Báo cáo khoa học: Molecular characterization of Osh6p, an oxysterol binding protein homolog in the yeast Saccharomyces cerevisiae pot

Molecular characterization of Osh6p, an oxysterol binding protein homolog in the yeast Saccharomyces cerevisiae Penghua Wang1, Wei Duan1, Alan L Munn2,3 and Hongyuan Yang1 Department of Biochemistry, Faculty of Medicine, National University of Singapore, Republic of Singapore Institute of Molecular and Cell Biology, A*STAR Biomedical Research Institutes, Singapore, Republic of Singapore Institute for Molecular Bioscience, University of Queensland, St Lucia, Queensland, Australia Keywords OSBP; OSH; Osh6p; oxysterol-binding protein; sterol homeostasis Correspondence H Yang, Department of Biochemistry, National University of Singapore, Singapore, 119260 Republic of Singapore Fax: +65 67791453 Tel: +65 68747996 E-mail: bchyangr@nus.edu.sg (Received 25 May 2005, revised 22 July 2005, accepted 28 July 2005) doi:10.1111/j.1742-4658.2005.04886.x Oxysterol binding protein (OSBP) and its homologs have been shown to regulate lipid metabolism and vesicular transport However, the exact molecular function of individual OSBP homologs remains uncharacterized Here we demonstrate that the yeast OSBP homolog, Osh6p, bound phosphatidic acid and phosphoinositides via its N-terminal half containing the conserved OSBP-related domain (ORD) Using a green fluorescent protein fusion chimera, Osh6p was found to localize to the cytosol and patch-like or punctate structures in the vicinity of the plasma membrane Further examination by domain mapping demonstrated that the N-terminal half was associated with FM4-64 positive membrane compartments; however, the C-terminal half containing a putative coiled-coil was localized to the nucleoplasm Functional analysis showed that the deletion of OSH6 led to a significant increase in total cellular ergosterols, whereas OSH6 overexpression caused both a significant decrease in ergosterol levels and resistance to nystatin Oleate incorporation into sterol esters was affected in OSH6 overexpressing cells However, Lucifer yellow internalization, and FM4-64 uptake and transport were unaffected in both OSH6 deletion and overexpressing cells Furthermore, osh6D exhibited no defect in carboxypeptidase Y transport and maturation Lastly, we demonstrated that both the conserved ORD and the putative coiled-coil motif were indispensable for the in vivo function of Osh6p These data suggest that Osh6p plays a role primarily in regulating cellular sterol metabolism, possibly stero transport The oxysterol binding protein (OSBP) and its related proteins (ORP) constitute a large conserved family of proteins in eukaryotes [1,2] OSBP homologs are present in many species including humans and the yeast Saccharomyces cerevisiae (the OSBP homolog in yeast is OSH) These proteins all share a conserved  400 amino acid OSBP related domain (ORD), which contains an ‘OSBP fingerprint’ ‘EQVSHHPP’ [1] Recent studies on the OSBP homologs of humans and Saccharomyces cerevisiae have demonstrated the importance of OSBP proteins in sterol and sphingolipid metabolisms The canonical OSBP is believed to play a role in regulating sterol biosynthesis, Abbreviations ACAT, acyl CoA:cholesterol acyl transferase; CPY, carboxypeptidase Y; DAPI, 4¢-6-diamidino-2-phenylindole; GFP, green fluorescent protein; GST, glutathione-S-transferase; LY, Lucifer yellow; MVB, multivesicular body; ORD, OSBP related domain; ORP, oxysterol binding protein related protein; OSBP, oxysterol binding protein; OSH, yeast gene encoding oxysterol binding protein; Osh6p, yeast OSBP homolog; PA, phosphatidic acid; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PH, pleckstrin homology; PtdIns, phosphatidylinositol; PtdInsP, phosphatidylinositol phosphate; SD, synthetic dropout; SE, sterol ester; TAG, triacylglycerol FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS 4703 Yeast OSBP and sterol homeostasis esterification and sterol-regulated gene transcription Lagace et al [3] reported that overexpression of OSBP in several cell lines reduced cholesterol ester synthesis by 50% and showed a 40–60% decrease in acylCoA:cholesterol acyltransferase (ACAT) activity and mRNA In contrast, overexpression of OSBP up-regulated the transcription of sterol-regulated genes and increased the rate of cholesterol biosynthesis Moreover, OSBP localization is inherently linked to sterol homeostasis [4,5] and sphingolipid metabolisms [6] A recent report suggested that OSBP may be involved in vesicle-dependent ceramide transport from the endoplasmic reticulum to the Golgi [7] Overexpression studies with other human ORPs have also provided further evidence Stable overexpression of ORP2 led to a significant reduction of ACAT activity and cholesterol esters [8] Overexpression of a splice variant of ORP4 (ORP4-S) caused a 40% reduction in esterification