Sphingolipid Requirement for Generation of a Functional V1 Component of the Vacuolar ATPase*

Ji-Hyun Chung, Robert L. Lester and Robert C. Dickson {ddagger}

From the Department of Molecular and Cellular Biochemistry and the Lucille P. Markey Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky 40536

Received for publication, January 28, 2003 , and in revised form, April 25, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
There has been no previous indication that vacuolar ATPases (V-ATPases) require sphingolipids for function. Here we show, by using Saccharomyces cerevisiae sur4{Delta} and fen1{Delta} cells, that sphingolipids with a C26 acyl group are required for generating V1 domains with ATPase activity. Sphingolipids in sur4{Delta} cells contain C22 and C24 acyl groups instead of C26 acyl groups whereas about 30% of the sphingolipids in fen1{Delta} cells have C26 acyl groups and the rest have C22 and C24 acyl groups. sur4{Delta} cells have several phenotypes (vacuolar membrane ATPase, Vma) that indicate a defect in the V-ATPase, and vacuoles purified from sur4{Delta} cells have little to no ATPase activity. These phenotypes are less pronounced in fen1{Delta} cells, consistent with the idea that the C26 acyl group in sphingolipids is necessary for V-ATPase activity. Other results show that the two V-ATPase domains, V1 and V0, are assembled and delivered to the vacuolar membrane in sur4{Delta} cells similar to wild-type cells. In vitro assembly studies show that V1 from sur4{Delta} cells associates with wild-type V0 but the complex lacks V-ATPase activity, indicating that V1 is defective. Reciprocal experiments with V0 from sur4{Delta} cells show that it is normal. We conclude that sphingolipids with a C26 acyl group are required for generating fully functional V1 domains.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vacuolar ATPases (V-ATPases)1 are found in all eukaryotes where they are required for receptor-mediated endocytosis, renal acidification, bone reabsoprtion, neurotransmitter accumulation, and activation of acid hydrolases (reviewed in Refs. 1 and 2). The Saccharomyces cerevisiae V-ATPase, the current paradigm for V-ATPases, moves protons from the cytosol to the lumen of the vacuole to establish a proton gradient that is necessary for vacuolar transport proteins to drive ions and small molecules, amino acids, and metabolites into the vacuole (reviewed in Ref. 3). Knowledge of the S. cerevisiae V-ATPase has grown rapidly but remains incomplete. One area where little is known is the influence of membrane lipids on V-ATPase activity. Here we show that sphingolipids are necessary for V-ATPase activity in S. cerevisiae.

S. cerevisiae V-ATPase contains two components or domains, V1 and V0, which associate to form an active V1V0 complex on the vacuolar membrane (reviewed in Refs. 35). The V1 domain has ATPase activity and is composed of 8 different proteins. It can exist free in the cytoplasm or complexed with V0 on the vacuolar membrane. The V0 domain contains 5 protein subunits and is imbedded in the vacuolar membrane where it serves as a proton pore. The V1 and V0 domains are assembled and associate in the ER to form functional V1V0 complexes, which are then transported from the Golgi to the vacuole (reviewed in Refs. 3 and 5). Alternatively, the two domains assemble independently and then associate once V0 reaches the vacuole.

The ER is also where sphingolipid synthesis begins (reviewed in Ref. 6) with generation of ceramide. Ceramide is transported to the Golgi where it is converted sequentially into the complex sphingolipids inositol-phosphoceramide, mannose-inositol-phosphoceramide, and finally to mannose-(inositol-phospho)2-ceramide. Complex sphingolipids are delivered to cellular compartments, particularly the plasma membrane (7) and to a lesser extent the vacuole (8).

One of the distinguishing features of sphingolipids in S. cerevisiae is the C26 acyl group. Fatty acids with 20 or more carbons, very long chain fatty acids (VLCFAs), are ubiquitous in nature, but little is known about their functions. VLCFAs are mostly found in the ceramide portion of sphingolipids. The importance of the C26 acyl component of S. cerevisiae sphingolipids was demonstrated by the isolation of mutant strains that do not make sphingolipids (9), but instead make a set of novel sphingolipid mimics in which ceramide is replaced by diacyl-glycerol (10). The presence of a C26 acyl group is the unique feature of these novel glycerolipids, which enables them to mimic some sphingolipid functions.

Further evidence for the essentiality of the C26 acyl group in sphingolipids comes from studies of the FEN1 (ELO2) and SUR4 (ELO3) genes. In fen1{Delta} cells only 29% of the sphingolipids contain a C26 acyl group, the rest contain C22 and C24 acyl groups. The sphingolipids in sur4{Delta} cells contain only C22 and C24 acyl groups and no C26s (11, 12). Fen1p and Sur4p are components of the enzyme system that elongates C16 and C18 fatty acids to form VLCFAs. The exact function of Fen1p and Sur4p are unclear because the elongation system has not been fully characterized in any organism. sur4{Delta} and fen1{Delta} cells are viable, although they have many mutant phenotypes (reviewed in Ref. 13) and deletion of both genes is lethal (14).

A fraction of sphingolipids in higher eukaryotes also contain VLCFAs (15) and the mouse genes Ssc1 and Cig30 complement a sur4{Delta} and a fen1{Delta} mutant, respectively (16). Interestingly, the mice mutants Quaking and Jimpy, which develop intense tremors at the age of about 2 weeks as a result of severe demyelination of the central nervous system, have reduced levels of Ssc1 mRNA and reduced fatty acid elongation activity (17). Cig30 mRNA has the interesting property of being induced in brown adipose tissue when animals are exposed to cold temperature. These results suggest that VLCFAs are performing important, but unknown functions in mammals, and that some of these functions may be evolutionarily conserved.

