ATP-binding cassette (ABC) transporters are well known for their roles as multidrug resistance determinants but also play important roles in regulation of lipid levels. In the yeast Saccharomyces cerevisiae, the plasma membrane ABC transporter proteins Pdr5 and Yor1 are required for normal rates of transport of phosphatidyethanolamine to the surface of the cell. Loss of these ABC transporters causes a defect in phospholipid asymmetry across the plasma membrane and has been linked with slowed rates of trafficking of other membrane proteins. Four ABC transporter proteins are found on the limiting membrane of the yeast vacuole and loss of one of these vacuolar ABC transporters, Ybt1, caused a major defect in the normal delivery of the phosphatidylcholine (PC) analog NBD-PC (7-nitro-2,1,3-benzoxadiazol-PC) to the lumen of the vacuole. NBD-PC accumulates on cytosolic membranes in an ybt1Δ strain. We demonstrated that Ybt1 is required to import NBD-PC into vacuoles in the presence of ATP in vitro. Loss of Ybt1 prevented vacuolar remodeling of PC analogs. Turnover of Ybt1 was reduced under conditions in which function of this vacuolar remodeling pathway was required. Our data describe a novel vacuolar route for lipid remodeling and reutilization in addition to previously described enzymatic avenues in the cytoplasm.
Control of lipid content of membranes is a feature that defines the many different membranes in a eukaryotic cell. Phospholipids represent the major component of membranes and are highly regulated in terms of their content and distribution across these membranes (reviewed in 1). The differential distribution of phospholipids across a membrane is referred to as asymmetry and is generated through the balanced movement of phospholipids from the outer leaflet to the inner leaflet (flip) and the inverse of this directional transport (flop) (see Refs. 2,3 for reviews). The best characterized contributors to this vectorial movement of phospholipids are P-type ATPases such as Drs2 from Saccharomyces cerevisiae(4). Drs2 is a Golgi-localized enzyme that acts as a flippase, moving phospholipids like phosphatidylethanolamine (PE) and phosphatidyserine (PS) into the cytoplasm (5,6). Less is known about enzymes catalyzing the flop activity but a number of studies implicate ATP-binding cassette (ABC) transporters as participants in this directional transport of phospholipids.
The first example of an ABC transporter protein exhibiting a potential floppase activity came from the demonstration that mouse Mdr2 (Abcb4) was required for secretion of phosphatidylcholine (PC) into biliary circulation (7). Later work in S. cerevisiae has demonstrated the role of the plasma membrane ABC transporters Pdr5 and Yor1 in flop of PC and PE (8,9). A strain lacking Pdr5 and Yor1 exhibits a reduction in levels of exofacially exposed PE (10), indicating that the normal plasma membrane asymmetry has been lost in response to loss of these two genes and their products. Importantly, analysis of a pdr5Δyor1 strain demonstrated that the membranes of this double mutant strain were unable to support normal plasma membrane accumulation of the tryptophan transporter Tat2, consistent with asymmetry being required to maintain wild-type (WT) protein trafficking (11).
Along with the ABC transporters found on the plasma membrane, S. cerevisiae typically expresses four of these proteins localizing to the limiting membrane of the vacuole. These include the cadmium resistance determinant Ycf1 (12,13), two different transporters shown to transport bile pigments and bile acids, Bpt1 and Ybt1 (14,15), and a recently characterized protein that exhibits a multidrug resistance phenotype, Vmr1 (16). All of these proteins are members of the ABCC class of ABC transporters (http://www.ncbi.nlm.nih.gov/books/NBK31/). Yor1 is the only known plasma membrane representation of the S. cerevisiae ABCC family (17,18). As Yor1 has been shown to be involved in flop of phospholipids at the plasma membrane, we wanted to examine if these vacuolar membrane-localized ABC transporters might also be involved in regulation of phospholipid homeostasis. To examine this possibility, we used the fluorescent PC analog, NBD-PC, to follow trafficking of this compound in cells engineered to lack different combinations of genes encoding these vacuolar ABC transporter proteins.
Genetic analysis indicated that loss of the YBT1 gene alone was sufficient to completely eliminate the usual import of NBD-PC into the vacuolar lumen. Earlier work had found that loss of the endosomal trafficking protein Vps27 also blocked luminal delivery of NBD-PC. Ybt1 localization, expression and function were undisturbed in a vps27Δ strain, indicating that these two components of NBD-PC transport operated independently. Using an in vitro assay, we found that isolated vacuoles containing Ybt1 were capable of transporting NBD-PC into the lumen. Removal of Ybt1 from a strain constructed to lack other routes of PC analog remodeling caused a severe growth defect. Together, these data support the view that Ybt1 is a key participant in a novel vacuolar pathway required for phospholipid remodeling.