of low density lipoprotein-derived cholesterol [9] Most interestingly, the role of OSBP in sterol homeostasis seems to be conserved from mammals to the unicellular yeast The yeast OSHs collectively are essential for cell viability and loss of all OSH function results in a drastic increase in total cellular sterol levels and accumulation of free sterols in the cytoplasm [10,11] More recently, in a landmark paper Wang et al [12] showed that in response to cholesterol binding OSBP interacts with two phosphatases: HePTP and PP2A These phosphatases are brought by OSBP to the vicinity of phosphorylated extracellular signal-regulated kinase ⁄ (ERK1 ⁄ 2) within the cell to promote its dephosphorylation The functions of OSBP homologs are not limited to sterol and sphingolipid metabolisms, they are also implicated in the metabolism of other lipids and in membrane trafficking ORP1 and ORP2 appear to play a role in the Golgi secretory function [13] Deletion of OSH4 ⁄ KES1 is able to bypass the essential requirement for Sec14p, which is a major phosphatidylinositol (PtdIns) ⁄ phosphatidylcholine (PtdCho) transfer protein [14] A recent study proposed that Osh4p ⁄ Kes1p interfaces lipid metabolism and vesicle biogenesis possibly via its regulation on the adenosine diphosphate-ribosylation factor cycle or Pik1p activity, a Golgi associated phosphatidylinositol kinase [15] Lastly, it appears that OSBP and its homologs may also participate in other cellular activities such as cell cycle [16], meiosis and mating [17], mitosis and tumor metastasis [18] The yeast genome encodes seven OSBP homologs, Osh1p–Osh7p Based on sequence homology, the Osh proteins can be further divided into four subfamilies: Osh1p and Osh2p; Osh3p; Osh4p ⁄ Kes1p and Osh5p; Osh6p and Osh7p [10] Among all individual OSH 4704 P Wang et al gene deletions, Osh6D and osh5D exhibited most elevated sterol levels, highlighting the importance of Osh6p and Osh5p in maintaining sterol homeostasis [10] Here, we characterize Osh6p in greater detail and show that deletion or overexpression of OSH6 causes sterol-related defects but does not affect endocytosis and endocytic trafficking of a marker, multivesicular body sorting (MVB) or carboxypeptidase Y (CPY) transport to the vacuole Results Osh6p binds phospholipids As a short Osh protein, Osh6p consists of an ORD for lipid binding and a putative coiled-coil motif for protein–protein interaction In this study, Osh6p was demonstrated to bind a pool of phosphatidylinositol phosphates (PtdInsP) including PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2 and PtdIns(3,5)P2 with the strongest binding to PtdIns(5)P (Fig 1) The ORD domain showed a very similar lipid binding pattern as the fulllength protein While the coiled-coil half and glutathione S-transferase (GST) alone (control) failed to bind any lipids As a positive control GST-EEA1-a FYVE protein preferably bound PtdIns(3)P [19], indicating the specificity and reliability of this assay Although Osh6p showed a higher affinity for PtdIns(5)P than for PtdIns(4)P in vitro, the physiological level of PtdIns(4)P is much higher than that of PtdIns(5)P [20], implying that PtdIns(4)P may be the native ligand of Osh6p under physiological conditions Interestingly, when more lipids and protein were loaded, both Osh6p and the ORD domain of Osh6p bound phosphatidic acid (PA) (Fig 2) The other lipids except for PtdIns(4)P, PtdIns(5)P, PtdIns(3, 4)P, and PtdIns(3,5)P in Fig were also tested under this condition, but no binding was observed (data not shown) Osh6pORD showed an eightfold higher affinity for PA than Osh6p did; indicating the N-terminal half containing the conserved ORD domain mediates lipid binding A weak binding to PtdIns(3)P by all proteins was also observed, which might be nonspecific Characterization of the cellular location of Osh6p The Saccharomyces cerevisiae yeast encodes seven OSBP homologs that share an essential overlapping function; however, each OSH performs distinct roles probably at different cellular locations [10] We hereby examined the cellular location of Osh6p using a C-terminal tagged green fluorescent protein (GFP) construct and examined its distribution by fluorescence FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS P Wang et al Yeast OSBP and sterol homeostasis Fig Osh6p binds phosphoinositides One hundred picomoles of desired lipids were spotted on Hybond-C membranes (Amersham Biosciences), dried and blocked The blot was incubated with GST fusion proteins or GST at a concentration of 30 nM or 60 nM, respectively, washed and then incubated with mouse anti-GST IgG and anti-mouse secondary antibody sequentially Protein-bound lipids were detected using ECL microscopy As shown in Fig 3A, Osh6p-GFP was predominantly cytosolic, with a minor pool associated with patch-like structures in the vicinity of plasma membrane