Recently Kohlwein et al. (12) reported that sur4{Delta} and fen1{Delta} cells contain small vacuoles called fragmented vacuoles that fail to properly fuse to form larger vacuoles. This observation suggested to us that sphingolipids with a C26 acyl group are needed for some vacuolar function(s). Here we show that V1 domains in sur4{Delta} cells lack ATPase activity even though they associate with V0 domains on the vacuolar membrane. Our data are the first to implicate sphingolipids with a C26 acyl group in the generation of a fully functional V1 domain.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Strains, Plasmids, and Culture Conditions—Strains used in these experiments are listed in Table I. The sur4-{Delta}1::KAN and the fen1-{Delta}1::KAN alleles have codons 1–560 replaced with a kanamycin resistance cassette, generated by using the PCR and pUG6 as the template (18). Diploid cells transformed with deletion alleles were selected for G418 resistance (19). The expected deletion event was verified by PCR analysis of chromosomal DNA. Haploid offspring were obtained by sporulation and tetrad dissection. VMA13 under the control of its own promoter and having a Myc epitope immediately following the methionine start codon was carried in pRS316 (20).


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TABLE I
Strains used in this work

 

Buffered medium was prepared by the addition of 50 mM MES and 50 mM MOPS to YPD, and the pH was adjusted to pH 5.5 with NaOH. YPD plates supplemented with 100 mM CaCl2,4mM CaCl2, or buffered to pH 7.5 with 100 mM Hepes were prepared as described previously (21). YPD medium contained 1% yeast extract, 2% Bacto-peptone, and 2% dextrose.

Quinacrine Staining and Semiquantitative Quinacrine Assay— Vacuolar accumulation of quinacrine was assessed by fluorescence microscopy as described by Roberts et al. (22) or by a semiquantitative quinacrine assay (21). Fluorescence measurements of cell suspensions were done in a Beckman spectrofluorometer (excitation = 419 nm, emission = 425 nm) and the OD at 600 nm was monitored in a spectrophotometer.

SDS-PAGE, Immunoblotting, and Antibodies—SDS-PAGE and immunoblotting were performed according to procedures recommended for the Bio-Rad Tray-Blot S.D. Semi-Dry transfer cell (Bio-Rad Inc.). Monoclonal antibodies against Vph1p, Vma1p, Vma2p, CPY, and ALP were from Molecular Probes. Anit-Myc antibodies were from Roach Applied Science. Dr. Patricia Kane provided anti-Vma5p. Nitrocellulose membranes (Bio-Rad) were washed four times after the first and second antibody reactions with 0.1% phosphate-buffered saline containing 0.1% Triton X-100. Secondary antibody was anti-mouse IgG conjugated to alkaline phosphatase (Sigma). Membranes were incubated for 5 min facedown in ECF substrate (Amersham Biosciences) and fluorescent signals were collected by using a Molecular Dynamics Storm Phosphorimager and quantified by using ImageQuant software (version 5.1).

HPLC Analysis of LCBs and LCBPs—Lipids were extracted from whole cells, converted to fluorescent derivatives and analyzed by HPLC as described previously (23).

Treatment of Purified Vacuoles with PHS—A 100x stock of PHS (10 mM PHS in 95% EtOH) was diluted into a suspension of purified vacuolar membranes suspended in buffer (10 mM Tris, 10 mM MES, pH 6.9, 5 mM MgCl2, 25 mM KCl) to give a final concentration of 100 µM. Samples were incubated on ice for 0, 30, 60, 90, and 120 min followed by centrifugation at 13,000 rpm for 10 min in a microcentrifuge (4 °C). Proteins in the supernatant fluid were precipitated by incubating with 5% trichloroacetic acid (final concentration) for 2 h on ice. Pellets and trichloroacetic acid-precipitated proteins were resuspended in 50 µl of cracking buffer (8 M urea, 5% SDS, 1 mM ethylenediamine tetraacetate, 50 mM Tris-HCl, pH 6.8, 5% {beta}-mercaptoethanol, Ref. 24) and equal volumes were analyzed by SDS-PAGE and immunoblotting.

Miscellaneous Procedures—Vacuolar membrane vesicles were purified by centrifugation on Ficoll gradients and Mg-ATPase activity was measured at 23 °C as described previously (22) except that membranes were not homogenized before centrifugation on the second Ficoll gradient.

Vacuolar membrane vesicles were also purified by sucrose density gradient centrifugation as previously described (25), except that the concentration of sucrose was increased to prevent membranes from pelleting on the bottom of the centrifuge tube. For these experiments, 4 ml of the membrane fraction was overlaid onto a 32-ml gradient composed of equal volumes of 10, 30, 50, and 60% (w/v) sucrose. The gradient was centrifuged for 35 min at 100,000 x g in a Sorvall AH629 rotor at 4 °C and then fractionated starting from the top into 9 fractions of 4, 7, 2, 6, 2, 6, 2, 7 ml and the resuspended pellet. Fractions were frozen in liquid nitrogen and stored at –80 °C. V1V0 complexes on vacuolar membranes prepared by sucrose gradient centrifugation were dissociated by treatment with KI as described previously (26, 27).

Cell-free protein extracts used for analysis of Vph1p (Fig. 3), were prepared as described by Kunz et al. (28). Cell-free protein extracts used for the analysis Vma1p, Vma2p, and Vma5p were prepared as described (29).



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FIG. 3.
Vacuoles isolated from sur4{Delta} cells by Ficoll density gradient centrifugation lack the V1 subunits Vma1p, Vma2, and Vma5p. Cell-free extracts and Ficoll-purified vacuolar membrane vesicles were examined by immunoblotting for the presence of the V0 subunit Vph1p and the V1 subunits Vma1p, Vma2p, and Vma5p. Samples (20 µg of cell-free protein extracts and 10 µg of purified vacuolar membrane vesicles) were incubated in cracking buffer for 10 min at 65 °C for analysis of Vph1p or in SDS sample buffer for 5 min at 95 °C for the analysis of other proteins.

