7. Protein extraction and Western blotting analysis 186
Yeast cells were grown in SC-glucose medium to exponential phase and protein extracts were 187
prepared as described [24] with minor modifications. Briefly, 9x108cells were collected, suspended 188
in 200 µL of water and 200 µL of 0.2 M NaOH. Samples were vortexed and incubated at room 189
temperature for 5 min, centrifuged and the pellet was suspended in sample buffer and boiled for 5 190
min at 95 ºC. Equal amounts of protein (BCATM Protein Assay Kit quantification) were loaded onto 191
SDS-PAGE and transferred onto a nitrocellulose membrane (GE Healthcare). Detection of the 192
proteins was performed with the primary antibodies: mouse anti-yeast phosphoglycerate kinase 193
(Pgk1) antibody (1:30000, Molecular Probes), rabbit anti-Sit4 antibody (1:5000, kindly provided by 194
Dr Yu Jiang), mouse anti-c-myc antibody (1:100, Roche), rabbit anti-Sch9 antibody (1:1000, kindly 195
provided by Dr Robert Dickson) or rabbit anti-P-T570-Sch9 antibody (1:10000, kindly provided by Dr 196
Robbie Loewith), and with the secondary antibody, anti-mouse IgG-peroxidase (1:5000, Molecular 197
probes) or anti-rabbit IgG-peroxidase (1:5000, Sigma). Immunodetection was performed by 198 chemiluminescence (Advansta). 199 200 2.8. Statistical analysis 201
The results were represented by mean and standard deviation values of at least three independent 202
experiments. Statistical analyses were carried out using GraphPad Prism Software v6.02 203
(GraphPad Software). 204
3. Results 206
207
3.1. Ceramide levels in Ncr1-deficient cells 208
In order to characterize changes in the levels of ceramide species associated with NCR1 deletion, a 209
lipidomic analysis was performed in cells grown to post diauxic shift (PDS) phase (respiratory 210
phase). At this phase, ncr1∆ mutant cells present mitochondrial dysfunction associated with the 211
fragmentation of the mitochondrial network [17]. The levels of long chain (LC; C12 to C22) and very 212
long chain (VLC; C24 to C26) dihydroceramides, phytoceramides and α-hydroxylated 213
phytoceramides were measured. 214
The total levels of dihydroceramides (dhCer) were 40% lower in ncr1∆ cells (Fig. 2A-B) due to a 215
decrease in the levels of most (C18- to C26) dhCer species (Fig. S1A-B). Concerning 216
phytoceramides (phytoCer), the levels of C14- to C20-species were 2-fold higher in ncr1∆ cells 217
comparing with parental levels (Fig. S1C-D), but no major differences were observed for VLC 218
phytoCer (Fig. 2C-D). Notably, the levels of C14- to C20 phytoCer also increased in ncr1∆ cells 219
grown to exponential phase (data not shown). These results show significant changes in ceramide 220
levels, including an increase of long chain phytoCer and a decrease of dhCer species in cells 221
lacking Ncr1. 222
We postulated that the mitochondrial defects of ncr1∆ cells could result from a decrease in α -223
hydroxylated phytoceramides (αOH-phytoCer), which are particularly enriched in mitochondria [63]. 224
However, the levels of LC αOH-phytoCer were slightly higher in Ncr1-deficient cells (Supplementary 225
Fig. S1E-F). Thus, the mitochondrial dysfunction displayed by ncr1∆ cells does not seem to result 226
from a decrease in αOH-phytoCer, but we cannot rule-out the hypothesis that a decrease of these 227
species may occur specifically in mitochondria of ncr1∆ cells. 228
To assess changes in the expression of genes related to ceramide synthesis and turnover, namely 229
ceramidases (YPC1 and YDC1) and ceramide synthases (LAG1 and LAC1), BY4741 and ncr1∆
230
cells were transformed with lacZ reporters under the control of those gene promoters. The ncr1∆