Ybt1 is required for NBD-PC transport across vacuolar membrane in yeast
S. cerevisiae expresses five ABCC (Mrp/CFTR subfamily) type ABC transporter proteins (recently reviewed in 19). While this type of ABC transporter is often found localized to the periphery of mammalian cells, only the Yor1 ABCC transporter is directed to the S. cerevisiae plasma membrane (18). The other four members of this group of ABC transporters are believed to localize to the limiting membrane of the vacuole. These proteins include Ycf1, Ybt1, Bpt1 and Vmr1. The best studied of these transporters is Ycf1, a protein required for resistance to cadmium that acts as a glutathione conjugate transporter (20). To explore the function of these related vacuolar membrane transporters, we took a genetic approach and constructed a series of isogenic strains that varied in the dosage of the genes encoding these ABC transporters. These strains were examined for a variety of phenotypes, and a particularly interesting result was obtained when evaluating the influence of these mutations on vacuolar accumulation of the PC analog, NBD-PC.
NBD-PC has been widely used to follow traffic of phospholipids through cells. Previous experiments in S. cerevisiae established that NBD-PC is taken up via a Dnf1/2-dependent flip across the plasma membrane and eventually trafficked through a endosomal pathway into the vacuolar lumen (10,21–23). Loss of the endosomal protein Vps27 prevented NBD-PC entry into the vacuolar lumen (24), while a strain lacking normal endocytosis (end3Δ) exhibited no defect in NBD-PC localization (23). The various vacuolar ABC transporter deletion strains were labeled with NBD-PC and then visualized by fluorescence microscopy to determine the subcellular distribution of this fluorescent PC analog (Figure 1). These strains were also labeled with the endocytic probe FM4-64 to test for potential endocytosis defects.
A highly specific failure to accumulate luminal NBD-PC was observed when any strain lacking Ybt1 was tested in this assay. Even a strain lacking the other three vacuolar transporter loci (ycf1Δbpt1Δvmr1Δ) and expressing only Ybt1 was found to normally accumulate NBD-PC inside the vacuole. Loss of the P-type ATPase protein Dnf2 reduced NBD-PC uptake and end3Δ mutants were normally labeled as seen before (23). In all cases, labeling of cells by FM4-64 was undisturbed. These results strongly suggested that Ybt1p is the sole NBD-PC translocator present on the yeast vacuolar membrane. To determine the specificity of Ybt1-dependent phospholipid transport, the effect of loss of Ybt1 was also evaluated using other NBD-labeled phospholipids (NBD-PE, NBD-PS). As seen earlier (reviewed in 25), these compounds were transported to the nuclear envelope and mitochondrial membrane in an Ybt1-independent manner, suggesting that trafficking defect seen in the ybt1Δ delete cells is specific toward NBD-PC.
Although FM4-64 endocytosis was normal in all the strains examined, including the ybt1Δ mutant, we tested several other measures of vacuolar function to ensure that the failure to accumulate NBD-PC was not due to some more general issue with vacuolar function.
Ste3 and Npc2 are membrane proteins that are targeted to the vacuolar lumen (26,27). Plasmids expressing fusions between each of these proteins and green fluorescent protein (GFP) were introduced into isogenic WT and ybt1Δ cells (Figure 2). Transformants were examined by fluorescence microscopy and both fusion proteins were found to be unaffected in their delivery to the vacuole by the presence of the ybt1Δ allele.
An important parameter of the vacuole is the maintenance of the pH gradient that is developed across its limiting membrane by the vacuolar ATPase complex (reviewed in 28). The pH-sensitive dye quinacrine was used to stain vacuoles from WT and ybt1Δ cells (Figure 2). This analysis indicated that Ybt1 did not influence formation of the characteristic pH gradient. Together, these results argue that ybt1Δ cells possess vacuoles that exhibit a specific defect in the accumulation of NBD-PC.