 

To measure the calcium-dependent ATPase activity of cytosolic V1 domains, cells were grown, lysed and a high speed supernatant fraction was prepared as previously described (5). Proteins were precipitated by treatment with 5% trichloroacetic acid as described above, resuspended in and dialyzed against buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM p-methylsulfonylfluoride, 10% glycerol), and centrifuged on a glycerol gradient (30). Fractions (750 µl) were collected from the top of the gradient. Fractions (912) containing V1 domains were located by immunoblotting for Vma1p, Vma2p, Vma5p, and Vph1p. The pooled V1-containing fractions were assayed for calcium-dependent ATPase activity as described previously (5). Values are expressed as the difference between assays performed with and without 1.6 mM CaCl2.

The subunit composition (Fig. 7) of V0 and the V1V0 complex were analyzed by using a published procedure to cross-link proteins before immunoprecipitation and SDS-PAGE analysis (31). The procedure was modified so that 1 OD of 600-nm units of spheroplasts were incubated with 50 µCi of Trans[35S] label (ICN Inc., 1175 Ci/mmol, 5100607). After pretreatment of the sample with protein A-Sepharose beads (Sigma Inc.), 400 µl a solution of 5% bovine serum albumin/phosphate-buffered saline containing 5 µl of antibody solution was added, and the sample was incubated overnight on ice with mixing. Protein A-Sepharose (40 µl of a 40% (v/v) suspension) was added to each sample and incubated for2honice with mixing. Immunoprecipitates were collected by centrifugation at 5,000 rpm for 5 min in a microcentrifuge. Pellets were washed four times in buffer (1% Triton X-100, 1% deoxycholic acid, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl), and precipitated proteins were eluted from the beads by incubation for 10 min at 95 °C in 50 µl of 4x SDS-loading buffer (50 mM Tris base, pH 6.8, 8% glycerol, 1.6% SDS, 4% {beta}-mercaptoethanol, 0.04% bromphenol blue). Half of each precipitate was subjected to SDS-PAGE and phosphorimager analysis.



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FIG. 7.
The V0 domain and the V1V0 complex in sur4{Delta} cells have a normal subunit composition. The subunit composition of the V0 domain and the V1V0 complex was examined by radiolabeling proteins with Trans [35S] label, treating the samples with a protein cross-linker, immunoprecipitating proteins and subjecting them to SDS-PAGE and phosphorimage analysis. A, V0 domains were precipitated with anti-Vph1 antibodies. Free V1 and V1V0 complexes were precipitated with anti-Vma1p (B) or with anti-Vma2p (C). The size of radiolabeled V-ATPase subunits present in the samples are indicated at the right of the panels. D, Myc-Vma13p was analyzed in vma13{Delta} or sur4{Delta} vma13{Delta}cells transformed with a plasmid expressing Myc-tagged Vma13p. Vacuolar membrane vesicles were isolated by sucrose density gradient centrifugation and the fractions were immunoblotted for Myc-Vma13p, Vma1p, Vma2p, and Vma5p (V1 domain marker) and Vph1p (V0 domain marker). V1V0 complexes are located primarily in fraction 5.

 

Cells were prepared for indirect immunofluorescent microscopy by using a published procedure (22). The secondary antibody was goat anti-mouse IgG labeled with FluoroLinkTM Cy3TM (Amersham Biosciences). Fluorescent images were obtained with a Nikon Àclipse E800 fluorescence microscope equipped with a Nikon 100X/1.3 plan fluor oil-immersion objective and a Diagnostic instruments Spot camera controlled by Adobe Photoshop software. For Cy3 fluorescence, samples were excited at 510–560 nm and viewed with a barrier filter of 570–650 nm. Adobe PhotoShop software was used to process images. Protein concentrations were determined with the Bio-Rad DC protein assay kit with bovine serum albumin as a standard.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
sur4{Delta} Cells Have a Unique Set of Vma Phenotypes That Indicate Reduced V-ATPase Activity—The presence of fragmented vacuoles in sur4{Delta} cells suggests that the C26 acyl group of sphingolipids is necessary for some vacuolar function. Functional vacuoles require a proton pump or V-ATPase to acidify their lumen. When the V-ATPase is defective a set of phenotypes, referred to as Vma, are produced. For example, vma mutants do not grow on YPD buffered to pH 7.5 (32, 33). We reasoned that sur4{Delta} cells might have Vma phenotypes if sphingolipids containing a C26 acyl group are needed for V-ATPase activity. Indeed, we found that sur4{Delta} cells grow very poorly at pH 7.5 just like the authentic vma mutant vma2{Delta} (Fig. 1) (34). In contrast, fen1{Delta} cells grow better at pH 7.5 (Fig. 1), indicating that they are able to acidify their vacuoles more effectively than sur4{Delta} cells.



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FIG. 1.
sur4{Delta} cells display Vma phenotypes. Phenotypes were examined by spotting 10-fold serial dilutions of cells onto agar plates containing YPD medium, YPD buffered to pH 7.5, YPD plus 100 mM CaCl2, or YPD plus 4 mM ZnCl2. Plates were incubated for 2 days at 30 °C.

 

Another Vma phenotype is failure to grow on YPD plates containing 4 mM ZnCl2 (34). We found that sur4{Delta} cells have this phenotype whereas fen1{Delta} cells do not since they grow almost as well as wild-type cells (Fig. 1). Other Vma phenotypes include failure to grow in the presence of 100 mM CaCl2 (35) or on medium containing a non-fermentable carbon source such as glycerol (36). We found that 100 mM CaCl2 inhibits growth of sur4{Delta} cells but does not inhibit growth of fen1{Delta} cells (Fig. 1), again supporting the idea that sur4{Delta} cells are less able to acidify their vacuoles than fen1{Delta} cells. In addition, sur4{Delta} cells grow slowly on YP-glycerol while fen1{Delta} cells grow slightly faster and both grow better than vma2{Delta} control cells (Table II). These data show that the Vma phenotypes of sur4{Delta} cells are nearly as pronounced as those in vma2{Delta} cells and that the phenotypes are less severe in fen1{Delta} cells. Based upon these phenotypes it appears that the V-ATPase is more impaired in sur4{Delta} cells than in fen1{Delta} cells.