However, the N-terminal half Osh6p(1– 254)-GFP showed strikingly different localization As seen in Fig 3A, Osh6p(1–254)-GFP was associated with one or two punctate structures without discernible peripheral staining These punctate structures were further found to colocalize with the FM4-64 staining endosomes (Fig 3B) Most interestingly, the C-terminal half Osh6p(255–448)-GFP was localized to the nucleoplasm (Fig 3A), which was further confirmed by 4¢-6-diamidino-2-phenylindole (DAPI) staining As Fig 3C shows, GFP staining structures were well colocalized with DAPI staining nucleus Osh6p is required for sterol homeostasis Osh proteins collectively play a crucial role in maintaining sterol homeostasis In addition, deletion of each individual OSH was also shown to affect sterol levels to some extent [10] Here we examined the role of Osh6p in sterol homeostasis by gene deletion and overexpression approaches First we checked the FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS OSH6 deletion and overexpression strains by Western blotting using anti-Osh6p serum Figure 4A shows that an  50 kDa band was recognized by anti-Osh6p serum from cell extracts of wildtype cells, which was absent from extracts of OSH6 knockout cells On the other hand, the amount of Osh6p protein overexproduced using the ADH1 promoter from a 2l plasmid (pADNSOSH6) was increased by more than 10-fold compared to vector only (pADNS) (Fig 4B) Next we analyzed the steady-state ergosterol levels in osh6D and overexpression cells Consistent with a previous report [10], deletion of OSH6 caused an increase in total ergosterol levels by  80% (Fig 4C) in a strain from the Euroscarf collection (Institute of Microbiology, Johann Wolfgang Goethe-University, Frankfurt, Germany) Introduction of OSH6-GFP on a CEN plasmid into osh6D corrected the ergosterol levels back to that of wildtype However, overexpression of OSH6 reduced the ergosterol levels (Fig 4C) and associated with nystatin-resistance (10-fold greater than vector control) (Fig 4D), indicating a reduction of free sterols in the plasma membrane To test the sensitivity and reliability of GC-MS analysis, we used arv1 and erg3 (ergosterol biosynthesis) mutants as controls 4705 Yeast OSBP and sterol homeostasis P Wang et al 103 500 250 125 62 31 pmol PA PC PE PS PI(3)P GST PA PI(3)P GST-Osh6p PA a dye that specifically stains neutral lipids As shown in Fig 4E, an average of six lipid droplets per cell (n ¼ 100) was observed in wildtype cells; while there were about four only in the OSH6 overexpressing cells Exposure times were equal for both strains and the brightness of lipid bodies was almost the same Deletion of OSH6 resulted in a significant increase of total sterol levels (Fig 4C); however, no change in the number of lipid droplets was observed (data not shown) In order to elucidate how Osh6p affected sterol levels, we tested sterol esterification and the rate of sterol biosynthesis in both osh6D and OSH6 overexpressing strains Sterol biosynthesis was slightly accelerated in osh6D but not affected in OSH6 overexpressing cells (data not shown) Sterol esterification in OSH6 deletion and overexpressing cells was decreased, but triacylglycerol biosynthesis was not significantly affected (Fig 5), indicating that fatty acid uptake and transport was normal As positive controls, deletion of the major sterol esterification gene ARE2 reduced 3H-labeled sterol esters by  75% [27], and the mutation of VPS4 (vacuolar protein sorting 4) caused a 40% decrease [34] (Fig S2) Osh6p is not essential for fluid-phase endocytosis or endocytic traffic of membrane markers from the cell surface to the vacuole PI(3)P GST-Osh6pCC PA PI(3)P GST-Osh6pORD Fig Osh binds PA as described in Fig except for the concentrations used for GST fusions and GST, which were 60 nM and 120 nM, respectively The amount of lipid spotted is indicated on the top of blots Deletion of ARV1 results in  50% increase in free sterols and  75% increase in sterol esters [32] and erg3D mutants cannot synthesize ergosterol [33] Consistent with these reports, we observed a 38% increase in total ergosterol in arv1D compared to wildtype and no ergosterol was detected in erg3D by GC-MS (Fig S1) To examine whether osh6 mutants affected sterol ester levels, cells were stained with Nile Red, 4706 We next investigated the role of Osh6p in certain membrane trafficking pathways Because endocytosis is severely impaired in cells with seven OSHs mutated, we first examined Lucifer yellow uptake in the osh6D and OSH6 overexpressing mutants Lucifer yellow (LY) is taken up and delivered to the lumen of the vacuole in wildtype cells As shown in Fig 6A, osh6D exhibited no deficiency in uptake or transport of LY to the vacuole In addition, even when OSH6 was highly overexpressed from the ADH1 promoter on a 2l plasmid, no significant defect was observed (data not