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Eukaryotic Cell, April 2004, p. 302-310, Vol. 3, No. 2
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.2.302-310.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

pdf1, a Palmitoyl Protein Thioesterase 1 Ortholog in Schizosaccharomyces pombe: a Yeast Model of Infantile Batten Disease

Steve K. Cho and Sandra L. Hofmann^*

Department of Internal Medicine and the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received 3 December 2003/ Accepted 18 January 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Infantile Batten disease is a severe neurodegenerative storage disorder caused by mutations in the human PPT1 (palmitoyl protein thioesterase 1) gene, which encodes a lysosomal hydrolase that removes fatty acids from lipid-modified proteins. PPT1 has orthologs in many species, including lower organisms and plants, but not in Saccharomyces cerevisiae. The fission yeast Schizosaccharomyces pombe contains a previously uncharacterized open reading frame (SPBC530.12c) that encodes the S. pombe Ppt1p ortholog fused in frame to a second enzyme that is highly similar to a previously cloned mouse dolichol pyrophosphatase (Dolpp1p). In the present study, we characterized this interesting gene (designated here as pdf1, for palmitoyl protein thioesterase-dolichol pyrophosphate phosphatase fusion 1) through deletion of the open reading frame and complementation by plasmids bearing mutations in various regions of the pdf1 sequence. Strains bearing a deletion of the entire pdf1 open reading frame are nonviable and are rescued by a pdf1 expression plasmid. Inactivating mutations in the Dolpp1p domain do not rescue the lethality, whereas mutations in the Ppt1p domain result in cells that are viable but abnormally sensitive to sodium orthovanadate and elevated extracellular pH. The latter phenotypes have been previously associated with class C and class D vacuolar protein sorting (vps) mutants and vacuolar membrane H⁺-ATPase (vma) mutants in S. cerevisiae. Importantly, the Ppt1p-deficient phenotype is complemented by the human PPT1 gene. These results indicate that the function of PPT1 has been widely conserved throughout evolution and that S. pombe may serve as a genetically tractable model for the study of human infantile Batten disease.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The neuronal ceroid lipofusinoses (NCLs, also known collectively as Batten disease) are a group of progressive inherited neurodegenerative disorders of children characterized by the accumulation of autofluorescent inclusion bodies in the brain and other tissues (19, 31). These disorders are characterized by declining mental abilities, severe intractable seizures, blindness, loss of motor skills, and premature death. Traditionally, NCLs have been divided into subtypes based on the age of onset and pathology. The three major forms (infantile, late infantile, and juvenile onset) are caused by autosomal recessive mutations in the CLN1, CLN2, and CLN3 genes, respectively. The infantile form of NCL is caused by mutations in the PPT1/CLN1 gene, which encodes a lysosomal palmitoyl protein thioesterase 1 (Ppt1p) (53). Ppt1p catalyzes the removal of the fatty acid palmitate from cysteine residues in posttranslationally lipid-modified proteins (29). In mammals, Ppt1p is a classical globular monomeric enzyme, which possesses an /ß hydrolase fold typical of lipases and a classical catalytic triad consisting of serine, aspartic acid, and histidine residues (4).

As is true of many lysosomal storage disorders, the mechanism whereby the underlying enzyme deficiency leads to organ pathology is poorly understood. The storage material (granular osmiophilic deposits) accumulating in affected Ppt1p-deficient brain and other peripheral tissues is heterogeneous and complex, largely defying attempts at characterization. However, dolichol pyrophosphoryl oligosaccharides (DolPP-OS) have been found to accumulate to high levels in the brain tissue of NCL patients, including those with infantile NCL (13-16, 23). This has led to the hypothesis that abnormal dolichol metabolism may be involved in the pathogenesis of Batten disease.

Schizosaccharomyces pombe is a single-celled free living archiascomycete fungus sharing many features associated with more-complex eukaryotic cells. Because the genome of S. pombe can be manipulated experimentally, it has served as an excellent model organism for many studies, particularly those involving cell cycle control, mitosis and meiosis (9), and DNA repair and recombination (20). S. pombe is also increasingly used for investigating the functions of human disease genes. About 50 genes (of a total complement of approximately 4,800) are orthologous to known human disease genes, including those underlying various metabolic, cardiac, renal, and neurological diseases and cancer. Most of these genes are also found in Saccharomyces cerevisiae (another common fungal model). However, two known disease-associated genes (SPAC630.13c and SPBC530.12c) are found only in S. pombe and not in S. cerevisiae (55). One of the genes, SPBC530.12c, encodes the S. pombe ortholog of the human PPT1 gene. The other encodes a gene associated with tuberous sclerosis (TSC2).