231
cells displayed an increase in reporter activity for YPC1 and YDC1 (Fig. 3A) as well as for LAC1 232
and LAG1 (Fig. 3B). However, the induction of LAG1 (10-fold) was much higher than those 233
observed for the other genes (3-4-fold). We also evaluated changes in Lag1 and Ypc1 levels in 234
BY4741 and ncr1∆ cells expressing 9Myc tagged LAG1 and YPC1. Consistent with the induction of 235
LAG1 expression, the levels of Lag1-9Myc increased significantly in ncr1∆ cells (Fig. 3C), but the 236
increase of Ypc1-9Myc levels was mild (Fig. 3D). These results suggest that upregulation of 237
ceramide synthases in exponential phase may contribute to the accumulation of phytoCer in ncr1∆
238
cells in log and PDS phase. The concomitant induction of ceramidases, which hydrolyze ceramides, 239
probably explains the accumulation of LCBs in this mutant [17]. 240
241
3.2. Activation of CAPP is associated with oxidative stress sensitivity, premature ageing and 242
mitochondrial dysfunctions of Ncr1 deficient cells 243
Besides structural functions, ceramide is known to regulate cellular processes through modulation 244
of signaling pathways [1]. Yeast cells present a CAPP composed of two regulatory subunits, Tpd3 245
and Cdc55, and the catalytic subunit Sit4 that is stimulated by ceramide [37, 38, 56]. Thus, we 246
postulated that accumulation of phytoceramide species in ncr1∆ cells could mediate cell responses 247
by activation of the Sit4 phosphatase [38]. Sit4 regulates the dephosphorylation of Gln3 248
transcription factor, leading to its translocation to the nucleus [64]. To assess Sit4 activation, we 249
measured the Gln3-dependent MEP2 expression in BY4741, ncr1∆, sit4∆, ncr1∆sit4∆, cdc55∆, and 250
ncr1∆cdc55∆ cells transformed with a MEP2-lacZ reporter [58]. In addition, Sit4 levels were 251
analyzed by Western blotting. At PDS phase, β-galactosidase activity increased 2.8-fold in ncr1∆
252
cells (relative to BY4741), and this effect was suppressed by SIT4 or CDC55 deletion (Fig. 4A). The 253
levels of Sit4 were similar in ncr1∆ cells and decreased by deletion of CDC55 (Fig. 4B). We also 254
assessed β-galactosidase activity in cells depleted for Sur2, a sphinganine C4-hydroxylase 255
responsible for conversion of dihydrosphingosine to phytosphingosine and dihydroceramide to 256
phytoceramide. Notably, deletion of SUR2 led to a decrease of β-galactosidase in ncr1∆ cells (Fig. 257
4A) but this decrease was not as marked as that associated with SIT4 deletion. These results 258
suggest that CAPP activation increases in Ncr1-deficient cells, in part due to an increase of 259
phytoceramide levels, with no major changes in Sit4 expression. 260
Next, we assessed if Sit4 activation by phytoceramides contributes to the oxidative stress sensitivity 261
and shortened CLS of ncr1∆ cells. As previously described [52], hydrogen peroxide resistance and 262
CLS were higher in sit4∆ as well as cdc55∆ single mutants in comparison with parental cells. 263
Notably, deletion of SIT4 or CDC55 suppressed the hydrogen peroxide sensitivity and reversed the 264
shortened CLS of ncr1∆ cells (Fig. 4C-D). 265
Sit4 negatively regulates mitochondrial function [42, 52] and ncr1∆ cells also present severe 266
mitochondrial defects [17]. Thus, we hypothesized that an increase of CAPP activity could be 267
associated with mitochondrial dysfunctions displayed by this mutant. To test this hypothesis, we 268
analyzed mitochondrial functionality in BY4741, ncr1∆, sit4∆, ncr1∆sit4∆, cdc55∆ and ncr1∆cdc55∆
269
cells. Our results show that deletion of SIT4 or CDC55 led to a reversal of the low oxygen 270
