DHE can functionally replace yeast ergosterol
DHE is a naturally occurring sterol, similar to yeast ergosterol and mammalian cholesterol (). To validate its use as a fluorescent reporter of intracellular ergosterol transport in living yeast cells, we tested whether DHE could functionally replace ergosterol. To facilitate DHE uptake into yeast, we made use of the sterol auxotrophy of cells that are unable to synthesize heme. The enzyme 5-aminolevulinate synthase, encoded by HEM1
, catalyzes the first step in the heme biosynthetic pathway (24
Δ strains can grow in media that is supplemented with 5-aminolevulinic acid (ALA), but in the absence of ALA a sterol supplement is required (25
). DHE supported the growth of hem1
Δ cells as effectively as ALA or ergosterol (). After shifting ALA-grown hem1
Δ cells to DHE-containing media for 48 hrs,>99% of the cellular sterol was DHE (). The DHE content of these cells (per OD600
unit) was similar to the ergosterol content of a parallel culture grown with an ergosterol supplement (). We conclude that DHE can replace the bulk function of ergosterol in vivo
and is therefore a suitable probe for the study of intracellular ergosterol traffic in yeast. A similar conclusion was recently reported (26
Intracellular sterol content of ergosterol- and DHE-loaded cells
DHE is incorporated into the PM of hypoxically grown yeast
Under normal growth conditions, yeast cells cannot import exogenously supplied sterols, a phenomenon termed `aerobic sterol exclusion', and rely entirely on ergosterol biosynthesis to meet their sterol requirements (27
). However, because ergosterol biosynthesis requires molecular oxygen, yeast cells grown under hypoxic or anaerobic growth conditions must import sterols to maintain viability (28
). The ability to import sterols depends on the PM-localized ABC transporters Aus1 and Pdr11 () that are expressed only when oxygen levels are low (29
We exploited the ability of yeast cells to take up sterols under hypoxic conditions to introduce DHE into the PM. We supplemented a yeast culture with DHE and incubated the cells for 36 hrs at 30°C in <0.7% (v/v) oxygen. When the cells were examined by fluorescence microscopy, a uniform signal was detected at the cell cortex, indicating PM-localized DHE ()(26
). No fluorescence was observed in cultures incubated with ergosterol instead of DHE, indicating that the fluorescence we observed was due to DHE rather than cellular autofluorescence (). As expected, hypoxic conditions were necessary for DHE incorporation, because no cell-associated fluorescence was observed in cultures that were grown aerobically in the presence of DHE. Reversed phase HPLC analysis established that fluorescence was due to DHE and not a metabolite such as DHE ester (). About 80% of the total cellular sterol was recovered as DHE, with the rest being ergosterol ().
Incorporation of DHE into the PM of cells grown under hypoxic conditions
We confirmed the PM localization of DHE by Renocal gradient fractionation of a yeast cell homogenate. This procedure separates the PM from the ER and other organelle membranes(30
). Consistent with the normal distribution of endogenous ergosterol, DHE levels were highest in gradient fractions containing the PM marker Gas1 and uniformly low in fractions containing the ER marker Sec61 (). The low DHE content of the Sec61-containing fractions indicates that high DHE levels in the PM fraction are unlikely to be due to contamination of that fraction with cortical ER.
Redistribution of DHE from the PM to LDs
To visualize sterol trafficking from the PM, cells were loaded with DHE as described above, and then incubated aerobically for 1 hr at 30°C prior to examination by fluorescence microscopy. Fluorescence was seen to be entirely intracellular, occupying a number of discrete foci that co-localized with the LD markers Nile Red () and Erg6 (not shown).
Redistribution of DHE from the PM to lipid droplets
HPLC analyses of lipid extracts prepared from the cells revealed that a significant percentage (~40%) of DHE had been converted to DHE esters (), consistent with localization of fluorescence to LDs. Furthermore, no intracellular foci were seen in are1
Δ double mutant cells that lack the ability to esterify sterols. Examination of the single mutants are1
Δ and are2
Δ suggested that esterification under these conditions was primarily due to Are2 which is the major catalytic isoform (31
)(, top panels).
DHE redistribution is likely linked to the initiation of ergosterol biosynthesis as oxygen becomes available (33
). Thus, we propose that newly synthesized ergosterol displaces DHE at the PM, resulting eventually in its esterification and sequestration in LDs. Consistent with this proposal, cellular ergosterol content increased ~6-fold, from ~140 ± 80 pmol (per OD600
unit of cells) at time zero to 840 ± 165 pmol after the 60 min aerobic incubation period.