Interestingly, examination of SPBC530.12c reveals that it is a fusion gene consisting of the S. pombe PPT1 ortholog followed by a single in-frame open reading frame (ORF), which is an ortholog of DOLPP1, a gene encoding dolichol pyrophosphate (DolPP) phosphatase-1 (Dolpp1p), an enzyme also implicated in dolichol metabolism. Dolpp1p is a multimembrane-spanning resident endoplasmic reticulum (ER) protein that catalyzes the conversion of DolPP to dolichol phosphate and is postulated to play a role in the recycling of dolichol utilized in the synthesis of the DolPP-OS. DolPP-OS are intermediates in the asparagine-linked glycosylation of proteins (43). Dolpp1p null mutant alleles in S. cerevisiae produce normal dolichol and dolichol-linked oligosaccharide intermediates at a reduced level (20%) compared to wild-type yeast. The presence of a putative fusion protein containing both PPT1 and DOLPP1 orthologs in S. pombe suggests coordinate regulation of the synthesis of the two polypeptides in S. pombe.

In the present work, we have ablated the SPBC530.12c gene (hereafter referred to as pdf1) and determined the consequences of inactivating mutations in the Ppt1p and Dolpp1p domains through plasmid complementation assays. We find that pdf1 ablation causes lethality and that Dolpp1p alone can rescue the lethal phenotype. However, cells containing no functional copy of Ppt1p are abnormally sensitive to sodium orthovanadate and elevated external pH, which are phenotypes associated with vacuolar dysfunction in yeast. By immunoblotting yeast extracts with antibodies that recognize the carboxyl terminus of Pdf1p, we provide evidence that Pdf1p is produced as a precursor protein that is cleaved to two separate polypeptides, probably as a result of a kex-related protease, Krp1. A human PPT1 cDNA is able to complement the phenotypes of Ppt1p deficiency, demonstrating that S. pombe provides a new genetic model for the study of Batten disease.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Yeast strains, media, and genetic methods. Genotypes of S. pombe strains used in this study are listed in Table 1. All strains were maintained on yeast extract plus supplement agar plates or under selection on Edinburgh minimal media (EMM) with appropriate supplements as described previously (32). Yeast cells were transformed with lithium acetate (1). Standard molecular biological techniques were employed (44).

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TABLE 1. S. pombe strains used in this study and their genotypes

Identification and subcloning of pdf1⁺. The SPBC530.12c gene of S. pombe was identified as containing sequences corresponding to a potential human PPT1 orthologue through a BLAST search of GenBank with the human PPT1 amino acid sequence (NP_000301) as the query. S. pombe cosmid 530 (GenBank accession number AL023634), which contains the SPBC530.12c gene, was obtained from the Sanger Center (http://www.sanger.ac.uk). PCR was carried out with Pfu polymerase (Stratagene) and cosmid 530 DNA as a template DNA with primers 5'-TAAGAATTCGACTCAAGACAACAATCACT-3' and 5'-CTTGGATCCCCTTGGGTAATTTTCTGAAC-3'. The amplified product was digested with EcoRI and BamHI (underlined in the forward and reverse primers, respectively) and subcloned into the plasmid vector pGEM-T-Easy (Promega) to create pGPD1. The entire ORF was sequenced on both strands and corresponds exactly to nucleotides 37983 through 39794 of AL023634.

Construction of the pdf1 disruption. Disruption of pdf1 was performed by removal of a large portion of the ORF (Fig. 1, corresponding to amino acids 69 through 565) of pGPD1 and insertion of a his3⁺ gene. Briefly, pGPD1 was digested with BbsI and BlpI to remove most of the ORF, and the remaining 3.5-kb fragment (containing vector and flanking sequence) was rendered blunt ended. The his3⁺ gene from pJB1 (a gift from Luis Rokeach, University of Montreal) (5) was amplified with Pfu polymerase and primers 5'-CAACGTTTTCTTTACTATTGCAC-3' and 5'-ACGCGTGAATGGACTGTTGGCTG-3' and ligated into pGEM-T-Easy (Promega). A 2.1-kb NotI fragment containing the his3⁺ gene was rendered blunt ended and ligated to the 3.5-kb fragment of pGPD. The resultant plasmid (pGPDH1) was digested with NotI, and the 2.6-kb insert was transformed into a wild-type diploid strain SC2478. His⁺ diploids were selected and designated SC41. Replacement of the wild-type allele by the disrupted gene was confirmed by Southern analysis with a randomly primed labeled probe generated by PCR amplification of a 300-bp portion of pGPD with primers 5'-CCCTTGGGTAATTTTCTGAAC-3' and 5'-GTCTTCAAATTCGTTGTCACC-3'. The correct targeting of the deletion cassette into the genomic locus was also verified by PCR. Primers were designed that were specific to the upstream, downstream, and internal regions of the ORF and to the internal region of the his3⁺ cassette (data not shown). To determine the effect of the gene disruption on spore viability, SC41 cells were plated on malt extract plates to induce sporulation. After 36 to 48 h, the presence of asci was confirmed under the light microscope, and tetrad analysis was performed by using a micromanipulator as described previously (1).