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TABLE II
Vma- phenotypes of sur4{Delta} and fen1{Delta} cells

 

To directly examine vacuolar acidification in vivo we used the lysosomotropic fluorescent dye quinacrine, which is taken up by cells and concentrated in acidified vacuoles; if vacuoles are not acidified the dye remains in the cytoplasm and gives a diffuse fluorescent signal (37). A strong fluorescent vacuolar signal was observed in wild-type cells (Fig. 2A). No fluorescent signal was seen in sur4{Delta} cells, which behaved like vma2{Delta} control cells that do not acidify their vacuole. The fluorescent signal in fen1{Delta} cells was lower than in wild-type cells but greater than in sur4{Delta} cells.



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FIG. 2.
Vacuolar acidification is defective in sur4{Delta} cells. A, vacuolar acidification was monitored by accumulation of the fluorescent dye quinacrine within vacuoles. Cells were viewed by differential interference contrast optics for observation of normal cell morphology (left panels) and by fluorescent microscopy with an FITC filter for observation of quinacrine uptake (right panels). B, quinacrine uptake was monitored by spectrofluorometric analysis of populations of cells. Mean values ± S.D. (n = 3) are expressed relative to the wild type.

 

The fluorescent microscopy results were verified by a semiquantitative spectrofluorometric assay of the relative fluorescence of populations of cells stained with quinacrine. By this technique, wild-type cells fluoresced strongly whereas sur4{Delta} cells fluoresced near the background level measured in vma2{Delta} cells (Fig. 2 and Table II). The fluorescent signal in fen1{Delta} cells was 60% of the wild-type level indicating that they are able to acidify their vacuoles better than sur4{Delta} cells but not as well as wild-type cells. Taken together these data show that sur4{Delta} cells have a unique set of Vma phenotypes and that they fail to acidify their vacuoles. These phenotypes are less severe in fen1{Delta} cells, which have some ability to acidify their vacuoles, although they do not acidify as well as wild-type cells.

V-ATPase Activity Is Reduced in sur4{Delta} Cells and Ficoll Dissociates the V1V0 Complex—The data presented in Figs. 1 and 2 and Table II suggest that vacuolar membranes isolated from sur4{Delta} cells should have less V-ATPase activity than vacuolar membranes isolated from fen1{Delta} cells and both should have less activity than those isolated from wild-type cells. In agreement with this prediction, vacuolar membranes isolated by Ficoll density gradient centrifugation from sur4{Delta} cells had only 10% of the wild-type V-ATPase activity while those isolated from fen1{Delta} cells had 25% of the wild-type activity (Table III).


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TABLE III
V-ATPase activity in purified vacuolar membranes

 

Reduced V-ATPase activity could be due to either a reduced specific activity or fewer molecules. To differentiate between these alternatives the concentration of Vph1p, a subunit of the V0 domain, and Vma1p, Vma2p, and Vma5p, subunits of the V1 domain, were measured by immunoblotting of whole cell protein extracts and of purified vacuolar membranes (Fig. 3). Whole cell protein extracts from wild-type, sur4{Delta}, and fen1{Delta} mutants contained a similar level of each of the four proteins, indicating that the steady-state level of the proteins is similar in mutant and wild-type cells. Likewise, Vph1p was present in similar amounts in vacuolar membranes purified from the three strains. In contrast, the level of Vma1p Vma2p, and Vma5p in vacuolar membranes isolated from sur4{Delta} and fen1{Delta} cells was greatly reduced (Fig. 3). These results suggest that there is a defect in the V1 domain in sur4{Delta} and fen1{Delta} cells or that the interaction between V0 and V1 is abnormal and V1 or some of its subunits dissociate during vacuolar purification.

The interaction of V1 with V0 has been examined by treating vacuolar membranes with a low salt buffer containing ethylenediamine tetraacetate and then determining if particular protein subunits remained in the vacuolar fraction (high speed pellet) or became soluble (38). Because Ficoll purification removed V1 subunits from vacuolar membranes, we used cell-free extracts in place of vacuolar membranes for these assays. Treatment with a low salt buffer did not reveal any difference in the level of Vma1p and Vma2p in the pellet and soluble fractions obtained with sur4{Delta}, fen1{Delta}, or wild-type cells (data not shown). We also determined if increasing concentrations of sodium carbonate (38) preferentially solubilized Vma1p and Vma2p in cell-free extracts made from sur4{Delta} and fen1{Delta} cells compared with wild-type cells. Vma2p but not Vma1p was more readily solubilized in the sur4{Delta} and fen1{Delta} samples (data not shown). Thus, whatever the nature of the abnormality in the interaction between Vma1p and Vma2p and V0 in sur4{Delta} and fen1{Delta} cells, it must be fairly subtle and unique since, as far as we are aware, sensitivity of the V1V0 complex to Ficoll has not been reported previously.

Vacuoles have also been partially purified by using sucrose density gradient centrifugation (25). We examined this procedure to see if V1 domains remained attached to vacuolar membranes isolated from sur4{Delta} cells. Sucrose gradient fractions were analyzed by immunoblotting for Vph1p to detect V0 domains and for Vma1p and Vma2p to detect V1 domains. The concentration of these three proteins peaked around fractions 5 and 6 in both the sur4{Delta} and wild-type sample (Fig. 4). Thus, unlike the results obtained when extracts from sur4{Delta} cells were centrifuged on Ficoll gradients, sucrose gradients yield a fraction in which the V1 and V0 domains remain associated.



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FIG. 4.
V1 associates with V0 on vacuolar membranes isolated from sur4{Delta} cells by sucrose density gradient centrifugation. Vacuolar membranes were isolated by centrifugation on sucrose gradients and fractionated into non-equal sized fractions as described in "Experimental Procedures." Fraction 1 is the top of the gradient and fraction 9 is the resuspended pellet. Part of each fraction (10 µg of protein) was incubated in SDS-sample buffer for 5 min at 95 °C prior to SDS-PAGE and then immunoblotted for the V0 subunit Vph1p and the V1 subunits Vma1p and Vma2p.