Specific role of Ybt1 in NBD-PC uptake into the vacuolar lumen
Earlier work has indicated the presence of redundancy and overlapping substrate specificity among the vacuolar membrane ABC transporters. For example, substrates of the ABC transporter Ycf1, such as cadmium, adenine pigment, bile pigment and glutathione, can also be transported by Bpt1p (29). Similarly, Ybt1 is also involved in transportation of the adenine pigment like Ycf1p and Bpt1p (30). To determine if the close Ybt1p homologs have the ability to transport NBD-PC, we tested the ability of Ycf1p, Bpt1p and Vmr1p to bypass the defective NBD-PC trafficking of an ybt1Δ delete strain by introducing each of these transporters into ybt1Δ null background on a high-copy-number plasmid. We believe these transporters are all overproduced relative to WT gene dosage on the basis of increased adenine pigmentation using a sensitive color assay (30, data not shown).
Transformants of isogenic WT or ybt1Δ cells carrying an empty high-copy-number vector plasmid (pRS426) or the same plasmid containing a gene encoding one of the vacuolar ABC transporters were grown to mid-log phase and tested for accumulation of NBD-PC. These transformants were also labeled with FM4-64 to label the vacuolar membrane (Figure 3).
Only introduction of the high-copy-number plasmid containing the YBT1 gene was able to correct the NBD-PC vacuolar accumulation defect of the ybt1Δ mutant strain. FM4-64 labeling was unaffected in all these strains. These data argue that Ybt1 provides a specific route of a PC analog, NBD-PC, which cannot be conferred by any of the other three vacuolar ABC transporters.
To provide support for the argument that Ybt1 was acting as an ABC transporter to drive vacuolar uptake of NBD-PC, a mutant form of this gene was constructed. A lysine residue corresponding to K735 in the first nucleotide-binding domain (NBD) of Ybt1 is critical for function of the analogous region present in ABC transporters. This lysine residue is in the Walker A domain of the NBD of these transporter proteins and is involved in ATP binding and hydrolysis (reviewed in 31). Mutation of this lysine to a methionine residue has been found to inactivate other ABCC-type transporters like multidrug resistance protein (32) or cystic fibrosis transmembrane conductance regulator (33). A K735M YBT1 mutation was constructed in the context of the high-copy-number plasmid and introduced into the ybt1Δ strain (Figure 3). This mutant form of Ybt1 lost the ability to complement the NBC-PC uptake defect of the ybt1Δ mutant, consistent with Ybt1 acting as a typical ABC transporter in driving the accumulation of this PC analog. To ensure that plasmid copy number was not influencing these results, we cloned the WT and K735M forms of YBT1 into the low-copy-number vector pRS316. Again, the WT YBT1 gene complemented the ybt1Δ strain while the K735M YBT1 gene did not (data not shown).
To confirm that the defect in NBD-PC accumulation of the K735M Ybt1 derivative was due to loss of activity, the level of expression and appropriate localization of this protein were compared to that of the WT transporter. An amino-terminal HA tag was inserted in plasmids expressing either the WT or K735M form of Ybt1, and whole cell protein extracts analyzed for expression of the epitope-tagged transporters (Figure 4A).
Two different forms of HA-Ybt1 proteins could be seen that were equivalently expressed in extracts from either WT or K735M forms of Ybt1: 180 or 115 kD. The defect in NBC-PC transport in cells containing K735M Ybt1 as their sole source of this protein was not due to defective expression of this mutant transporter.
Similarly, a C-terminal GFP cassette was inserted into plasmids expressing the WT or K735M forms of Ybt1. Transformants were visualized by fluorescence microscopy and both forms of Ybt1 were found on the limiting membrane of the vacuole (Figure 4B). These data indicate that localization of both forms of Ybt1 is normal and support the view that the K735M mutant is defective in transporter activity.
Vps27 acts upstream of Ybt1 in NBD-PC trafficking
Previous studies have characterized the itinerary followed by NBD-PC as it travels to the lumen of the vacuole. NBD-PC and other fluorescent phospholipid analogs are transported into the cell via the action of P-type ATPases such as Dnf1/2 at the plasma membrane (10). These analogs then transit to the vacuolar lumen via a route that depends on function of the late endosomal sorting determinant Vps27 (34). Mutants lacking Vps27 were found to accumulate NBD-PC in the mitochondria and endoplasmic reticulum (ER) membranes. Vps27 is a late-acting endosomal sorting determinant known to influence protein trafficking to the vacuole (34). As we have found that mutants lacking Ybt1 exhibit NBD-PC trafficking defects that resemble those previously described for vps27Δ and other class E mutant strains (23,24), we explored the possible interactions between vps27Δ and ybt1Δ strains in terms of trafficking this fluorescent PC analog.