consumption rate, low cytochrome c oxidase (COX) activity and growth defect on a non-fermentable 271
carbon source (glycerol) exhibited by ncr1∆ cells (Fig. 5A-C). Notably, the deletion of SUR2 in 272
ncr1∆ cells also restored oxygen consumption and growth on glycerol plates and increased CLS 273
(Fig. S2). This is consistent with the hypothesis that CAPP activation by phytoceramides contributes 274
to mitochondrial dysfunction in ncr1∆ cells. 275
To get further insights on mitochondrial function, we also assessed the integrity of the mitochondrial 276
network by fluorescence microscopy using cells expressing a mitochondria matrix-targeted peptide 277
fused to DsRed (Fig 5D). Our results show that the punctate pattern exhibited by ncr1∆ cells, 278
indicative of mitochondrial network fragmentation, was suppressed by SIT4 or CDC55 deletion, with 279
double mutant cells displaying a normal tubular mitochondrial network. The overall findings indicate 280
that CAPP activation in ncr1∆ cells contributes to an impairment of mitochondrial dynamics and 281
functionality, leading to a decrease in oxidative stress resistance and chronological lifespan. 282
283
3.3. Levels of sphingolipids in sit4∆ and ncr1∆sit4∆ cells 284
The regulation of sphingolipid metabolism is highly complex, and it may involve feedback 285
mechanisms controlled by signaling proteins modulated by bioactive sphingolipids. Thus, we 286
postulated that the suppression of ncr1∆ phenotypes upon SIT4 deletion could be associated with 287
the modulation of sphingolipid homeostasis. To test this hypothesis, ceramide levels were 288
measured in sit4∆ and ncr1∆sit4∆ cells grown to PDS phase. 289
Our results show that the levels of LC dhCer decreased about 50 % in sit4∆ cells when compared 290
to parental cells (Fig. 2A). Although there was an increase of C16-dhCer species, the levels of C18:1-, 291
C20-, C20:1-, C22- and C22:1-dhCer, which are the most abundant dhCer species, decreased about 2-292
fold in sit4∆ cells (Fig. S1A). These results are consistent with a previous report showing that total 293
dhCer levels decrease in sit4∆ cells [56]. Interestingly, deletion of SIT4 in ncr1∆ cells increased C18- 294
to C22-dhCer content to parental levels. Concerning VLC dhCer, sit4∆ cells displayed an increase in 295
the levels of C24-dhCer (Fig. S1B) and SIT4 deletion suppressed the lower levels of VLC dhCer 296
exhibited by ncr1∆ cells (Fig. 2B). 297
Notably, SIT4 deletion had major effects on phytoCer. The levels of LC (C14- to C22-) phytoCer were 298
significantly higher (more than 3-fold) in sit4∆ and ncr1∆sit4∆ cells compared to parental or ncr1∆
299
cells (Fig. 2C; Fig. S1C). In contrast, the levels of C26- and C26:1-phytoCer (the most abundant 300
species) decreased almost 3-fold in sit4∆ cells (Fig. 2D; Fig. S1D), which is consistent with the 301
reduction of total phytoCer previously described for this mutant [56]. Regarding αOH-phytoCer, 302
similar changes were observed: the levels of most LC species increased in sit4∆ and ncr1∆sit4∆
303
cells whereas αOH-phyto-C26-Cer decreased (Supplementary Fig. S1E-F). 304
These findings suggest that Sit4 plays a role in the regulation of sphingolipid metabolism. Since the 305
accumulation of long chain sphingoid bases (LCBs) and the LCB-activated protein kinase Pkh1 306
have been implicated in ncr1∆ phenotypes [17], we also investigated how SIT4 deletion affects the 307
levels of dihydrosphingosine (DHS), phytosphingosine (PHS) and its phosphorylated forms (DHS-1-308
P and PHS-1-P). LCBs increased in sit4∆ vs parental cells, due to the accumulation of DHS (in the 309