shown) We then examined whether Osh6p was important for FM4-64 uptake and transport FM4-64 is a lipophilic dye that intercalates into cell membranes and is delivered to the vacuolar limiting membrane via the endocytic pathway [21] The osh6D cells were labeled with FM4-64 at 15 °C and warmed to 30 °C (chase), then assessed for distribution of FM4-64 at different time points At early time points of chase FM4-64 labels small punctate structures representing early endosomes, and at later time points it accumulates in large late endosomal ⁄ prevacuolar structures adjacent to the vacuole Finally FM4-64 reaches the vacuole limiting membrane in wildtype cells As shown in Fig 6B, several small punctate structures representing early endosomes were seen at FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS P Wang et al Yeast OSBP and sterol homeostasis A C GFP DAPI Merge DIC GFP Osh6p (1-254) -GFP Osh6p (255-448) -GFP Osh6p -GFP GFP DIC B GFP FM4-64 Merge Fig Localization of Osh6p (A) Exponentially growing cells (Y10000) expressing GFP fusions from YEplac181 vector were mounted on a glass slide and visualized using a Leica fluorescence microscope (B) Colocalization of Osh6p(1–254)-GFP with FM4-64 positive compartments Cells (Y10000) were labeled with FM4-64 in ice-water for 30 and then shifted to 15 °C for 20 allowing FM4-64 to be internalized Cells were immediately put back on ice and washed thoroughly to remove excess dye GFP and FM4-64 images were acquired via a GFP filter and Texas Red filter, respectively Arrows indicate some colocalization (C) Colocalization of Osh6p(255–448)-GFP with DAPI stained nucleus Scale bar: lm DIC, differential interference contrast of chase At of chase, one or two large dots were observed around the vacuole, which represent late endosomal ⁄ prevacuolar structures At 10 min, punctate staining diminished and vacuolar staining increased, and finally punctate staining was almost lost with predominant vacuolar staining at 30 Compared with wildtype, no delay of FM4-64 transport was observed in osh6D In addition, FM4-64 transport was not affected in OSH6 overexpressing cells (data not shown) Osh6p is not essential for carboxypeptidase Y maturation or multivesicular body sorting Although it has been recently demonstrated that loss of all OSH function did not affect carboxypeptidase Y (CPY) maturation [11], the role of individual Osh proteins in CPY sorting has not been investigated It is likely that members of the Osh protein family may have FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS antagonistic effects on each other [10]; therefore, we tested whether Osh6p play a role in biosynthetic protein trafficking of the soluble CPY to the vacuole CPY is modified by core glycosylation in the endoplasmic reticulum (p1 form with molecular mass of 67 kDa), and transported to the Golgi for further glycosylation (p2 form with molecular mass of 69 kDa) The p2 form of CPY traverses the Golgi and is packaged into vesicles destined for the vacuolar protein sorting pathway which delivers it to the vacuole Upon delivery to the vacuole it is processed to its mature form (M form with molecular mass of 61 kDa) Deficiency in this process leads to accumulation of and secretion of p2 CPY As shown in Fig 7, at of chase, p1 CPY was the predominant form The p2 form was seen at the time point At a later time (10 min), mature CPY was observed together with some remaining p2 and p1 precursors At the 30 time point, most CPY was in the mature 61 kDa form Compared with wildtype, 4707 Yeast OSBP and sterol homeostasis P Wang et al osh6 WT 1µg/ml nystatin Osh6p Dpm1p pADNS pADNS pADNS OSH6 pADNSOSH6 No nystatin pADNS Osh6p pADNS OSH6 Ergosterol (µ g/mg dry weight) VATPase60 16 pADNS 12 pADNS OSH6 WT osh6∆ vector vector osh6∆ WT WT OSH6-GFP pADNS pADNSOSH6 Nile Red DIC Fig Osh6p is required for maintaining sterol homeostasis (A,B) Western blotting Osh6p was detected using rabbit anti-Osh6p serum at : 300 dilution Dpm1p and VATPase60 were used as internal loading controls pADNS represents the Y10000 strain harboring vector (pADNS); pADNSOSH6 shows Y10000 strain transformed with pADNSOSH6 overexpressing OSH6 from the ADH1 promoter (C) Total sterols (free and esterified) were extracted with hexane and blow-dried under a stream of nitrogen Ergosterol was identified and quantified by GC-MS Results were obtained from two independent experiments (n ¼ 3) The x-axis denotes strain genotype Wildtype (WT) vector: Y10000 cells transformed with YCplac111-scGFP; osh6D vector: Y15074 cells transformed with YCplac111-scGFP; osh6D OSH6-GFP: Y15074 cells carrying YCpOSH6GFP; WT pADNS ⁄ WT pADNSOSH6: Y10000 cells harbouring pADNS ⁄ pADNSOSH6 (D) Nystatin assay Cells at mid log phase were serially diluted by 10-fold and grown on SD solid medium lacking leucine with (upper) or without (lower) lgỈmL)1 nystatin at 30 °C for 