An isogenic set of strains lacking VPS27, YBT1 or both genes was prepared. These strains were grown along with the parental WT cells and then labeled with NBD-PC or 4′,6-diamidino-2-phenylindole (DAPI). Labeled cells were then visualized by fluorescence microscopy (Figure 5).
As described earlier (23,24), NBD-PC was found to accumulate in the mitochondrial and ER membranes of the vps27Δ strain. Both the ybt1Δ and ybt1Δvps27Δ strains exhibited this same localization defect.
A possible unifying explanation for the failure to accumulate NBD-PC in the vacuolar lumen in vps27Δ cells could be provided by a defect in expression, localization or function of Ybt1. To determine if Ybt1 localization required Vps27, a GFP cassette was placed at the C-terminus of the chromosomal YBT1 locus in WT and vps27Δ cells. Appropriate clones were grown to early log phase and localization of Ybt1-GFP assessed by fluorescence microscopy (Figure 6A). A tandem affinity purification (TAP)-tagged form of Ybt1 was prepared by integrating a cassette encoding the TAP tag into the C-terminus of the chromosomal YBT1 gene in isogenic WT and vps27Δ cells. Transformants were grown to mid-log phase and levels of Ybt1-TAP assessed by western blotting using the anti-TAP antiserum (Figure 6B). Expression of Ybt1-TAP was unaffected by removal of the VPS27 gene.
Ybt1-GFP was found on the limiting membrane of the vacuole, irrespective of the presence of Vps27. We also carried out this same experiment in a vps4Δ strain and again found that Ybt1-GFP localized to the vacuolar membrane (data not shown). FM4-64 trafficking was disturbed in the vps27Δ strain as detailed before (24). Lack of an effect of loss of Vps27 on a polytopic vacuolar integral membrane protein like Ybt1 is not without precedent as other membrane proteins can be delivered to the vacuolar limiting membrane in vps27Δ cells, while lumenal, soluble proteins fail to appropriately traffic (35).
To determine if Ybt1 was non-functional in vps27Δ cells, we used an additional phenotype that has been associated with Ybt1. Resistance to the heavy metal nickel has been linked with the presence of Ybt1 (36). We tested our series of single vacuolar transporter disruption mutant strains for their level of nickel resistance (Figure 6C). As seen with NBD-PC, only cells lacking Ybt1 were sensitive to nickel, consistent with the data reported by another group. Importantly, an ycf1Δbpt1Δvmr1Δ triple mutant exhibited WT nickel resistance, fully agreeing with the conclusion that Ybt1 represents the sole important vacuolar ABC transporter required for nickel tolerance (data not shown).
Having linked Ybt1 activity to nickel tolerance, we used this phenotype to examine the functional status of this vacuolar ABC transporter in vps27Δ cells. Isogenic WT, ybt1Δ or vps27Δ cells were transformed with a high-copy-number vector plasmid or the same plasmid containing the YBT1 gene. Transformants were then placed on medium containing nickel (Figure 6D).
The presence of WT YBT1 on the high-copy-number plasmid consistently elevated nickel tolerance in all three strains when compared to the empty vector plasmid alone. Importantly, loss of Vps27 did not significantly alter the level of nickel resistance seen. Together, these data support the view that Vps27 influences vacuolar accumulation of NBD-PC at a step other than Ybt1, expression, localization or function.
Ybt1 transports NBD-PC into the vacuolar lumen in vitro
While the data above support the idea that Ybt1 acts to transport NBD-PC into the vacuolar lumen, many possible explanations are consistent with these findings. To strengthen the argument that Ybt1 acts directly on NBD-PC, we developed a cell-free NBD-PC translocation assay using isolated vacuoles. This was accomplished by growing WT and ybt1Δ cells to early log phase in rich medium in the presence of the fluorescent compound monochlorobimane (MCB). MCB has previously been described as a substrate of the Ycf1 vacuolar ABC transporter that accumulates in the lumen of the vacuole (20). After confirming the vacuolar localization of MCB, vacuoles were prepared from MCB-labeled WT and ybt1Δ cells using Ficoll density gradient centrifugation as described (13). Isolated vacuoles were then incubated with NBD-PC in the presence or absence of ATP. The limiting membrane of the vacuoles was also stained with FM4-64 to provide a measure of the relative intact nature of these isolated organelles along with their luminal MCB fluorescence. After incubation at 30°C for 30 min, vacuoles were washed with cold buffer and fatty acid-free BSA to remove excess dyes and then inspected under a fluorescence microscope (Figure 7).