exponential phase) or both DHS and PHS (in the PDS phase). In both growth phases, the levels of 310
DHS-1-P and PHS-1-P also increased in sit4∆ cells (the increase was even higher, comparing to 311
LCBs). Notably, ncr1∆sit4∆ cells exhibited similar changes (Table 3). Thus, SIT4 deletion seems to 312
suppress ncr1∆ phenotypes by means unrelated with a decrease in LCBs. 313
3.4. SIT4 deletion suppresses the activation of Pkh1-Sch9 pathway in ncr1∆ cells 315
Since Pkh1 is activated by LCBs and the levels of DHS (in the exponential phase) or both DHS and 316
PHS (in the PDS phase) further increased in ncr1∆sit4∆ double mutants, compared with ncr1∆
317
cells, we decided to investigate the effect of SIT4 deletion on the activation of the Pkh1-Sch9 318
pathway in ncr1∆ cells. As previously shown [17], the levels of Sch9 and Sch9-phospho-T570 319
(Pkh1-dependent phosphorylation) increased in ncr1∆ cells. Notably, they greatly decreased in both 320
sit4∆ and ncr1∆sit4∆ cells, when compared with parental and ncr1∆ cells (Fig. 6). 321
In ncr1∆sit4∆ double mutants, the increase of LCB-1-Ps was higher to the observed for LCBs. 322
Thus, we postulated that LCB-1-Ps could mediate the cellular effects of SIT4 deletion in ncr1∆ cells. 323
To test this hypothesis, the LCB4 gene, which encodes for the major LCB kinase in yeast, was 324
deleted. Our results show that the levels of Sch9 and Sch9-phospho-T570 increased in sit4∆lcb4∆ 325
and ncr1∆sit4∆lcb4∆ mutants, compared with sit4∆ or ncr1∆sit4∆ cells, although to levels 326
significantly lower to the observed in parental or ncr1∆ cells (Fig. 6). Consistently, ncr1∆sit4∆lcb4∆ 327
mutants were still able to grow on glycerol medium (data not shown), suggesting that SIT4 deletion 328
improves ncr1∆ phenotypes by a LCB-1-P-independent mechanism. Moreover, the deletion of 329
DPL1, which encodes for the LCB-1-P lyase, did not suppress the mitochondrial dysfunction of 330
ncr1∆ cells (data not shown), suggesting that the mitochondrial dysfunction of ncr1∆ cells is not 331
related with the decrease of LCB-1-P levels. The overall results indicate that the protective effect of 332
SIT4 deletion in ncr1∆ cells is mainly associated with downregulation of the Pkh1-Sch9 pathway. 333
334
4. Discussion 335
The Niemann-Pick type C1 disease is a lysosomal storage disorder caused by mutations in the 336
NPC1 protein, which seems to be involved in the transport of cholesterol through the 337
endolysosomal system. Several studies have shown a high degree of complexity of lipids that 338
accumulate in NPC1 cells, including cholesterol, complex lipids such as glycosphingolipids and 339
sphingomyelin [65], as well as sphingosine, which was suggested to be the first lipid that 340
accumulates in these cells [14]. 341
We have previously reported that, similarly to NPC1 cells, yeast cells lacking Ncr1 accumulate 342
LCBs. This may contribute to the activation of the Pkh1-Sch9 pathway that ultimately contributes to 343
mitochondrial dysfunctions, oxidative stress sensitivity and a shortened CLS [17]. Here we show 344
that yeast ncr1∆ cells grown to PDS (respiratory) phase also exhibit significant changes in ceramide 345
levels. These include a decrease in LC dhCer and an increase in LC phytoCer. Our data also 346
demonstrate that both ceramide synthases and ceramidases genes are upregulated in ncr1∆ cells, 347
with the LAG1 ceramide synthase being highly induced. These changes seem to favor phytoCer 348
accumulation in ncr1∆ cells and may also contribute to the increase of LCBs promoted by the 349
ceramidases. However, the mitochondrial defects exhibited by this mutant was not suppressed by 350