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FIG. 1. Amino acid sequence alignment (A) and domain structure (B) of proteins encoded by S. pombe pdf1, human PPT1, and mouse DOLPP1. Gaps in the sequences are indicated by dashes. Identical amino acid residues are shaded dark gray; similar residues are light gray. The linker region contains two distinct clusters (*) of basic amino acids, which are putative cleavage sites of serine endopeptidase Krp1. Amino acids that participate in catalysis and that were mutated to catalytically inactive residues for the purposes of this study are indicated (). An acidic linker region of 76 amino acids between the Ppt1p and Dolpp1p domains is indicated by a shaded bar.

Expression plasmids. The ORF of pdf1⁺ was amplified by PCR with pGPD1 as a template, and the amplified product was subcloned into three plasmid vectors—pREP1, pAAU (a gift from Steven Marcus, M. D. Anderson Cancer Center), and pAAL. The pAAL vector was created by replacing the ura4⁺ marker with LEU2⁺ in pAAU by digestion with HindIII and ligation. Point mutations were introduced in the pdf1⁺ ORF by using the QuikChange site-directed mutagenesis kit (Stratagene). Oligonucleotide sequences used in the construction of point mutations are available upon request. The truncation mutation (373 to 602) was constructed by PCR with pGPD1 as the template.

Isolation of haploid pdf1 strains complemented by pdf1⁺. SC41 cells were transformed with pAAU-pdf1⁺, and His⁺ and Ura⁺ transformants were selected on medium lacking adenine, histidine, and uracil. Transformants were induced to sporulate on malt extract plates, the ascus walls were digested with glusulase (Sigma), and the individual spores were germinated on plates lacking histidine and uracil. The resulting colonies were examined as His⁺ Ade^– haploids only if they were also Ura⁺, indicating plasmid rescue of the disruption phenotype. Loss of the wild-type allele was confirmed by Southern analysis. The haploid strains were designated SC41R, SC41U, or SC41L, for strains complemented by pREP1-pdf1⁺, pAAU-pdf1⁺, and pAAL-pdf1⁺, respectively.

Structure-function analysis of pdf1⁺ by plasmid shuffling. SC41U cells were transformed with wild-type pAAL-pdf1⁺ or with plasmids containing mutations, as indicated in the figure legends. Transformants were selected on a medium lacking leucine and uracil. Each transformant was then streaked onto medium containing 0.1% 5-fluoroorotic acid (5-FOA) and 50 µg of uracil/ml (28) and was grown at 30°C for 4 days to promote the loss of the ura4⁺ plasmid. The growth of cells in the presence of 5-FOA was scored to assess the ability of the wild-type or mutated pAAL-pdf1⁺ to promote viability of the haploid pdf1 strain.

Vanadate and extracellular pH sensitivity. Sodium orthovanadate (Sigma) was added to medium from a filter-sterilized stock solution (100 mM) after autoclaving. For pH sensitivity experiments, 50 mM morpholinepropanesulfonic acid (MOPS) and 50 mM morpholineethanesulfonic acid (MES) was included in the medium and the pH was adjusted by dropwise addition of 10 N NaOH.

Affinity-purified polyclonal antibodies. Three New Zealand White rabbits were each immunized with 300 µg of a synthetic peptide corresponding to amino acid residues 583 to 603 of Pdf1p that was coupled to keyhole limpet hemocyanin by using methods described previously (43). The antigen was injected intradermally in Freund's complete adjuvant, and rabbits were boosted three times at 3-week intervals with 300 µg of peptide in Freund's incomplete adjuvant. An immunoglobulin G (IgG) fraction was prepared from preimmune and immune serum by specific binding to protein A-Sepharose CL-4B (Pharmacia). The IgG was affinity purified by specific binding to a column consisting of the peptide cross-linked to SulfoLink coupling gel (Pierce).