Vacuoles isolated from WT cells accumulated NBD-PC in the lumen but only in the presence of ATP. When the same reaction was performed on vacuoles isolated from ybt1Δ cells, no luminal uptake of NBD-PC could be detected. These data are consistent with the idea that vacuoles containing Ybt1 in their limiting membrane are capable of importing NBD-PC into their lumen.
Ybt1 is required for growth under PC depletion
While NBD-PC represents a useful model for trafficking of PC in cells, this compound is still only an analog of PC. To confirm that Ybt1 transport of NBD-PC into the vacuolar lumen faithfully reflected a facet of endogenous PC metabolism, we examined the role of this vacuolar ABC transporter in utilization and remodeling of PC analogs that can be used to support cell growth. To genetically isolate the presumptive vacuolar role of Ybt1 in PC metabolism, we constructed a strain lacking the de novo (PEM1 and PEM2) and remodeling pathways (ALE1 and NTE1) for PC biosynthesis. Pem1 and Pem2 are PE methyltransferases required for the methylation pathway of PC biosynthesis from PE (37). ALE1 (acyltransferase for lyso-phosphatidylethanolamine) is highly enriched at mitochondria-associated ER membranes and accounts for the majority of acyl-CoA-dependent lyso-PtdEtn acyltransferase (LPEAT) activity in yeast (38). Nte1p is an ER-localized phospholipase B that degrades PC to generate glycerophosphocholine and free fatty acids (39). A quadruple pem1Δpem2Δale1Δnte1Δ strain was constructed with the aim of eliminating all currently described routes of PC de novo biosynthesis (pem1Δpem2Δ) as well as remodeling of PC analogs (ale1Δnte1Δ). An ybt1Δ allele was introduced into this quadruple disruption strain to determine the contribution of Ybt1 to PC production in this strain. This strain lacking five genes (pem1Δpem2Δale1Δnte1Δybt1Δ) will be referred to as the pentuple disruption. We also evaluated the growth of pem1Δpem2Δ and pem1Δpem2Δybt1Δ strains to assess the possible role of Ybt1 in the presence of the remodeling pathways but absence of de novo biosynthesis.
To impose choline limitation on these strains, cultures were grown to saturation overnight in minimal media lacking choline. The next morning, cultures were diluted into fresh minimal media containing various sources of choline and growth measured over the next 48-h period while incubating at 30°C (Figure 8A).
Supplementation of minimal media with the PC analogs lyso-PC or short acyl chain PC (diC8-PC) supported growth of all mutant strains except the pentuple disruption mutant. The best growth for each PC analog was shown by the pem1Δpem2Δ strain. Introducing either the ybt1Δ or the ale1Δnte1Δ alleles into this background reduced growth roughly equally. Only the pentuple mutant failed to resume growth on media containing these PC analogs. This same general trend was seen when these strains were incubated in the absence of choline. This is likely reflective of the relative internal pools of PC in each of these four different genetic backgrounds. Finally, addition of 1 mm choline to the media allowed equal and robust growth of all these strains.
These data support the view that Ybt1 is required for normal metabolism of PC analogs in cells, possibly through supporting a vacuolar PC analog remodeling pathway. Given this metabolic link between Ybt1 and PC analog utilization in cells, we examined if Ybt1 might exhibit a regulatory response when cells were in need of its role in vacuolar PC reclamation. First, a chromosomal TAP-tagged version of Ybt1 was constructed in WT and pem1Δpem2Δale1Δnte1Δ cells. This Ybt1-TAP tag was fully functional as assessed by nickel resistance and NBD-PC uptake (data not shown). The WT and quadruple disruption Ybt1-TAP strains were grown in minimal media containing limiting choline and then resuspended in media containing or lacking exogenous choline. After 2 h of growth, cycloheximide was added to each culture, time-points removed and whole cell protein extracts prepared. Equal aliquots of these extracts were analyzed by western blotting using antibodies against TAP or the ER marker Kar2 (Figure 8B).
Imposition of choline starvation led to the dramatic stabilization of Ybt1-TAP as this protein was not detectably degraded over an 8-h time–course in a quadruple mutant background. Addition of choline returned the degradation of Ybt1-TAP back to a rate equivalent to that seen in WT cells. Ybt1-TAP exhibited no significant stabilization in WT cells, irrespective of choline supplementation, as these cells contained their full complement of choline biosynthetic enzymes. Kar2 degradation was unaffected by these regimens. These data strengthen the view that Ybt1 represents a normal component of the machinery cells use to maintain normal phospholipid levels.