deletion of the LAG1 ceramide synthase or ceramidases (YPC1, YDC1 or both) genes (data not 351
shown). It is to be expected that modulations in any of the sphingolipid enzymes cause ripple 352
effects that alter the concentrations of many sphingolipids. So, it is possible that the downregulation 353
of Lag1 or ceramidases increases the levels of LCBs and ceramides, respectively, leading to further 354
activation of downstream signaling pathways, which are known to promote mitochondrial 355
dysfunctions [17, 52]. The inhibition of SPT (catalyzes the first step in sphingolipid biosynthesis) 356
with low doses of myriocin also did not alleviate ncr1∆ phenotypes [17]. However, myriocin induces 357
autophagy in ncr1∆ cells, which is probably detrimental since this process is already increased in 358
untreated cells (data not shown) as described in NPC1 cells [66]. 359
Ceramide constitutes the metabolic hub in sphingolipid metabolism since it is the core lipid that 360
drives the synthesis of more than 50 distinct molecular species. Ceramides with different fatty acids 361
have distinct roles as signaling molecules, acting through modulation of specific downstream 362
components of signaling pathways, including CAPP [1]. Here we show that Sit4, the catalytic 363
subunit of the CAPP related to type 2A family of protein phosphatases, was more activated in Ncr1-364
deficient cells. Moreover, the deletion of genes encoding for the catalytic (Sit4) or regulatory subunit 365
(Cdc55) of CAPP abolished the shortened CLS, H2O2 hypersensitivity and mitochondrial 366
dysfunctions displayed by ncr1∆ cells. Also, deletion of SUR2, which encodes for C4-hydroxylase 367
responsible for generation of phytosphingosine and phytoceramides (Fig.1), suppressed the 368
shortened lifespan and mitochondrial defects of ncr1∆ cells. These findings support our hypothesis 369
that phytoceramides are key mediators of sphingolipid signaling in ncr1∆ cells, promoting, at least 370
in part, CAPP/Sit4 activation. 371
Previous studies have shown that Sit4 is involved in catabolic repression and negatively regulates 372
mitochondrial function [42, 43]. In addition, sit4∆ cells exhibit a higher resistance to oxidative stress 373
and an increased lifespan [52]. Thus, we propose that Sit4 activation mediates the mitochondrial 374
defects of ncr1∆ cells, decreasing hydrogen peroxide resistance and CLS. It is likely that Sit4 375
affects mitochondria function through modulation of mitochondrial proteins or other proteins related 376
to mitochondrial function and dynamics. Consistent with this hypothesis, published work has shown 377
that Sit4 interacts with Atp3 (subunit of the ATP synthase) and Nde1 (external NADH 378
dehydrogenase), and a mutation in Atp3 or NDE1 deletion increase CLS [67, 68]. The identification 379
of Sit4 direct targets will provide new hints to the elucidation of how Sit4 regulates mitochondria and 380
lifespan. 381
Importantly, our results suggest that Sit4 may also function as a modulator of sphingolipid 382
homeostasis and dynamics. The deletion of SIT4 gene suppressed the lower levels of dhCer in 383
ncr1∆ cells. This effect may contribute to the improvement of mitochondrial fitness in ncr1∆sit4∆
384
cells, since dhCer can exert protective effects by inhibiting the assembly of ceramide channels in 385
mitochondria [69]. In contrast, long chain phytoceramides species were significantly increased in 386
sit4∆ and ncr1∆sit4∆ cells. Notably, similar effects were observed upon deletion of SCH9 (data not 387
shown), which also suppresses ncr1∆ phenotypes [17]. More studies are needed to uncover 388