48 h (E) Visualization of lipid droplets Cells at stationary phase were stained with Nile Red and visualized via a Texas Red filter using a Leica fluorescence microscope Scale bar: lm DIC, differential interference contrast osh6D showed no delay in CPY maturation Although even wildtype cells secret a minute fraction of CPY precursors, we could not detect CPY in the extracellular extracts from either wildtype or osh6D cells in this study This discrepancy may be due to insufficient proteins loaded for detection We next checked the role of Osh6p in the multivesicular body (MVB) pathway using GFP fusions of the surface receptor-Ste3p and vacuolar hydrolase-carboxypeptidase S MVB is a process whereby the limiting membrane of late endosomes invaginates and buds into the lumen of the organelle, and is responsible for the biosynthetic delivery of lysosomal hydrolases and the down-regulation of numerous activated cell surface receptors However, no defect was 4708 observed in either osh6D or OSH6 overexpressing cells (data not shown) Characterization of the functional domains of Osh6p As a short Osh protein, Osh6p consists of a conserved ORD domain that is believed to mediate lipid binding and a putative coiled-coil motif for protein interaction We were also interested to know whether these domains were important for the function of Osh6p We expressed GST fusion proteins in JRY6326 strain (pMET2-OSH2, osh1-osh7D) In the presence of methionine, OSH2 expression is suppressed, leading to cell FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS TAG (cpm/mg dry weight) SE (cpm/mg dry weight) P Wang et al Yeast OSBP and sterol homeostasis 2500 2000 1500 1000 500 WT osh6 ∆ WT osh6 ∆ pADNS pADNSOSH6 8000 6000 4000 2000 pADNS pADNSOSH6 Fig Oleate incorporation Sterol esterification (A) or triacylglycerol (TAG) synthesis (B) was determined by incorporation of [3H]oleic acid into sterol esters (SE) or TAG and expressed as 3H-labeled SE or TAG per mg of dry cells Results were obtained from two independent experiments (n ¼ 4) The x-axis denotes strain genotype cpm, counts per minute; WT, Y10000; osh6D, Y15074; pADNS (vector control) and pADNS OSH6 indicate Y10000 cells transformed with pADNS ⁄ pADNSOSH6 growth arrest Reintroduction of any OSH gene can restore cell growth except in OSH1 for which overexpression is required [10] When the coiled-coil domain, ORD domain and full-length Osh6p were overexpressed in JRY6326 (Fig 8), only full-length Osh6p protein could rescue JRY6326 in the presence of methionine, which suggested the ORD and coiledcoil domains were both essential for Osh6p function Discussion Osh proteins collectively play a crucial role in maintaining sterol homeostasis Recent studies have largely focused on the function of all seven OSH gene products [10,11] Those studies provided invaluable insights into understanding the function of this family of proteins in yeast and offered guidance to future research On the other hand, although the entire Osh protein family shares at least one essential function, each individual Osh protein possesses unique roles One prominent example is KES1, the mutation of FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS which, but none of the other OSH genes, bypasses the essential requirement for SEC14 [14] Therefore, it is necessary to analyze individual OSH genes to gain further insights into the function of this family of proteins The lipid binding properties have long been established for OSBP and its homologs OSBP binds oxysterols via its C-terminal half containing the conserved ORD domain and phosphoinositides via its pleckstrin homology (PH) domain [22–24] Here we show that the non-PH containing Osh6p can also bind phosphoinositides and PA (Figs and 2) with different affinity In addition, bacterially expressed Osh6p shows no affinity for ergosterol, diacylglycerol (DAG) or ceramide in our assay system (data not shown) Our results represent a novel and exciting finding given the fact that the bypass sec14 mutants require a basal PA level to exert their suppression effects [25,26] Consistent with this, the three OSBP homologs: Kes1p, ORP1and ORP2 which are potentially involved in the Golgi secretory function are able to bind PA with varying affinity [13–15] Further, our results demonstrate that the N-terminal half (amino acids 1–300) containing the conserved OSBP domain is sufficient and indispensable for Osh6p binding to phospholipids Unlike the long Osh proteins, Osh6p contains no canonical PH domain that mediates phospholipid binding [23,24] However, it is possible that Osh6p may bind phospholipids through a domain other than PH In support of this, the short Kes1p containing no PH domain was also shown to bind PtdIns (4,5)P [15] Although the role of the conserved ORD domain is unclear, our results imply that it could recognize specific lipid ligand(s) As an effort