 

Fractions 5 and 6 obtained from wild-type cells contain functional V1V0 complexes because they have V-ATPase activity which has a slightly higher specific activity than that measured in vacuolar membranes isolated by Ficoll density gradient centrifugation (Table III). Even though the immunoblot of the sur4{Delta} sample (Fig. 4) indicates that fractions 5 and 6 have both V1 and V0 domains, the fractions have only 20% as much V-ATPase activity as the wild type.

The data presented in this section show that V1 domains do associate with V0 on the vacuolar membrane in sur4{Delta} cells but the association is abnormal because the Ficoll gradient procedure causes Vma1p, Vma2p, and Vma5p and possibly the entire V1 domain to dissociate from V0 domains. In addition, vacuolar membranes isolated from sur4{Delta} cells by either Ficoll or sucrose density gradients have reduced V-ATPase activity.

V1 Associates with the Vacuolar Membrane in sur4{Delta} and fen1{Delta} Cells—Both density gradient procedures for isolating vacuolar membranes subjects the sample to non-physiological conditions and could create artifacts such as by proteolysis. To try and avoid these potential complications, we examined the association of V1 with V0 on the vacuolar membrane of intact cells by using indirect immunofluorescence microscopy with anti-Vma1p or anti-Vma2p antibodies (33). In wild-type cells both antibodies localized to the vacuolar membrane as expected (Fig. 5). A similar localization is seen in sur4{Delta} and fen1{Delta} cells except that the vacuoles are fragmented and do not stain as uniformly as do vacuoles in wild-type cells (Fig. 5). Control cells lacking the vma2 gene show diffuse staining throughout the cytoplasm with anti-Vma1p antibody (data not shown) because V1 subunits are not formed and no staining with anti-Vma2p is observed because the protein is absent (Fig. 5). These data verify those obtained by sucrose density gradient centrifugation and together the two sets of data establish two critical points about the V-ATPase in sur4{Delta} and fen1{Delta} cells. First, V1 domains are assembled and, second, at least some of them do associate with V0 on the vacuolar membrane.



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FIG. 5.
V1 domains associate in vivo with vacuoles in sur4{Delta} cells. The location of the V1 domain in cells was determined by indirect immunofluorescence staining with anti-Vma2p primary antibody followed by Cy3-tagged secondary antibody (red images). Images taken by differential interference contrast optics are shown in black and white.

 

V0 Domains Are Functional in sur4{Delta} Cells but V1 Domains Lack ATPase Activity—The nature of the V-ATPase defect in sur4{Delta} cells was further elucidated by determining whether V1, V0 or both domains are defective. For these experiments vacuolar membranes were isolated by sucrose density gradient centrifugation then treated with KI to dissociate V1 (27). Samples were then centrifuged to give a vacuolar membrane pellet (P) containing V0 and a supernatant fraction (S) containing V1. It has been shown previously that upon mixing the P and S fractions and dialyzing away the KI, V1 and V0 associate, as determined by immunoblotting, and V-ATPase activity is partially restored (35–40%) (27). We observed very similar results using the P and S fractions derived from wild-type cells. By immunoblotting, Vma1p and Vma2p (V1 subunits) were more concentrated in the P fraction following dialysis compared with the non-dialyzed sample (Fig. 6A, sample 2) showing that V1 associates with V0. In addition, 35% of ATPase activity was restored in the dialyzed sample compared with only 9% in the non-dialyzed sample. The control experiment using wild-type V1V0 complexes that were not treated with KI showed that all of the Vma1p and Vma2p were in the pellet along with Vph1p (Fig. 6A, sample 1). Dialysis reduced ATPase activity to 68% of the non-dialyzed sample. Similar mixing experiments with the P (V0) and the S (V1) fractions from sur4{Delta} cells showed that dialysis does promote association of Vma1p and Vma2p with the P fraction, but it does not restore any ATPase activity (Fig. 6A, sample 4). The control experiment using sur4{Delta} V1V0 complexes that were not treated with KI showed that all of the Vma1p and Vma2p were in the pellet along with Vph1p and that dialysis reduced ATPase activity slightly from 18 to 13% (Fig. 6A, sample 3). Results from other control reactions are shown in samples 7 and 8 of Fig. 6.



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FIG. 6.
V0 domains are functional in sur4{Delta} cells but V1 domains lack ATPase activity. A, vacuolar membranes were isolated by sucrose density gradient centrifugation and treated (reactions 2, 4–8) or not treated (reactions 1 and 3) with 300 mM KI to dissociate V1 from V0. Reactions treated with KI were centrifuged to generate a pellet fraction containing V0 domains and a supernatant fraction containing V1 domains that were mixed (reactions 2, 4–6) and dialyzed to promote domain association on not dialyzed to prevent association. To determine if the V1 and V0 associated, the reactions were centrifuged to yield a pellet (P) fraction containing V0 domains and V1V0 complexes (detected by immunoblotting for Vph1p) and a supernatant (S) fraction containing V1 domains (detected by immunoblotting for Vma1p and Vma2p). V-ATPase values represent the average of three determinations ± S.D. Reactions 1, 3, 7, and 8 are controls. B, cytosolic V1 domains were isolated from RCD390 (wild-type, •) and RCD410 (sur4{Delta}, {circ}) cells on sucrose density gradients as described in "Experimental Procedures" and 200 µg of protein was assayed for Ca-stimulated release of Pi from ATP (i.e. ATPase activity). Values represent the average of three determinations ± S.D.

 

Mixing the S (V1) fraction from wild-type cells with the P (V0) fraction from sur4{Delta} cells followed by dialysis resulted in association of Vma1p and Vma2p with the P fraction and restoration of 36% of the ATPase activity (Fig. 6A, sample 5). A reciprocal mixing experiment with the P (V0) fraction from wild-type cells the S (V1) fraction from sur4{Delta} cells showed that Vma1p and Vma2p associate with the P fraction but there was no restoration of ATPase activity (Fig. 6A, sample 6). These experiments demonstrate that V0 domains in sur4{Delta} cells are fully functional and can associate with wild-type V1 domains to generate ATPase activity whereas the V1 domains are capable of association with V0 on the vacuolar membrane but they do not have ATPase activity.