downstream effects mediated by these specific ceramide species as well as its subcellular 389
localization. 390
We have previously shown that SCH9 deletion suppresses the high levels of LCBs displayed by 391
ncr1∆ cells [17]. Notably, sit4∆ and ncr1∆sit4∆ cells exhibited major changes in LCBs. The levels of 392
PHS and DHS increased in these mutants concomitantly with a higher increase of their 1-393
phosphate forms, leading to an increase of DHS-1-P/DHS and PHS-1-P/PHS ratios. Although the 394
increase of LCB-1-phosphates has been associated with inhibition of cell death pathways [2], our 395
results suggest that it does not contribute to cellular effects of SIT4 deletion. LCBs are known 396
activators of the Pkh1 and Pkh2 protein kinases [70] that activate the Sch9 kinase by 397
phosphorylating a T570 residue [71, 72]. Our results show that Sch9 and Sch9-phospho-T570 398
levels decrease in ncr1∆sit4∆ cells, suggesting that the protective effect of SIT4 deletion is, at least 399
in part, mediated by downregulation of the Pkh1-Sch9 pathway. This crosstalk between Sit4 and 400
Pkh1/2-Sch9 seems to be conserved in mammals. Indeed, mammalian Akt/PKB, a homologue of 401
yeast Sch9, is inhibited by ceramide by a CAPP-dependent mechanism [73, 74]. More studies are 402
needed to uncover the molecular mechanisms underlying the intricate regulation of these pathways 403
and its relation with other protein kinases and protein phosphatases that control sphingolipid 404
biosynthesis, e.g. through modulation of Orm1/2 by Ypk1 [75-77]. 405
In summary, our results suggest that the accumulation of phytoceramide species leading to 406
activation of the ceramide activated protein phosphatase Sit4 and the crosstalk between Sit4 and 407
Sch9 mediate the mitochondrial defects, premature ageing and oxidative stress sensitivity displayed 408
by the yeast model of Niemann-Pick type C1 disease (Fig. 7). To our knowledge, this is the first 409
report implicating ceramide signaling in NPC1 phenotypes. 410
Competing interests 412
No competing interests declared. 413
414
Acknowledgements 415
We are grateful to Bruno André (Université Libre de Bruxelles, Belgium), Paula Ludovico (ICVS, 416
Universidade do Minho, Portugal), Joris Winderickx (Inst. Botany and Microbiology, K.U. Leuven, 417
Belgium), Robert Dickson (University of Kentucky College of Medicine, Lexington, Kentucky, 418
USA),Robbie Loewith (University of Geneva, Switzerland) and Yu Jiang (University of Pittsburgh, 419
USA) for generously providing plasmids and antibodies used in this study. We would like to thank 420
Paula Sampaio (ALM, IBMC) for technical assistance and data treatment on fluorescence 421
microscopy. This work was funded by the project Norte-01-0145-FEDER-000008 - Porto 422
Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal 423
Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership 424
Agreement, through the European Regional Development Fund (FEDER), and in part by National 425
Institutes of Health Grant GM063265 (to Y.A.H.), the Lipidomics Shared Resource, Hollings Cancer 426
Center, Medical University of South Carolina (P30 CA138313) and the Lipidomics Core in the SC 427
Lipidomics and Pathobiology COBRE, Department Biochemistry, MUSC (P20 RR017677). 428
429
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[77] M. Shimobayashi, W. Oppliger, S. Moes, P. Jeno, M.N. Hall, TORC1-regulated protein kinase 641
Npr1 phosphorylates Orm to stimulate complex sphingolipid synthesis, Mol Biol Cell, 24 (2013) 870-642
881. 643
644 645
FIGURE LEGENDS 646
Figure 1. Schematic representation of sphingolipid metabolism in yeast. Metabolic 647