Protein extraction and immunoblotting. Cells were grown to mid-log phase in EMM with appropriate amino acids. The harvested cells were washed twice with distilled water, and cell lysates were prepared by glass bead lysis in homogenization buffer (20 mM HEPES [pH 7.0], 50 mM potassium acetate, 5 mM magnesium acetate, 100 mM sorbitol) with the addition of 1 mM dithiothreitol and a cocktail of protease inhibitors (2 µg [each] of leupeptin, pepstatin, and aprotinin/ml and 1 mM phenylmethylsulfonyl fluoride) (28). Lysates were cleared by centrifugation at 500 x g for 1 min at 4°C, and resulting supernatants were centrifuged at 20,000 x g for 30 min at 4°C in a Beckman Optima TLX ultracentrifuge. The supernatant fraction was reserved and the membrane pellet was washed once with homogenization buffer and resuspended in 8 M urea for immunoblotting. Samples were analyzed by electrophoresis in sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gel electrophoresis (PAGE) gels and immunoblotting essentially as described previously (43). Filters were blocked for 1 h with Sea Block (East Coast Biologics) and washed in PBS-T (0.25% [vol/vol] Tween 20 in phosphate-buffered saline [PBS]) followed by incubation for 1 h with rabbit antipeptide polyclonal antibody (0.1 µg/ml). The filters were washed with PBS-T and incubated for 45 min in a solution containing horseradish peroxidase-conjugated secondary antibody (sheep anti-rabbit IgG; Amersham) diluted 1:2,500 in PBS-T. The filters were washed and developed with ECL chemiluminescence reagents (Amersham).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of the fission yeast pdf1 gene. A BLAST search of the S. pombe genome database with the human PPT1 amino acid sequence yielded a gene (SPBC530.12c) that encodes a 603-amino-acid-residue protein that contains a Ppt1p domain (31% identical to human Ppt1p), a linker region, and a domain that shares 33% sequence identity to mouse Dolpp1p (Fig. 1). Residues of human Ppt1p and mouse Dolpp1p known to participate in catalysis (i.e., catalytic triad residues Ser115, Asp233, and His289 of human PPT1) (4) and the consensus lipid-phosphate phosphatase motif of mouse Dolpp1p (Lys455, His478, and His530) (43) are conserved in Pdf1p (Fig. 1A). The linker region (Fig. 1B) contains sequences that conform to cleavage recognition sites for a kex-related processing protease, Krp1 (40).

Disruption of fission yeast pdf1 is lethal. To investigate the function of pdf1, we constructed and examined the phenotypes of S. pombe strains in which pdf1 was deleted. The pdf1 gene is 1,812 bp in length and contains no predicted intron sequences (Fig. 2A). One copy of the pdf1 ORF (comprising amino acids 69 to 565) was replaced with the his3⁺ selectable marker in a diploid S. pombe strain, and chromosomal deletion of the gene was confirmed by Southern analysis (Fig. 2B), which yields a 2.2-kb band in the wild type (lane 1), 2.2- and 1.8-kb bands in heterozygous diploids (lane 2), and a single band of 5.5 kb in the disruption strain rescued by a plasmid (pREP1-pdf1⁺) in the haploid state (lane 3). Correct targeting of the deletion cassette into the genomic locus was also confirmed by PCR as described in Materials and Methods (data not shown). Sporulation and tetrad analysis of independently derived heterozygous diploid strains ( pdf1::his3⁺/pdf1⁺) resulted in only two viable spores, showing a 2:0 segregation ratio (Fig. 2C). All of the viable spores were his^– and pdf1⁺, indicating that pdf1 is an essential gene in S. pombe.

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FIG. 2. Disruption of pdf1+. (A) Restriction map and schematic of the pdf1+ locus. The direction and location of the pdf1+ ORF are indicated. The position of the probe used for Southern blot analysis is indicated as a shaded box. The location of the his3+ gene in the pdf1::his3+ disruption construct is shown. An additional HindIII site is located within the his3+ gene. (B) Southern blot analysis of the chromosomal pdf1+ deletion. Genomic DNA was digested with HindIII, resolved on a 1% agarose gel, and probed with the 0.3-kb fragment indicated in panel A. Lane 1, homozygous wild type pdf1+/pdf1+; lane 2, heterozygous pdf1/pdf1+ strain; lane 3, homozygous pdf1/pdf1 strain rescued with a plasmid bearing pdf1+. (C) Tetrad analysis of the pdf1+ gene disruption. A 2:0 segregation ratio resulted from microdissection of heterozygous diploid (pdf1::his3+/pdf1+) cells after sporulation. The spores that formed colonies in any tetrad were his– and hence pdf1+.