Several experiments established the characteristics of DHE redistribution from the PM to LDs. If the samples were kept on ice or treated with energy poisons immediately after DHE loading, the PM fluorescence pattern persisted for several hours and no intracellular foci were observed (). We examined DHE redistribution in end4-1ts
cells that are defective for endocytosis at 37°C (34
). Whereas transport of the lipid dye FM4–64 (35
) from the PM to vacuoles was blocked in these cells at 37°C as expected (not shown), redistribution of DHE from the PM to intracellular foci was not (, bottom panels). Thus, redistribution of DHE from the PM to LDs does not require vesicular transport from the PM. Our data suggest that a fraction of DHE moves from the PM to the ER during the aerobic incubation period. At the ER, it is esterified by Are2 and packaged into LDs. The overall redistribution process occurs by a non-endocytic mechanism that requires metabolic energy. The molecular basis of the metabolic energy requirement is unknown: energy may be required directly for transport, e.g
., to activate an STP by phosphorylation, or indirectly for the assay read-out, e.g.
, synthesis of fatty acyl CoA for steryl ester production.
Kinetics of DHE redistribution
To evaluate the rate at which DHE redistributes from the PM to LDs during aerobic incubation, we analyzed samples taken at different time points over a 60-min period. The samples were preserved for analysis by adding energy poisons and incubating on ice. Three complementary analyses were performed.
First, fluorescence images were recorded and scored manually by binning the fluorescence pattern into three categories: PM-staining, intracellular foci and an intermediate category (). More than 200 cells were scored per time-point. Data compiled from 3 independent experiments are shown in . The results reveal that DHE fluorescence begins to disappear from the cell periphery immediately on shifting the cells to aerobic conditions and starts to be detectable in intracellular foci after a lag period of ~20 min. The entire redistribution process is complete by ~60 min as assessed by this method ().
Quantification of DHE redistribution from the PM to LDs
We also prepared lipid extracts from the cells at each time point and analyzed these by HPLC to determine the extent of conversion of DHE to DHE esters (). These results corroborated the data obtained by fluorescence microscopy. DHE esterification commenced with a lag period of ~20 min, coincident with the appearance of intracellular fluorescent foci. However, only ~40% of the DHE was converted to DHE ester after 60 min (>70% conversion of DHE to DHE ester occurred with an incubation of 120 min) whereas manual scoring suggested that all of the observable DHE had relocated from the PM to LDs. Closer examination of the fluorescence images suggested that a significant proportion of DHE was diffusely distributed throughout cells that also had intracellular foci (), accounting for the difference in the estimates obtained by manual scoring and ester analysis. We therefore assessed total intracellular DHE by an image processing protocol. We generated a mask from the DIC image of each cell, and used it to define fluorescence at the cell cortex and within the cell interior (). Quantification of all intracellular fluorescence by this method revealed that ~25% of the DHE was intracellular at time zero (this is likely an overestimate because of the contribution of out-of-plane fluorescence from PM-localized DHE at early time points) whereas 75% of the DHE was intracellular at the 60-min time point (). Thus after 60 min, greater than half of the internalized DHE is localized in LDs in the form of DHE esters. We conclude that DHE redistributes from the PM to the cell interior during the aerobic incubation period, on a time scale of minutes, eventually localizing to LDs.
DHE redistribution occurs robustly in cells lacking functional Osh proteins
We used the DHE redistribution assay to test the hypothesis that Osh proteins are required for sterol transport between the PM, ER and LDs. Because yeast cells require the expression of at least one member of the Osh protein family for viability, we used a haploid strain (osh
) that lacks all seven chromosomally encoded Osh proteins but contains a plasmid expressing a temperature-sensitive Osh4 mutant in which a glycine residue (G183) is replaced with aspartic acid (36
). At 23°C the osh4-1
allele is functional ensuring cell viability at ambient temperature, but it is non-functional at 37°C. shows that the temperature-sensitive growth phenotype of this strain is preserved under hypoxic conditions.