intermediates and genes encoding the most important enzymes involved in each step of 648
sphingolipid synthesis and turnover are shown. 649
650
Figure 2. Levels of dihydroceramide and phytoceramide species. S. cerevisiae BY4741, ncr1∆, 651
sit4∆ and ncr1∆sit4∆ cells were grown in SC-glucose medium to post-diauxic shift (PDS) phase. 652
Levels of long chain ceramides (C14 to C22) and very long chain ceramides (C24 to C26) were 653
measured by HPLC-MS/MS as described in Material and Methods. Data are expressed as pmol of 654
lipid per total cell number (1.9x109) and are mean ± variance of three independent experiments. 655
656
Figure 3. Expression of ceramidases and ceramide synthases in Ncr1-deficient cells. (A,B) S. 657
cerevisiae BY4741 and ncr1∆ cells were transformed with lacZ reporter plasmids expressing the 658
promotors of ceramidases genes YDC1 and YPC1 (A) and ceramide synthases genes LAG1 and 659
LAC1 (B). Cells were grown in SC-glucose medium lacking uracil to log phase and the 660
transcriptional activation of these genes were measured by β-galactosidase activity. ****p<0.0001 661
relative to BY4741; Two-way ANOVA and Bonferroni test. (C,D) Analysis of Lag1 and Ypc1 levels 662
in BY4741 and ncr1∆ cells expressing 9Myc tagged LAG1 and YPC1. Protein extracts were 663
separated by SDS–PAGE and blotted into a nitrocellulose membrane, and tagged proteins were 664
detected using an anti-Myc antibody. Pgk1 was used as loading control. One representative out of 665
three is shown for (C) Lag1-9Myc and (D) Ypc1-9Myc. The quantification of Myc band intensities 666
(normalized for Pgk1) is shown. 667
668
Figure 4. Sit4 activation increases in Ncr1 deficient cells and mediates oxidative stress 669
sensitivity and premature ageing. (A) S. cerevisiae BY4741, ncr1∆, sit4∆ and ncr1∆sit4∆, 670
cdc55∆ and ncr1∆cdc55∆ cells expressing a Sit4-Gln3-dependent MEP2-LacZ reporter were grown 671
to post-diauxic shift phase in SC-glucose medium lacking uracil. (A) β-galactosidase activity was 672
measured as described in Material and Methods. ***p<0.001; One-way ANOVA and Bonferroni test. 673
(B) Immunoblot analysis of Sit4 levels in cells grown to PDS. Pgk1 was used as loading control. A 674
representative experiment out of three is shown. (C) Cells were grown to exponential phase 675
(O.D.600nm=0.6) in SC-glucose medium and exposed to 1.5 mM H2O2 for 1 h. Cellular viability was 676
measured as the percentage of the colony-forming unit (treated cells vs non-stressed cells). 677
***p<0.001, relative to ncr1∆; One-way ANOVA and Bonferroni test. (D) Cells were grown to PDS 678
phase and maintained in the growth medium overtime. Cellular viability was measured at 2 to 3 679
days intervals and was expressed as % colony forming units (aged vs day 0). 680
681
Figure 5. SIT4 and CDC55 disruption suppresses mitochondrial dysfunctions exhibited by 682
ncr1∆ cells. (A,B) S. cerevisiae cells were grown in SC-glucose medium to post-diauxic shift 683
phase. Oxygen consumption rates (A) and cytochrome c oxidase (COX) specific activity (B) were 684
measured. **p<0.01 and ***p<0.001, relative to ncr1∆; One-way ANOVA and Bonferroni test. (C) 685
Cells were grown to exponential phase and fivefold serial dilutions were plated in SC solid medium 686
containing glucose or glycerol as carbon source. One representative experiment out of three is 687
shown. (D) Yeast cells transformed with pYX222-mtDsRed (expressing mitochondrial DsRed) were 688
visualized by fluorescence microscopy. One representative experiment out of three is shown. Scale 689
bar: 5 µm. 690
691
Figure 6 – Sch9 and Sch9-phospho-T570 levels decrease drastically upon SIT4 deletion. 692