ABC transporters are well known for their actions as drug transporters and arguably, this is likely their best known role in eukaryotic cells. A role of growing importance is the activity of these proteins to traffic lipids in the cell. The importance of ABC transporters in lipid trafficking is illustrated by the observation that of the 18 ABC transporters implicated in human disease, 8 of these diseases represent defects in lipid transport (reviewed in 40). One of the puzzles that remains unsolved in the study of ABC transporter activity is their astounding substrate range, which can reach into the hundreds of different substrates (see Ref. 41 for example). Recent structural information suggests that their active sites may have unprecedented capacity for accommodation of different substrates (reviewed in 42) but understanding of this feature is still a work in progress. An attractive speculation is that these transporters might exert their final phenotypic influence in part through direct drug transport but also as a consequence of their activity to control membrane composition.
Transport of phospholipids across the yeast plasma membrane has been extensively studied (reviewed in 25). Outward movement or flop of phospholipids has been linked to the function of two ABC transporters that are also involved in drug resistance: Pdr5 and Yor1 (10). Interestingly, Yor1 is the only ABCC-type ABC transporter found in the yeast plasma membrane (18). All other members of this class of transporters are represented by the four vacuolar ABC transporters. Yor1 (along with Pdr5) is required for movement of phospholipids out of the cytoplasm which is the same direction we propose Ybt1 transports PC. In the case of Ybt1, PC is localized inside the lumen of the vacuole. We suspect that the other vacuolar ABC transporters may also participate in lipid trafficking, perhaps by regulating the uptake of other phospholipids into the vacuole.
Previous studies on trafficking of NBD-PC provided clear evidence that Vps27 function is required for delivery of this PC analog into the vacuolar lumen (43). As Vps27 also plays important roles in trafficking of membrane proteins to the vacuole (34), we examined if the NBD-PC trafficking defect of cells lacking this protein might be due to compromised Ybt1. The data reported here argue that Ybt1 has a selective defect in transport of NBD-PC into the vacuolar lumen but other substrates of this transporter (like nickel) are unaffected in vps27Δ cells. From this result, we suggest that Vps27 is required to deliver PC and its analogs in an appropriate form to support Ybt1-dependent translocation into the vacuole. No difference could be detected in delivery of Ybt1 to the vacuolar limiting membrane in comparison with WT and vps27Δ strains (Figure 6), indicating that Ybt1 traffic is normal even in this strong vacuolar mutant background. We conclude that loss of Vps27 was unlikely to globally interfere with Ybt1 action and was restricted to phospholipid transport.
A simple model for vacuolar accumulation of NBD-PC comes from the role of Vps27 in the formation of multivesicular bodies (43). As vesicles and their cargo arrive at the late endosome, Vps27 participates in a process through which material destined for delivery into the lumen of the vacuole is sorted from other components. A straightforward route for delivery of NBD-PC into the vacuolar lumen would be to passively follow membranes into the intralumenal vesicles forming in the late endosome. These intralumenal vesicles enter the vacuolar lumen upon fusion with the vacuole. This mechanism is unlikely to explain luminal accumulation of NBD-PC as even in cells with WT Vps27 and no apparent defects in overall protein traffic to the vacuole, loss of Ybt1 prevents NBD-PC uptake. We suggest that NBD-PC is sorted in a Vps27-dependent fashion, allowing this phospholipid derivative to be transported by Ybt1 when NBD-PC reaches the limiting membrane of the vacuole. Our findings are most consistent with a model in which Vps27 (and other class E Vps proteins) is necessary but not sufficient for accumulation of NBD-PC into the vacuolar lumen.
While our data have provided an explanation for vacuolar accumulation of PC, important questions still remain concerning remodeling of these phospholipids. Ybt1 provides the route of entry of PC analogs but the enzyme(s) responsible for either release of the choline headgroup or reconstruction of appropriate acyl chains have yet to be determined. One candidate gene that was examined was the phospholipase B homolog Plb3. Use of a GFP fusion provided evidence that this protein is localized to the vacuolar lumen (44). We constructed a plb3Δ disruption mutant but could find no effect on recycling of PC analogs (data not shown), suggesting that either this phospholipase is not involved in this process or that redundant enzymes exist. Finally, the protein(s) involved in extracting the remodeled PC/choline from the vacuolar lumen must be identified.