DHE redistribution from the PM to LDs in cells lacking functional Osh proteins
We incubated oshΔ osh4-1ts cells under hypoxic conditions in the presence of DHE for 36 hrs at 23°C, then shifted the temperature to 37°C for 60 min before transferring the cells to aerobic conditions at 37°C. Samples were withdrawn every 20 min and DHE redistribution was examined. Fluorescence images of cells taken at the 0 and 60-min time point () show clearly that DHE redistributes from the PM to LDs effectively in oshΔ osh4-1ts cells at the non-permissive temperature indicating that Osh proteins are dispensable for this process.
Manual scoring of fluorescence images indicated that DHE redistribution occurred with similar kinetics in both oshΔ osh4-1ts and wild-type cells (). However, whereas both cell types showed clear fluorescent foci at the 60-min time point, the oshΔ osh4-1ts cells had a higher level of diffuse fluorescence. This suggested that the rate of intracellular transport and/or esterification of DHE might be slightly reduced as a result of Osh deficiency. Analysis of fluorescence images taken at different time points revealed that intracellular accumulation of DHE occurs ~2.5-fold more slowly in the Osh-deficient strain than in wild-type cells (). As indicated above, this value is an overestimate, more so as the characteristic flocculation of the Osh-deficient cells increases the contribution of out-of-plane fluorescence, especially at early time-points. We also carried out HPLC analyses to measure DHE esters. shows that the rate of DHE esterification is ~2-fold lower in the oshΔ osh4-1ts mutant than in wild-type cells. The reduction in the rate of esterification was not due to ACAT deficiency as ACAT activity in the mutant cells was ~2-fold higher than in wild-type cells (2.13 ± 0.27 vs 0.89 ± 0.02 nmol/mg/min). We conclude that redistribution of DHE from the PM to intracellular compartments and LDs is slowed ~2-fold in the absence of the Osh proteins (). This is a small effect, possibly a downstream consequence of the loss of Osh protein function. We conclude that members of this protein family are not required for direct transfer of sterols from the PM to internal membranes and, in turn, to LDs.
Transport-coupled conversion of cholesterol to cholesteryl esters in Osh-deficient cells
Our results with DHE are quantitatively different from those of Raychaudhuri et al. (18
) who reported that cholesterol transport from the PM to LDs is slowed by ~7-fold in the absence of Osh proteins. Possible reasons for the difference include distinct methods of sterol loading (hypoxic conditions versus use of upc2-1
strains), and the use of cholesterol versus DHE.
To explore the latter possibility we tested cholesterol in our uptake assay. We discovered that, different from the situation with DHE, cholesterol esterification occurs to some extent even under hypoxic conditions. We therefore incubated oshΔ osh4-1ts cells under hypoxic conditions at the permissive temperature, shifted the culture to 37°C for 60 min, then incubated aerobically for 60 min at 37°C in YPD medium supplemented with [14C] cholesterol. The wild-type parental strain was processed alongside. shows that cholesteryl esters are formed ~1.5-fold more slowly in the Osh-deficient cells compared with wild-type cells. These data reinforce the conclusions of the DHE redistribution assay (cumulative results are summarized in ), i.e., that Osh protein deficiency has only a modest effect on retrograde transport of sterol from the PM to the ER and LDs.
The reasons for the discrepancy between our results and those of Raychaudhuri et al. are unclear. We note, however, that under our assay conditions ACAT activity is ~2-fold higher in the osh
cells than in comparably treated wild-type cells (see above) whereas Raychaudhuri et al. report that the ACAT activity of Osh-deficient (osh
Δ osh4-1ts upc2-1
) cells is 2-fold lower than that of wild-type cells (18
). If esterification is the rate-limiting step in the movement of sterols from the PM to LDs, then the rate of cholesterol movement from the PM to the ER would be ~3-fold (the present study) and ~3.5-fold (Raychaudhuri et al.) lower in Osh-deficient cells compared with wild-type cells, making the results of the two studies comparable.
Newly synthesized ergosterol is transported rapidly from the ER to the PM in Osh-deficient cells
We next tested whether Osh proteins play a role in moving ergosterol from the ER to the PM. For this we incubated oshΔ osh4-1ts cells for 90 min at the non-permissive temperature (37°C), before pulse-labeling them with [3H-methyl]-methionine for 4 min at the same temperature to generate a pool of newly synthesized [3H]ergosterol in the ER. We then tracked the movement of this pool to the PM over a chase period of 90 min at 37°C. Wild-type cells were analyzed in parallel. Transport was assessed by measuring [3H]ergosterol in the PM by two different methods.