Yeast cells were grown in SC-glucose medium to exponential phase. Sch9 and phospho-T570-693
Sch9 levels were analyzed by immunoblotting, as described in Material and Methods. Pgk1 levels 694
was used as loading control. A representative experiment out of three is shown. 695
696
Figure 7. Activation and cross-talk of LCB and ceramide signaling pathways in the NPC1 697
yeast model. Deletion of yeast NPC1 related protein induces high levels of LCBs, particularly 698
phytosphingosine (PHS) [17], and long chain phytoceramide species (this study). Ceramide 699
signaling targets the ceramide-activated protein phosphatase (CAPP) Sit4 that mediates 700
mitochondrial dysfunction and decreases cellular lifespan in ncr1∆ cells. Sit4 also regulates Sch9 701
levels and Pkh1-dependent phosphorylation which also contributes to ncr1∆ phenotypes, by an 702
unknown mechanism [17]. 703
Table 1. S. cerevisiae strains used in this work. 704
Strain Genotype Source
BY4741 Mata, his3∆1, leu2∆0, met15∆0, ura3∆0 EUROSCARF
ncr1∆::KanMX4 BY4741 ncr1∆::KanMX4 [17]
ncr1∆::URA3 BY4741 ncr1∆::URA3 [17]
sit4∆ BY4741 sit4∆::KanMX4 EUROSCARF
ncr1∆::URA3 sit4∆::KanMX4 BY4741 ncr1∆::URA3 sit4∆::KanMX4 This study
sit4∆::HIS3 BY4741 sit4∆::HIS3 This study
ncr1∆::KanMX4 sit4∆::HIS3 BY4741 ncr1∆::KanMX4 sit4∆::HIS3 This study
cdc55∆ BY4741 cdc55∆::KanMX4 EUROSCARF
ncr1∆::URA3 cdc55∆::KanMX4 BY4741 ncr1∆::URA3 cdc55∆::KanMX4 This study
cdc55∆::HIS3 BY4741 cdc55∆::HIS3 This study
ncr1∆::KanMX4 cdc55∆::HIS3 BY4741 ncr1∆::KanMX4 cdc55∆::HIS3 This study
sur2∆ BY4741 sur2∆::KanMX4 EUROSCARF
sur2∆::KanMX4 ncr1∆::URA3 BY4741 sur2∆::KanMX4 ncr1∆::URA3 This study
sur2∆::HIS3 BY4741 sur2∆::HIS3 This study
sur2∆::HIS3 ncr1∆::KanMX4 BY4741 sur2∆::HIS3 ncr1∆::KanMX4 This study
BY4741 LAG1-9MYC BY4741 LAG1-9MYC::hphNT1 [55]
ncr1∆ LAG1-9MYC ncr1∆::URA3 LAG1-9MYC::hphNT1 This study
BY4741 YPC1-9MYC BY4741 YPC1-9MYC::hphNT1 [55]
ncr1∆ YPC1-9MYC ncr1∆::URA3 YPC1-9MYC::hphNT1 This study
lcb4∆ BY4741 lcb4∆::KanMX4 EUROSCARF
ncr1∆ lcb4∆ BY4741 ncr1∆::URA3 lcb4∆::KanMX4 This study
sit4∆lcb4∆ BY4741 sit4::HIS3 lcb4∆::KanMX4 This study
ncr1∆ sit4∆ lcb4∆
BY4741 ncr1∆::URA3 sit4::HIS3
lcb4∆::KanMX4
This study
705 706
Table 2. Plasmids used in this work. 707
Strain Insert Marker Reference
YCpMEP2-lacZ MEP2 promotor (-828/+27) URA3 [58] pYX222-mtDsRed Mitochondrial matrix targeted-DsRed HIS3 [59] YEp357-LAC1- lacZ LAC1 promoter (-780/+18) URA3 [55] YEp357-LAG1- lacZ LAG1 promoter (-780/+30) URA3 [55] YEp357-YDC1- lacZ YDC1 promoter (-750/+30) URA3 [55] YEp357-YPC1- lacZ YPC1 promoter (-750/+30) URA3 [55] 708
Table 3: LCBs and LCB-1-Ps levels in cells grown to exponential and PDS phase. 710
Yeast cells were grown to exponential or post-diauxic shift (PDS) phase and sphingolipid levels 711
were measured as described in Methods. Data are expressed as pmol/total cell number (mean ± 712
SD of three independent experiments). The quantification in BY4741 and ncr1∆ cells [17] 713
(reproduced here with permission) was paired with the quantification in sit4∆ and ncr1∆sit4∆ 714
mutants (this study). 715
Exponential phase PDS phase
Strain DHS* DHS-1-P PHS PHS-1-P* DHS* DHS-1-P PHS* PHS-1-P* BY4741 123.54± 13.96 2.95± 0.93 17.08± 0.29 0.63± 0.05 50.53± 3.85 0.73± 0.13 28.84± 2.91 1.35± 0.18 ncr1∆ 153.35± 32.35 11.51± 0.94 24.05± 6.47 1.62± 0.09 129.03± 4.58 1.01± 0.29 55.90± 6.28 2.01± 0.28 sit4∆ 195.74± 11,70 21.44± 1.13 18.96± 2.08 1.87± 0.10 266.79± 124.48 6.46± 2.15 101.34± 5.87 5.19± 0.45 ncr1∆sit4∆ 228.47± 4.42 11.85± 0.46 22.90± 2.49 1.31± 0.10 261.11± 27.14 7.50± 0.78 89.54± 9.76 6.58± 1.12 * p<0.05, One-way ANOVA, non parametric test.
716 717