V1 Domains from sur4{Delta} Cells Lack Ca-ATPase Activity— Free V1 domains do not show ATPase activity when assayed in the presence of Mg2+, but do show activity when assayed in the presence of Ca2+ (20). Thus, to verify that V1 domains in sur4{Delta} cells lack ATPase activity we partially purified V1 complexes by velocity centrifugation on glycerol gradients and assayed them for ATPase activity in the presence Ca2+. To remove background ATPase activity that was not stimulated by Ca2+, the assay was also done in the absence of Ca2+ and this value was subtracted from the value obtained in the presence of Ca2+ to give the Ca-stimulated activity. In preliminary experiments increasing concentrations of protein in the pooled V1-containing sucrose gradient fractions were assayed for Ca-stimulated ATPase activity (release of Pi). Activity was linear with increasing protein concentration (data not shown). A protein concentration that gave easily measured activity using V1 domains from wild-type cells was chosen for kinetic analyzes. The kinetics of Ca-stimulated ATP hydrolysis for wild-type V1 domains were linear up to about 5 min and then reached a plateau whereas the V1 domains from sur4{Delta} cells showed no ATP hydrolysis over the entire 30 min incubation period (Fig. 6B). These results verify those shown in Fig. 6A and based upon the combined data we conclude that V1 domains in sur4{Delta} cells lack ATPase activity.

The Subunit Composition of the V1 and V0 Domains Appears Normal in sur4{Delta} Cells—V1 domains in sur4{Delta} cells may have reduced ATPase activity and dissociate from the vacuolar membrane in the presence of Ficoll because a subunit is missing. To examine the subunit composition of V1 and V0 domains, cells were converted to spheroplasts, metabolically labeled with [35S]amino acids, gently lysed, and then proteins were cross-linked with a reversible cross-linker. Samples were immunoprecipitated with monoclonal antibodies specific for Vph1p, Vma1p, or Vma2p and radioactive proteins in the immunoprecipitate were analyzed by SDS-PAGE and phosphorimaging.

The anti-Vph1p antibody used in these experiments only recognizes V0 domains that are not complexed with V1 (24) and is, therefore, useful for examining the subunit composition of V0 domains. Radiolabeled proteins of 100 (Vph1p), 36 (Vma6p), 19 (identity unknown, Refs. 30 and 31) and 17 (Vma11p) kDa were immunoprecipitated by the Vph1p antibody in wild-type sur4{Delta}, fen1{Delta}, and vma2{Delta} cells (Fig. 7A). We observed small variations in the relative intensity of bands from experiment to experiment, and the samples shown in Fig. 7 were chosen to represent the average of the results of three independent experiments. The data shown in Fig. 7A are in agreement with published results (e.g. Refs.30 and 31) and indicate that the subunit composition of the V0 domain is normal in sur4{Delta} and fen1{Delta} cells.

The anti-Vma1p and anti-Vma2p antibody used in these experiments recognize free V1 and V1V0 complexes (24, 30). Radioactive proteins immunoprecipitated by anti-Vma1p and anit-Vma2p are shown in Fig. 7, B and C, respectively. Again, there were small variations is the intensity of some radioactive bands from experiment to experiment, but overall our results indicate that the subunit composition of free V1 and the V1V0 complex in sur4{Delta} and fen1{Delta} cells is similar if not identical to that in wild-type cells and to published data. The vma2{Delta} cells served as a control for cells lacking V1 and V1V0 complexes (30).

A limitation of these data is that not all V1 subunits are readily detected including Vma7p, Vma10p, and Vma13p. Vma7p and Vma10p are probably present in the V1 domains we examined because if either protein were absence then V1 and V0 would not associate (3942). Vma13p is not necessary for V1-V0 association and could be missing. To determine if it was present in vacuolar membranes isolated from sur4{Delta} cells, sur4{Delta} vma13{Delta}, and vma13{Delta} cells were transformed with a vector carrying a VMA13 allele having a Myc epitope inserted immediately downstream of the methionine start codon (20). Vacuolar membranes were isolated by sucrose density gradient centrifugation and fractions from the gradient were immunoblotted with anti-Myc antibody. The concentration of Myc-Vma13p in the peak fraction containing vacuolar membranes (Fig. 7D, fraction 5) was similar in the sur4{Delta} vma13{Delta} and vma13{Delta} samples, and the overall distribution of Myc-Vma13p was very similar in the two gradients. Immunoblots of the total cell-free extracts showed that the concentration of Myc-Vma13p was the same in the two strains as were the other Vma subunits examined (data not shown). We conclude that Vma13p associates with V1V0 complexes in sur4{Delta} cells.

Elevated Levels of LCBs and LCBPs Do Not Correlate with Reduced V-ATPase Activity—It has been noted previously that sur4{Delta} and fen1{Delta} cells accumulate LCBs but the levels have not been quantified nor have the species that accumulate been determined (11, 12, 43). Likewise, the concentration of long chain base phosphates (LCBPs) has not been measured.

We quantified LCBs and LCBPs by tagging them after extraction with a fluorescent reagent followed by HPLC (23). The analysis was done in two different strain backgrounds to see how similar or different they might be and if any differences correlated with mutant phenotypes. The five species of LCBs are at nearly identical levels in the two wild-type strains (Table IV). In the two sur4{Delta} strains all five species are elevated and their levels are similar in the two strain backgrounds except for C18-DHS and C20-DHS, which are less elevated in strain RCD410 (the W303 background). All five species are also elevated in the two fen1{Delta} strains but there is more variability between strains and only the C16-DHS species have similar values.


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TABLE IV
LCBs are elevated in sur4{Delta} and fen1{Delta} cells

 

LCBP values are also very similar in the two wild-type strains (Table V) and are quite low as we have reported for a wild-type strain related to RCD390 (44). All LCBPs show similar elevations in the two sur4{Delta} strains except for C18-DHSP and C20-DHSP. All LCBPs are also elevated in the two fen1{Delta} strains but the values vary between the strains.