1. Assay using subcellular fractionation
Cell samples were collected at the end of the pulse and chase periods, chilled, treated with energy poisons and homogenized. After low speed centrifugation to remove cell debris, the homogenate was subjected to sucrose gradient centrifugation to resolve subcellular fractions (37
). The protocol does not involve a prior direct pelleting of the membranes as this could conceivably facilitate ergosterol exchange through membrane contacts (39
). As shown in and consistent with previous reports (38
), the fractionation procedure effectively resolves the PM from ER and other internal membranes (vacuole, endosomes, Golgi membranes). The PM is mainly recovered near the bottom of the gradient as indicated by the profile of ergosterol and the PM marker protein Gas1, whereas the ER fractionates in the top half of the gradient as seen by the distribution of the ER protein translocon Sec61 and the mannosyl-transferase Dpm1 (not shown). As Sec61 is known to be distributed throughout the ER, including the cortical ER region that lies adjacent to the PM (37
), it is clear from that the PM-enriched fractions are not contaminated by ER membranes. Fractionation profiles of osh
cells and wild-type cells were similar; also, there was no difference in the fractionation profiles of samples taken at the end of the pulse period and after a 90 min chase (the data in correspond to osh
cells at the end of the 90 min chase period).
Delivery of newly synthesized ergosterol from the ER to the PM assayed by sub-cellular fractionation
The specific radioactivity (SR = cpm ÷ absorbance units) of ergosterol was determined for subcellular fractions as well as for whole cells, and the relative specific radioactivity (RSRfrac = SRfrac ÷ SRcell) of fractions pooled pairwise from the top of the gradient was calculated. We first analyzed wild-type cells. At the end of the pulse period, ER-enriched membranes (fractions 1–10) had an RSRfrac >1 whereas the PM (fractions 13–14) had an RSRfrac <1 (; blue squares). At the end of the chase period all gradient fractions had an RSRfrac ~1 (; yellow circles). These data indicate that at the end of the labeling pulse [3H]ergosterol is mainly located in the ER-enriched fractions as expected, but by the conclusion of the chase period it has equilibrated completely with the PM and other cellular membranes.
The spontaneous movement of ergosterol between membranes during the fractionation procedure is predicted to be very slow (t1/2
>100 hr at 4°C (40
)) and so it is unlikely to be a factor in our analysis. Moreover, if ergosterol moved spontaneously between membranes at an appreciable rate during the fractionation procedure, then RSRfrac
would have been equal to 1 for all fractions in the end-of-pulse sample. As RSRfrac
is not equal to 1 in almost all fractions from the end-of-pulse sample (; blue squares), it is clear that spontaneous transfer of ergosterol between membranes during this analysis is negligible.
shows the RSRfrac profiles for Osh-deficient and wild-type cells at the end of the pulse period and after a 90 min chase. The profiles at each time point are essentially the same, indicating that Osh-deficiency does not affect movement of ergosterol from the ER to the PM. Of note, RSRfrac of the PM (fractions 13–14) was ≥ 0.5 at the end of the pulse period for both strains indicating that the t1/2 for ergosterol movement between the ER and PM is ≥ 4 min, irrespective of Osh protein function. It is possible that non-vesicular anterograde sterol transport - like retrograde transport () - is slightly slower in Osh-deficient cells compared with wild-type cells and that this is masked by a low level of vesicular sterol transport.
2. Assay using DRMs
We next analyzed the movement of ergosterol from the ER to the PM by exploiting the fact that newly synthesized ergosterol is largely soluble in ice-cold Triton X-100 whereas PM-localized ergosterol is found in DRMs (9
). In a previous report (9
) we showed that the RSR of the DRM fraction (RSRDRM
) was ~0.5 at the end of the 4 min pulse radiolabeling period and rose to 1 by the end of a 60 min chase, consistent with movement of ergosterol from the ER to the PM. We carried out a similar analysis, comparing wild-type and Osh-deficient cells. The cells were prepared as described for the fractionation experiment except that samples withdrawn at the end of the pulse and chase periods were treated with ice-cold Triton X-100. DRMs were separated from detergent-soluble material by centrifugation. Recovery of bulk, unlabeled ergosterol was ~62.5 ± 5.4% for all samples, consistent with previous reports (9
). shows that RSRDRM
is ~0.5 and ~1 for the end-of-pulse and end-of-chase samples, respectively, for both wild-type and Osh-deficient cells. These data suggest that the movement of newly synthesized ergosterol from the ER to the PM, here operationally defined as the DRM fraction, does not require Osh proteins.