Our finding of the growth defect caused by loss of YBT1 from the pem1Δpem2Δale1Δnte1Δ strain was critical in establishing that Ybt1-dependent uptake of NBD-PC into the vacuolar lumen represents a physiologically relevant feature of phospholipid metabolism. Other members of the ABCC class of ABC transporters, such as multidrug resistance protein (Mrp1), are known to act as broad specificity drug transporters (45,46). This raises the concern that the action of Ybt1 on NBD-PC might simply be an illustration of its ability to sequester drugs into the vacuole, much as Mrp1 can efflux drugs out of mammalian cells. The requirement of Ybt1 for vacuolar reutilization of naturally occurring PC analogs like lyso-PC, coupled with Ybt1 stabilization in choline-limited cells, supports the belief that this function of Ybt1 reflects a fundamental role for this ABC transporter in phospholipid homeostasis.
Control of the distribution of derivatized phospholipids across organellar membranes has previously been described in two different human diseases: respiratory distress syndrome and Stargardt's disease. Respiratory distress syndrome (RDS) occurs upon loss of an ABC transporter normally found in the limiting membrane of lamellar bodies in alveolar cells in the lung (47). This ABC transporter, called ABCA3, is involved in exporting surfactant which contains large amounts of dipalmitoyl-PC into airspaces of the lung (48). Stargardt's disease is caused by defects in the ABCA4 gene (49). Loss of this ABC transporter protein causes the accumulation of a PE derivative in the luminal leaflet of the disc membrane in eye photoreceptor cells (50). Normally, this PE-based compound is flipped into the cytoplasmic leaflet out of the lumen of the disc. Interestingly, this activity of ABCA4 is in the opposite direction (into the cytoplasm) than described for either ABCA3 or Ybt1. Co-ordination of the activities of these organellar ABC transporters with their plasma membrane counterparts is critical for establishing normal physiology. Ybt1 and the other vacuolar ABC transporters provide an important model system for understanding how eukaryotic cells regulate phospholipids and their derivatives.
Materials and Methods
Yeast strains and media
Yeast strains were routinely grown in rich yeast extract/peptone/dextrose (YPD) liquid media containing 2% yeast extract, 1% peptone and 2% glucose for nonselective conditions or complete synthetic medium (CSM) media lacking desired amino acids (51). The choline auxotrophic strains were grown in the presence of 1 mm choline, unless otherwise indicated. The drug resistance assays were performed on solid agar plates containing indicated concentration of nickel sulfate. Yeast transformation was performed by using the lithium acetate technique (52). Plasmids are listed in Table 2.
Table 2. Plasmid list
Yeast strains are listed in Table 1. Generation of strains carrying single deletions of vacuolar ABC transporters in SEY6210 was accomplished by transformation with PCR disruption cassettes constructed as follows: YBT1 disruption cassettes with KanMX6 and His3MX6 markers were amplified from plasmids pFA6a-KanMX6 and pFA6a-His3MX6 (53) by using primers Ybt1 Del-For and Ybt1-Del-Rev to generate KGS69 and KGS70. For disruption of VMR1, vmr1::KanMX4 and vmr1::TRP1 cassettes were amplified from plasmids pFA6a-KanMX6 and pFA6a-TRP1 by using primers Vmr1 Del-For and Vmr1 Del-Rev to generate KGS72 and KGS73. The bpt1::KanMX4 deletion cassette was amplified by using primers Bpt1-Del-For and Bpt1-Del-Rev and transformed in SEY6210 to generate KGS71. The ybt1::His3MX6, ale1::hphMX4 and nte1::HIS3MX6 deletion cassettes were transformed in SEY6210 pem1::natR-MX4Δpem2Δ::KanMX4 strain to generate KGS75, KGS80 and KGS78, respectively. To generate the single nte1Δ deletion strain, nte1::HIS3MX6 deletion cassettes were transformed in SEY6210 to generate KGS77. Construction of the quadruple knock-out strain was accomplished by using the SEY6210 pem1::natR-MX4Δpem2Δ::KanMX4 strain and then sequentially transforming with deletion cassettes ale1Δ::hphMX4 and nte1Δ::HIS3MX6 to generate KGS79. The strain KGS79 was then transformed with an ybt1Δ::URA3 deletion cassette yielding KGS81. The strain KGS79 was marker swapped by changing nte1Δ::HIS3MX6 to nte1Δ::TRP1 to generate KGS84. The YBT1 C-terminal TAP tag fusion construct YBT1-TAP was chromosomally integrated in BY4742 vps27Δ::KanMX4, SEY6210 pem1Δ::natR-MX4 pem2Δ::KanMX4 and KGS84 strains yielding KGS76, KGS83 and KGS85, respectively.