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TABLE V
LCBPs are elevated in sur4{Delta} and fen1{Delta} cells

 

The elevated levels of LCBs and LCBPs could be responsible for the lack of ATPase activity. However, if this were the case we would expect fen1{Delta} cells (RCD393), not sur4{Delta} cells (RCD389), to have a greater loss of V-ATPase activity because they have a higher total level of LCBs and LCBPs (Tables IV and V).

To determine directly if LCBs dissociate Vma1p and Vma2p from V0, vacuolar membranes purified from wild-type cells were incubated with increasing concentrations of PHS. After incubation, samples were centrifuged to give a pellet and a supernatant fraction, which where analyzed by immunoblotting for Vma1p and Vma2p. Because PHS has detergent-like properties and could release proteases from vacuoles, we also immunoblotted for CPY to control for proteolysis. In the presence, but not in the absence of PHS, the level of Vma1p and Vma2p in the pellet fraction gradually decreased over the 120-min incubation period (Fig. 8). However, the two proteins did not appear in the supernatant as would be expected if they were dissociating from V0. This result plus the fact that they disappeared (Fig. 8) at the same rate as CPY indicates that they are being digested by proteases. We conclude that LCBs do not dissociate Vma1p and Vma2p from purified vacuolar membranes.



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FIG. 8.
Purified V-ATPase is resistant to PHS treatments. A, vacuolar membranes purified from wild-type cells (RCD390) were treated or not treated with 100 µM PHS various lengths of time and then separated by centrifugation into a pellet (P) and supernatant (S) fractions. Supernatants were precipitated by treatment with trichloroacetic acid. All pellets were suspended in the same volume of SDS sample buffer and equal volumes were subjected to Western blotting. B, data in A were quantified by phosphorimage analysis. The values represent means ± S.D. (n = 3).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Our analysis of sur4{Delta} and fen1{Delta} cells shows that sphingolipids containing a C26 acyl group are necessary for generation of a V1 domain that is capable of hydrolyzing ATP. This conclusion is based upon an analysis of sur4{Delta} cells, whose sphingolipids lack a C26 acyl group, and fen1{Delta} cells, whose sphingolipids have about 30% of the wild-type level of C26 acyl groups (11, 12). We predicted that cellular functions dependent upon sphingolipids with a C26 acyl group would be more disrupted in sur4{Delta} than in fen1{Delta} cells. Several results support this line of reasoning and our conclusion. First, we found that sur4{Delta} cells have some but not all Vma phenotypes, indicating a defective V-ATPase, and that some of these phenotypes are more pronounced in sur4{Delta} than in fen1{Delta} cells. For example, growth is more inhibited in sur4{Delta} than in fen1{Delta} cells on YPD plates buffered to pH 7.5 or when the plates contain 100 mM CaCl2, 4 mM ZnCl2, or 2% glycerol as the carbon source (Fig. 1 and Table II). Second, V-ATPase activity in sur4{Delta} cells is as defective as that in vma2{Delta} cells, which lack V-ATPase activity, when measured by uptake of the vacuolar dye quinacrine (Fig. 2). In contrast, uptake in fen1{Delta} cells is about 4-fold higher than in sur4{Delta} cells or about 60% of the wild-type level. Finally, the ATPase activity of vacuoles purified from sur4{Delta} cells by Ficoll gradient centrifugation is only 10% of the wild type while fen1{Delta} vacuoles have 25% of the wild-type activity (Table III). These data show that V-ATPase activity is more impaired in sur4{Delta} than in fen1{Delta} cells.

To understand why sphingolipids with a C26 acyl group are needed for V-ATPase activity we determined if V1 domains could associate with V0 domains on the vacuolar membrane. Vacuolar membranes isolated on a Ficoll gradient were examined by immunoblotting to determine if V0 and V1 domains were present. The results showed that vacuolar membranes from sur4{Delta} and fen1{Delta} cells had very low, barely detectable levels of Vma1p, Vma2p, and Vma5p, indicating that the V1 domain was not associated with the V0 domain (Fig. 3). Since these three V1 subunits are present in cell extracts (Fig. 3) their absence from purified vacuoles suggests that they mislocalize, do not assemble into a functional V1 domain or V1 associates abnormally with V0. The finding that V1 and V0 are found together in fractions of a sucrose density gradient where vacuolar membranes are located (Fig. 4, fractions 5 and 6), supports either of the latter two possibilities. In addition, indirect immunofluorescent microscopy on intact sur4{Delta} and fen1{Delta} cells showed that the V1 subunit Vma2p was bound to vacuoles (Fig. 5). Together these data show that V1 is able to associate with V0 on the cytoplasmic face of the vacuolar membrane to form V1V0 complexes in sur4{Delta} cells, but the complexes are not stable in Ficoll (Fig. 3), have low ATPase activity (Table III), and do not acidify vacuoles (Figs. 1 and 2).

Reduced ATPase activity in sur4{Delta} cells suggested that the V1 domain was defective. To test this hypothesis, V1 and V0 domains were isolated and tested in vitro for association and for restoration of ATPase activity. Wild-type V1 and sur4{Delta} V0 associated to produce V1V0 complexes with ATPase activity. Wild-type V0 and sur4{Delta} V1 also associated but the V1V0 complexes had no ATPase activity (Fig. 6). We also partially purified cytosolic V1 domains from wild-type and sur4{Delta} cells and assayed them for calcium-dependent ATPase activity. V1 domains from sur4{Delta} cells completely lacked activity (Fig. 6B). These results establish that V0 domains in sur4{Delta} cells are normal, but the V1 domains are defective and lack ATPase activity.

To understand why V1 domains lack ATPase activity, we examined the subunit composition of V1 and V0 and found that they are the same in sur4{Delta}, fen1{Delta}, and wild-type cells (Fig. 7). Separate analysis of Myc-tagged Vma13p showed that it was present in V1 domains in sur4{Delta} cells (Fig. 7). The procedures used by us would not have detected the V1 subunits Vma7p or Vma10p, although we infer that they are present because if either protein was missing from cells, V1 domains would not associate with V0 (3942). Thus, lack of a protein subunit is not likely to be the cause of the defect in ATPase activity in V1 domains present in sur4{Delta} cells.