to understand the role of Osh6p in the vesicular transport, we examined the cellular location of Osh6p Although the cellular location of the full-length protein could not be pinpointed in this study, the patch-like structures might possibly represent endoplasmic reticulum Interestingly, the N terminus (1–254) is localized to endosomes and the C terminus (255–448) to the nucleoplasm To our knowledge, Osh6p may be the first of the OSBP homologs to be shown to localize to the nucleoplasm and the physiological relevance is worthy of further investigation In addition, our data suggest that like long Osh proteins, Osh6p may be able to associate with multiple membranes through different functional domains Consistent with the result from lipid binding analysis (Fig 1), the ORD domain may bind phospholipids on the endosomal membranes thus targeting Osh6p to membranes; whereas the C terminus containing the coiled-coil motif could interact with a protein or protein complex in the other compartment 4709 Yeast OSBP and sterol homeostasis P Wang et al WT FITC DIC Fig Osh6p is not essential for Lucifer yellow (LY) uptake and FM4-64 transport (A) Wildtype (Y10000) or osh6D (Y15074) cells at early log phase were allowed to internalize LY for h and images were captured using a Leica fluorescence microscope DIC, differential interference contrast; FITC, fluorescent image (B) Cells at early log phase were labeled with FM4-64 at 15 °C for 20 After removal of excess FM4-64, cells were chased for 0, 5, 10, and 30 at 30 °C FM4-64 staining was visualized using a Leica fluorescence microscope equipped with a Texas Red light filter Upper panels: DIC and FM4-64 images of Y10000 (wildtype) Lower panels: DIC and FM4-64 images of Y15074 (osh6D) Time of chase is indicated at the bottom Scale bar: lm 4710 FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS P Wang et al Yeast OSBP and sterol homeostasis P2 P1 OSH6 WT M P2 P1 osh6∆ M 10 30 Intracellular OSH6ORD OSH6CC 10 30 Extracellular Fig Osh6p is not essential for CPY maturation Cells were radiolabeled with [35S]methionine ⁄ cysteine and chased for various times with cold methionine ⁄ cysteine Cells were lysed and CPY was immunoprecipitated from cell lysate (intracellular) and extracellular growth medium ⁄ periplasm (extracellular) Proteins were resolved by SDS ⁄ PAGE and detected by radiophotography p1, core glycosylated endoplasmic reticulum form; p2, fully glycosylated Golgi form; M, mature vacuolar form; wildtype, SEY6210; osh6D, SEY6201 Times of chase are indicated under the blot (for example the nucleoplasm or endoplasmic reticulum membrane) Excess intracellular sterols are normally converted to sterol esters, a process that is catalyzed by Are1p and Are2p in yeast [27] Sterol esterification is primarily regulated by the availability of sterol substrates However, excess ergosterol observed in osh6D does not result in enhanced sterol esterification (Fig 5A) or increased lipid droplets as assessed by Nile Red staining (not shown) It is therefore possible that osh6D cells accumulate free sterols in intracellular compartments other than the ER, due to defects in lipid trafficking In support of this, it was recently reported that filipin staining sterols accumulated in the internal membranes of OSH null mutants [11] In addition, although some OSH mutants including osh6D have increased total ergosterol levels, they exhibit no nystatin sensitivity [10], indicating a possible disruption of sterol transport and accumulation of free sterols in membranes other than the plasma membrane In support of this idea, two recent studies suggested OSBP and its homologs are potentially involved in vesicleindependent intracellular lipid transport [9,28] More recently, Wang et al [12] showed that upon binding to cholesterol OSBP served as a scaffold and recruited two phosphatases, thus regulating the cholesterol-mediated ERK1 ⁄ signaling Although deletion of all OSH genes impaired endocytosis and the vacuole morphology [11], the results presented here suggest that Osh6p is not essential for some membrane trafficking pathways Neither FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS Vector Fig Functional domains of Osh6p pYEX4T-1, pYEXOSH6, pYEXOSH6CC, and pYEXOSH6ORD were introduced into JRY6326 cells and grown on YM solid medium containing mM methionine for 72 h at 30 °C endocytic (Fig 6) nor biosynthetic transport to the vacuole (Fig 7) is affected in the absence of Osh6p or the presence of a high level of Osh6p The discrepancy may be due to the fact that a more drastic disruption of sterol homeostasis is incurred upon loss of all Osh function For instance, total cellular ergosterol increase by 3.5-fold in OSH mutant cells compared with an 80% increase in osh6D cells In summary, this study provides a detailed characterization of Osh6p, one of the seven OSBP homologs in yeast We