Table 1. Strain list
In vitro NBD-PC translocation assay
The WT and ybt1Δ null mutant strains were grown in YPD containing 500 µm MCB for 6 h. MCB is imported into the vacuolar lumen (20) and its accumulation inside the vacuoles was observed under fluorescence microscope in intact cells. Vacuoles were prepared from the MCB loaded cells using a Ficoll density gradient as described earlier (13) and kept at 4°C until initiation of the assay. The NBD-PC uptake assay was performed on freshly prepared vacuolar membranes at 30°C in Tris/sucrose (TS) buffer (250 mm sucrose, 25 mm Tris–MES, pH 8.0) containing either no ATP or 5 mm ATP, 10 mm creatine phosphate, 20 units/mL creatine kinase, 2.5 µm NBD-PC and 50 µm FM4-64 dye. The uptake reaction was initiated by addition of isolated vacuoles (25 µg of protein) and allowed to proceed. Time-points were typically taken every 30 min. Vacuoles were then washed twice by ice-cold TS buffer containing 3% fatty acid-free BSA. Washed vacuoles were then visualized under fluorescence microscope. Intact vacuoles were selected on the basis of MCB fluorescence and FM4-64 labeling. Only vacuoles with an intact structure as judged by positive luminal staining for MCB and limiting membrane staining with FM4-64 were used for observing NBD-PC accumulation.
Wild-type and pem1Δpem2Δale1Δnte1Δ null mutant strains carrying chromosomally integrated C-terminal YBT1-TAP tagged alleles were grown in rich media until saturation. These cells were then extensively washed with water and transferred to fresh synthetic dextrose (SD) media without choline and allowed to grow for 12 h to deplete the intracellular pools of PC. These cultures were then shifted to limiting choline conditions (0.1 mm choline) and again grown to saturation. Cells from these cultures were then diluted to an optical density at 600 nm (OD600 nm) of 0.1 in fresh media containing 0.05 mm choline and allowed to reach OD600 nm of 0.8. Equal samples from these cultures were then placed in SD media containing or lacking choline and allowed to grow for 2 h before addition of 100 µg/mL cycloheximide. Samples were collected at indicated time-points, protein extracts were made and subjected to western analysis using anti-TAP and anti-Kar2 antibodies.
The strains carrying indicated genomic deletion backgrounds were first grown in YPD media overnight. These cells were then extensively washed with water and transferred to fresh SD media without choline and allowed to grow for 12 h to deplete the intracellular pools of PC. The cultures were reinoculated in fresh media containing no choline, 1 mm choline or 0.2 mm lyso-PC to an OD600 nm of 0.2. The growth rates of these strains were measured by taking OD600 nm at different time periods over 27 h. Averaged OD600 nm values from four independent experiments were plotted against the indicated time period.
NBD-PC transport assay
To label cells with NBD-PC, cultures were grown to early log phase in SD medium. Cells were incubated with NBD-PC solubilized in dimethyl sulphoxide (DMSO) to a final concentration of 10 µm for 25 min at 30°C. Cells were harvested and washed twice with SD media at room temperature. For visualization of nuclear and mitochondrial DNA, cells were resuspended in 2 mL of SD media and 2 µL of a 1 mg/mL stock of DAPI was added. Cells were incubated at 30°C for 15 min, washed twice with ice-cold SC+NaN3 and visualized under fluorescence microscope.
Yeast strains carrying GFP-tagged proteins were grown to early log phase, washed twice with water and analyzed for GFP fluorescence. For FM4-64 labeling, cells were incubated with FM4-64 for 20 min or the indicated time period, washed twice with dye-free media and then analyzed by fluorescence microscopy. Isolated vacuoles were visualized at 2.5× aperture settings. Fluorescent images were taken by placing a few microliters of a culture or isolated vacuoles on a glass slide, overlaying with a cover slip, and visualizing under the 100× oil objective of an Olympus BX60 fluorescence microscope.
We thank Drs. Todd Graham, Pam Hanson and Rob Piper for helpful discussions. We also thank Dr. Soraya Johnson for providing plasmids. This work was supported by NIH grant GM75120.