Others have reported that sur4{Delta} and fen1{Delta} cells contain high levels of LCBs (11, 12, 43), but the levels have not been quantified nor have the species been identified. We were concerned that the high level of LCBs might impair ATPase activity. It was recently shown that the reduced level of glucan synthase activity in sur4{Delta} and fen1{Delta} cells is due to elevated levels of PHS and DHS (43). Our analysis of LCBs shows that all five species are elevated in both sur4{Delta} and fen1{Delta} mutants in two different strain backgrounds, W303 and JK9-3d (Table IV). In the JK9-3d strain background, which we used for the majority of our experiments, there is a 44-fold increase in total LCBs in sur4{Delta} cells and a 63-fold increase in fen1{Delta} cells. The same trends hold for the mutants in the W303 background but the increases are smaller. If elevated LCBs were responsible for disruption of V-ATPase activity, then we would expect the activity to be reduced more in fen1{Delta} than in sur4{Delta} cells because fen1{Delta} cells have a higher level of LCBs. Our results are just the opposite of this prediction and argue that elevated LCBs are not responsible for reduced ATPase activity. However, such arguments cannot eliminate the possibility that LCBs are interfering with V1 function.

We also compared the total LCB content of fractions from a Ficoll gradient to see if there was any correlation between reduced V-ATPase activity in sur4{Delta} and fen1{Delta} cells and the level of these compounds. The vacuolar membrane fraction of wild-type cells had a very low, barely detectable level of LCBs, as did the two fractions below the membrane fraction (data not shown). The pellet at the bottom of the gradient had the highest level of LCBs, but the concentration was still very low. The level of LCBs was higher in the gradient fractions obtained from sur4{Delta} and fen1{Delta} cells. As with the total LCB values (Table IV), the levels in the Ficoll gradient fractions are higher in fen1{Delta} cells, suggesting that it is not the LCBs that are responsible for reduced V-ATPase activity. LCBPs were not detectable in any of the Ficoll gradient fractions nor in the cell-free extracts. Most likely they were degraded during the incubation period when cells were converted to spheroplasts.

We also determined if PHS added in vitro to purified vacuolar membranes could mimic what occurs in sur4{Delta} and fen1{Delta} cells and selectively release Vma1p and Vma2p from membranes. We found that neither protein was selectively released. In fact, the membrane V-ATPase was quite resistant to disruption by PHS and only at higher concentrations did the two proteins start to disappear from the membrane pellet (Fig. 8). Their disappearance, however, was not accompanied by their appearance in the soluble fraction. Rather their concentration decreased as a function of increasing PHS at the same rate as the luminal vacuolar protein CPY, indicating that protein loss was probably due to disruption of vacuolar membrane integrity and subsequent degradation by vacuolar proteases. Thus, wild-type V1V0 complexes are very resistant to dissociation by treatment in vitro with PHS.

We also measured LCBPs, which have not been measured, and found that all five species were elevated in sur4{Delta} and fen1{Delta} cells. The total level is similar in both wild-type strains and in both sur4{Delta} mutants, but is different in the fen1{Delta} strains (Table V). The differences may be due to strain-specific variation in the activity of metabolic pathways that make and degrade LCBPs. Again, it seems unlikely that LCBPs are disrupting V-ATPase activity because in the W303 strain background their level is higher in fen1{Delta} than in sur4{Delta} cells, yet the Vma phenotypes are less serve in fen1{Delta} than in sur4{Delta} cells (Table II). However, further work will be necessary to eliminate the possibility that elevated LCBPs are responsible for the defective V1 domain in sur4{Delta} cells.

How might sphingolipids with a C26 acyl group affect the ATPase activity of V1? One possibility is that ceramides, which are made in the ER (reviewed in Ref. 6), play a role in the assembly of V1 in the ER (45, 46). Another possibility is that complex sphingolipids in the Golgi influence maturation of V1 as it transits to the vacuolar membrane. In sur4{Delta} cells the ceramides and sphingolipids with C22 and C24 acyl groups would not substitute for the normal C26 groups and V1 domains would assemble incorrectly. The ATPase defect would be less severe in fen1{Delta} cells because about one-third of the ceramides and complex sphingolipids have C26 acyl groups. Alternatively, the RAVE protein complex (21) has recently been shown to be necessary for assembly of the V-ATPase (47) and some step in the action of RAVE may require sphingolipids with a C26 acyl group. Ficoll, a polymer of sucrose, may dissociate V1 from V0 by interacting with one or more V1 subunits that are not correctly folded or assembled in sur4{Delta} cells.

The results presented here are the first to indicate a role for C26 acyl groups and for sphingolipids in V-ATPase function. Their exact role will require further characterization of V1 domains in sur4{Delta} cells. Our results suggest that sphingolipids may be important for the activity of V-ATPases and related ATPases in other organisms. S. cerevisiae contains another type of V-ATPase located in the Golgi/endosomal compartments that is identical to the V-ATPase except that the V0 domain contains Stv1p in place of Vph1p (48). Our results suggest that the functionality of this V-ATPase may also require sphingolipids with a C26 acyl group.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM41302 (to R. C. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, 800 Rose St., Lexington, KY 40536. Tel.: 859-323-6052; Fax: 859-257-8940; E-mail: bobd{at}uky.edu.

1 The abbreviations used are: V-ATPase, vacuolar ATPase; DHS, dihydrosphingosine; LCB, long chain base; LCBP, long chain base phosphate; PHS, phytosphingosine; VLCFA, very long chain fatty acid; V1, peripheral domain of V-ATPase; Vma, vacuolar membrane ATPase; V0, membrane domain of V-ATPase; ER, endoplasmic reticulum; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high pressure liquid chromatography; FITC, fluorescein isothiocyanate. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Patricia Kane for sharing reagents with us and Dr. Lois Weisman for advice on indirect immunofluorescent microscopy.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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