show, for the first time, a direct binding of PA by the ORD domain and the cellular location of Osh6p We further demonstrate that deletion or overexpression of OSH6 affects sterol homeostasis but not endocytosis or CPY secretion Our results suggest that the primary molecular function of Osh6p is likely to be in sterol metabolism, probably sterol transport Experimental procedures Materials Mouse anti-CPY, Dpm1p, VATPase60 and rabbit antiGST IgGs were obtained from Molecular Probes (Eugene, OR, USA) Mouse anti-GST IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) YPD medium contained 2% (w ⁄ v) dextrose, 2% (w ⁄ v) peptone and 1% (w ⁄ v) yeast extract (Gibco-BRL ⁄ Life Technologies, Paisley, UK) Synthetic dropout medium (SD) was comprised of 0.67% (w ⁄ v) yeast nitrogen base, 2% (w ⁄ v) 4711 Yeast OSBP and sterol homeostasis P Wang et al dextrose plus dropout powder YM (yeast minimal) medium was made of 2% (w ⁄ v) dextrose and 0.67% (w ⁄ v) yeast nitrogen base LB broth contained 1% (w ⁄ v) tryptone, 0.5% (w ⁄ v) yeast extract, and 1% (w ⁄ v) sodium chloride Construction of plasmids and strains Construction of expression plasmids were performed according to the universal protocols [29] For plasmids and strains used in this study, see Tables and Biochemical assays Expression and purification of GST fusion protein was performed essentially according to a method of Dowler et al [30] Fluorescence microscopy FM4-64 staining was performed according to a previously described method [21] Cells at early log-phase (D600 ¼ 0.2) were stained with FM4-64 at a final concentration of 1.2 lgỈmL)1 at 15 °C for 20 for preliminary labeling and the excess dye was removed by washing with ice-cold labeling medium Cells were then resuspended in fresh labeling medium and incubated at 30 °C An aliquot was removed at 0, 5, 10 and 30 time points and endocytosis was stopped by addition of ice-cold NaF ⁄ NaN3 to a final concentration of 12 mm All samples were thoroughly washed with ice-cold wash buffer (1· NaCl ⁄ Pi, 10 mm NaF ⁄ NaN3) to remove excess dye Lucifer yellow and DAPI staining was performed essentially following a method of Yeo et al [31] and Levine et al [35] Nile Red staining was done following the method of Yang et al [27] with minor modification Cells were grown to stationary phase at 30 °C and mL of cells was harvested by brief centrifugation Cells were washed twice with 1· NaCl ⁄ Pi and resuspended in mL of staining solution (1· NaCl ⁄ Pi, lgỈmL)1 Nile Red) Nile Red staining pattern was visualized using a Leica DMLB fluorescence microscope (Chatsworth, CA, USA) via a Texas Red filter Table Plasmids Unless specified, all the constructs were made in this study Plasmids Description Ref or source pGEXOSH6 pGEXOSH6ORD pGEXOSH6CC pYEXOSH6 pYEXOSH6CC pYEXOSH6ORD pGEX4T-1 pYEX4T-1 pADNSOSH6 pADNS YCpOSH6-GFP YEpOSH6(1–254)-GFP YEpOSH6(255–448)-GFP YEpOSH6-GFP YCplac111-scGFP YEplac181-scGFP YEp-pOSH1-GFP pGEX4T-1, GST, OSH6(1–448) pGEX4T-1, GST, OSH6(1–300) pGEX4T-1, GST, OSH6(301–448) pYEX4T-1, GST, OSH6(1–448) pYEX4T-1, GST, OSH6(301–448) pYEX4T-1, GST, OSH6(1–300) Ptac, GST, AmpR Pcup1, GST, URA3, leu2-d, 2l pADNS, OSH6(1–448) PADH1, LEU2, 2l YCplac111, OSH6 promoter, OSH6(1–448), GFP YEplac181, OSH6 promoter, OSH6(1–254), GFP YEplac181, OSH1 promoter, OSH6(255–448), GFP YEplac181, OSH6 promoter, OSH6(1–448), GFP YCplac111, GFP, CEN, LEU2 YEplac181, GFP, 2l, LEU2 YEplac181, OSH1 promoter, GFP Amersham BD biosciences [31] [31] Table Strains Strain name Genotype Ref or source Y10000 Y15074 JRY6326 BY4742, MATa his3D1 leu2D0 lys2D0 ura3D0 BY4742, MATa his3D1 leu2D0 lys2D0 ura3D0 osh6D::KanMX4 SEY6210, TRP1::PMET3-OSH2 osh1D::kan-MX4 osh2D::kan-MX4 osh3D::LYS2 osh4D::HIS3 osh5D::LEU2 osh6D::LEU2 osh7D::HIS3 MATa ura3-52 his3D200 lys2-801 leu2-3,112 trp1D901 suc2D9 SEY6210, osh6D::LEU2 BY4742, MATa his3D1 leu2D0 lys2D0 ura3D0 erg3D::KanMX4 BY4742, MATa his3D1 leu2D0 lys2D0 ura3D0 arv1D::KanMX4 BY4742, MATa his3D1 leu2D0 lys2D0 ura3D0 vps4D::KanMX4 BY4742, MATa his3D1 leu2D0 lys2D0 ura3D0 are2D::KanMX4 Euroscarf Euroscarf [10] SEY6210 SEY6201 Y12667 Y15151 Y15588 Y15394 4712 [10] [10] Euroscarf Euroscarf Euroscarf Euroscarf FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS P Wang et al Pulse-chase radiolabeling and immunoprecipitation The CPY maturation experiment was carried out according to a method of Yeo et al [31] with minor modifications Twenty units at D600 of overnight cell culture in SDYE [SD medium containing 0.2% (w ⁄ v) yeast extract and minimum supplements] was harvested and washed with 50 mL of SD containing necessary supplements Cells were resuspended in 2.5 mL labeling mix (SD containing necessary supplements and mgỈmL)1 BSA) and incubated with 50 lL of [35S]methionine ⁄ cysteine (7.9 mCiỈmL)1 PerkinElmer, (Wellesley, MA, USA)) for 10 Fifty microliters of 50· chase solution (0.1 m Na2SO4, mgỈmL)1 