Transport of newly synthesized ergosterol measured by sampling PM ergosterol with MβCD
We previously described an assay in which delivery of newly synthesized [3
H]ergosterol to the PM was measured by sampling PM ergosterol in intact cells with MβCD (9
). As non-vesicular transport exchanges lipids between the cytoplasmic leaflets of cell membranes, newly synthesized ergosterol must first be delivered to the cytoplasmic leaflet of the PM before it can move to the MβCD-accessible outer leaflet. Thus, the MβCD sampling assay measures the end result of at least two transport steps. In contrast, the fractionation-based assays described in the previous section measure delivery of [3
H]ergosterol to the PM as a whole and do not distinguish ergosterol pools within the PM.
To assay transport of newly synthesized synthesized ergosterol to the PM by MβCD sampling, cells were removed at different times during the chase period and incubated with MβCD on ice for 20 min. After incubation, the sample was centrifuged and the cell-free supernatant containing MβCD–bound sterols (termed `MβCD–extract') was analyzed by organic solvent extraction and HPLC to determine its content of [3H]ergosterol and non-labeled ergosterol. An untreated cell sample was analyzed in parallel.
Under our standard conditions the amount of ergosterol extracted by MβCD from wild-type cells was ~0.25% of total cellular ergosterol as previously reported (9
), reminiscent of the poor extractability of DHE from large unilamellar vesicles composed of phosphatidylcholine with saturated acyl chains (43
). In contrast, the amount of ergosterol extracted from Osh-deficient cells under the same conditions was 6.3 ± 0.4% of total cellular ergosterol, corresponding to a ~25-fold greater extraction efficiency. The increased extractability may reflect a change in ergosterol organization at the PM in Osh-deficient cells (see Discussion).
shows the amount of [3H]ergosterol in the MβCD-extract from wild-type and Osh-deficient cells as a function of time, corrected for the 1.7-fold lower level of [3H]ergosterol synthesis in the latter. For wild-type cells, the [3H]ergosterol content of the MβCD-extract increased mono-exponentially (t1/2 ~20 min), whereas for Osh-deficient cells the amount of [3H]ergosterol in the MβCD-extract increased linearly at a rate ~2.5-fold greater than the initial rate measured for wild-type cells ().
Delivery of newly synthesized ergosterol from the ER to the PM assayed by MβCD-sampling
If ergosterol transport (measured by MβCD sampling) occurs at the same rate in wild-type and Osh-deficient cells then the 25-fold higher efficiency of MβCD-mediated ergosterol extraction from the latter should correlate with a 25-fold increase in the initial rate at which [3H]ergosterol is recovered in the MβCD-extract (; compare the measured initial rate for wild-type cells indicated by the line labeled `IR wt' with that predicted for Osh-deficient cells indicated by the line labeled `IR predicted for oshΔ'). This is not what we observed. Instead, the experimentally measured rate of ergosterol transport for Osh-deficient cells was 10-fold lower than the predicted rate.
To facilitate comparison, the transport data () were normalized to correct for the efficiency of MβCD-mediated ergosterol extraction. This yielded a graph of the relative specific radioactivity of the MβCD-extract (RSR = SRMβCD
) as a function of time (), similar to graphs described in earlier publications (9
). It is evident from that [3
H]ergosterol is transferred to the MβCD-extractable pool of PM sterol much more slowly in Osh-deficient cells compared with wild-type cells. These data suggest that Osh-deficiency affects either the delivery of ergosterol from the ER to the PM, or the subsequent movement of ergosterol within the PM to an MβCD-accessible location, or both.
The results from the MβCD sampling assay contrast with those obtained with the fractionation-based assay (). Firstly, the timing (t1/2) of ergosterol delivery to the PM in wild-type cells is different for the two assays: ≤ 4 min (fractionation assay) and ~20 min (MβCD sampling assay). Secondly, for Osh-deficient cells, the corresponding t1/2 values are ≤ 4 min (as for wild-type cells) and ~200 min (10-fold slower than for wild-type cells). This suggests that ergosterol moves rapidly from the ER to the PM, then more slowly within the PM to an MβCD-accessible location. Osh proteins affect only the latter transport step. This model is presented in more detail in the Discussion.