cysteine, mgỈmL)1 methionine) was added, and 0.5 mL sample was transferred immediately to a microfuge tube containing 50 lL stop solution (0.2 m NaF and NaN3) on ice (labeled as min) Subsequently, at times of 5, 10 and 30 min, 0.5 mL sample was removed and treated with stop solution as previously Cells were converted to spheroplasts and then lysed CPY was immunoprecipitated from the growth medium ⁄ periplasm (extracellular) or spheroplast lysate (intracellular) using anti-CPY IgG and Protein A sepharose beads After stringency wash, bound CPY was eluted and separated by SDS ⁄ PAGE, and finally detected by radiophotography GC-MS analysis of ergosterol Sterol lipids were extracted using a method of Beh et al [10] For triplicate analysis of a same culture, 150 mL of exponentially growing yeast (D600 ¼ 0.5–0.8) were split into three equal volumes and harvested by brief centrifugation (5 mL was removed for dry weight analysis) Cells were washed once with 50 mL distilled water The cell pellets were resuspended in 1.25 mL of 0.1 m HCl and boiled for 20 Cells were washed twice with mL of distilled water and then the cell pellets were resuspended in 0.5 mL of 67% (v ⁄ v) methanol Cells were lyzed with glass beads by vigorous vortexing for For total sterol analysis (free and esterified), 1.25 mL of methanol and 0.63 mL of 60% KOH were added to the slurry followed by heating at 70 °C for 90 Sterols were extracted with mL of hexane four times Ergosterol was identified and quantified by GC-MS QP5000 (Shimadzu Corporation, Kyoto, Japan) Oleate incorporation Sterol esterification or triacylglycerol (TAG) synthesis was measured by incorporation of [3H]oleic acid into sterol esters or TAG following a method of Yang et al [27] Ten milliliters of exponentially growing cells (D600 ¼ 0.7–0.8) were labeled with lL of [3H]oleic acid 5.0 mCiỈmL)1, Amersham Biosciences (Uppsala, Sweden) for h at 30 °C Cells were washed twice with mL of ice-cold wash buffer [0.5% (v ⁄ v) NP-40, 20 mm NaN3] and once with distilled water Cells FEBS Journal 272 (2005) 4703–4715 ª 2005 FEBS Yeast OSBP and sterol homeostasis were split into three equal aliquots and lyophilized overnight The dried cells were lyzed in 50 lL of lysis buffer [1700 mL)1 lyticase, 10% (v ⁄ v) glycerol, and 0.02% (w ⁄ v) sodium azide] at 37 °C for 15 The cell suspension was then subject to one freeze-thaw cycle As an internal standard, 0.1 lL of [14C]cholesterol (0.1 mCiỈmL)1, Amersham) in 200 lL isopropanol was added to the cell suspension Lipids were extracted from the cell suspension with mL of hexane by vigorous vortexing Phase separation was induced by addition of mL of KCl ⁄ methanol (2 m KCl ⁄ methanol ¼ : 1, v ⁄ v) The hexane extracts were blow-dried under a nitrogen stream Sterol esters or TAG were separated by TLC and quantified using a scintillation counter Protein lipid overlay assay Protein lipid overlay assay was carried out following a method of Dowler et al [30] Phosphatidylinositol phosphate (PtdInsP) strips containing indicated lipids were blocked in blocking solution [3% (w ⁄ v) fatty acid free BSA in TBST (50 mm Tris ⁄ HCl, pH 7.5, 150 mm NaCl and 0.1% (v ⁄ v) Tween-20)] at room temperature for h The PtdInsP strips were then incubated with GST fusion proteins at a concentration of 30–60 nm for fusion proteins or 60–120 nm for GST only for h at room temperature After washing with six changes of TBST over 0.5 h, the PtdInsP strips were incubated with anti-GST monoclonal antibody (1 : 3000) for h, washed again as previously, and then incubated with mouse anti-IgG horseradish peroxidase conjugate (1 : 4000) for h The PtdInsP strips were finally washed with 12 changes of TBST over h The protein-bound lipid ligands were detected by enhanced chemiluminescence (Amersham Biosciences) Acknowledgements This work was supported by a Young Investigator Award from the National University of Singapore, a grant from the National Medical Research Council, Singapore (to H.Y.) 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GC-MS Fig S2 Oleate incorporation Sterol esterification or TAG synthesis was determined by incorporation of [3H]oleic acid into sterol esters (SE) or TAG and expressed as 3H-labeled sterol esters or TAG per mg of dry cells 4715 ... Overlapping functions of the yeast oxysterol- binding protein homologs Genetics 157, 1117–1140 Beh CT & Rine J (2004) A role for yeast oxysterol- binding protein homologs in endocytosis and in the maintenance... provided invaluable insights into understanding the function of this family of proteins in yeast and offered guidance to future research On the other hand, although the entire Osh protein family shares... detailed characterization of Osh6p, one of the seven OSBP homologs in yeast We show, for the first time, a direct binding of PA by the ORD domain and the cellular location of Osh6p We further demonstrate