Dolpp1p domain of Pdf1p is essential for viability whereas Ppt1p domain is dispensable. To gain a further insight into the role of two domains of pdf1, we analyzed a pdf1 strain complemented by various plasmids as shown in Fig. 3. Of note, the analysis was complicated by an inability to express the S. pombe Dolpp1p domain alone in the absence of upstream sequences, presumably due to the need for a signal sequence or other sequences needed for proper Dolpp1p membrane insertion and folding.

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FIG. 3. Plasmid shuffling by counterselection on 5-FOA reveals that the Dolpp1p domain is essential for viability. The ability to grow in the presence of 5-FOA indicates complementation of pdf1 by the indicated plasmid in SC41U haploid cells. The schematic (1 to 8) indicates the amino acid sequences encoded by the complementing plasmid with inactivating point mutations, deletions, or nonsense mutations as indicated: 1, wild-type Pdf1p; 2, Pdf1p 373 to 602; 3, Pdf1p (S106A); 4, Pdf1p (D226A); 5, Pdf1p (K302Stop); 6, Pdf1p (H478A); 7, human Ppt1p; 8, S. cerevisiae Dolpp1p. The transformants were streaked to EMM plates supplemented only with adenine (–5'FOA) or adenine, uracil, and 5-FOA (+5'FOA). The growth of each transformant on the plates was scored. +, growth; –, no growth.

A structure-function analysis was performed by using a plasmid shuffling technique. First, we isolated a haploid strain SC41U which contains the pdf1 deletion and harbors the wild-type pdf1⁺ gene within a complementing plasmid (pAAU). Because pdf1 is an essential gene, only haploid cells containing the plasmid were viable. The SC41U cells were transformed with a second plasmid (pAAL) containing a series of mutant constructs or S. cerevisiae DOLPP1 or human PPT1 cDNAs. Those plasmids included specific point mutants and deletion and nonsense mutants within the pdf1 coding sequences on the plasmid. The growth of each transformant in the presence and absence of 5-FOA was scored (Fig. 3). As a result, we observed that plasmids bearing inactivating mutations in the Ppt1p domain (18) (S106A and D226A) that contained an intact Dolpp1p domain complemented the lethal phenotype (Fig. 3, sectors 3 and 4), whereas plasmids truncated before the Dolpp1p domain or bearing a mutation in a highly conserved residue in the phosphate binding pocket of Dolpp1p (47) (H478A) did not rescue the phenotype (Fig. 3, sectors 2, 5, and 6). Therefore, it is clear that loss of the Dolpp1p domain is responsible for the lethal phenotype. Interestingly, neither human Ppt1p nor S. cerevisiae Dolpp1p expression plasmids alone complemented the lethal phenotype.

Phenotype of Ppt1p-deficient mutants. We observed that while pdf1 mutants rescued by functional Dolpp1p alone were viable, the growth rate of these cells was somewhat slower than that of the haploid cells containing the wild-type pdf1⁺ gene (Fig. 4, left panel) This growth retardation phenotype was reversed by a second plasmid encoding a wild-type Ppt1p domain (Fig. 4, right panel) and an inactivated Dolpp1p domain, suggesting that the growth retardation was caused by the lack of Ppt1p function. Therefore, we have further examined the phenotype of these strains. Of note, Ppt1p enzymatic activity in these strains could not be measured due to interfering activities or substances in the yeast that raised background signals in the assay.

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FIG. 4. Growth retardation of cells harboring point mutants in the Ppt1p domain. (Left panel) pdf1::his3+ haploids were rescued by plasmids containing wild-type pdf1+ or plasmids bearing mutations in active site residues of the Ppt1p domain (S106A and D226A). (Right panel) Rescue of growth retardation by a second plasmid containing a wild-type Ppt1p domain and catalytically inactive Dolpp1p domain. Serial 10-fold dilutions were spotted onto plates containing the relevant amino acid supplement.

Ppt1p-deficient mutants are sensitive to vanadate and elevated extracellular pH. As Ppt1p is a lysosomal enzyme in mammalian cells and as the fungal vacuole is the equivalent organelle of the mammalian lysosome, we thought that it would be of interest to see whether deficiency of Ppt1p function would lead to phenotypes normally associated with vacuolar mutants in fungi, such as sensitivity to sodium orthovanadate or external pH (3, 21, 24, 25, 49). As shown in Fig. 5A, cells harboring a wild-type pdf1 gene were not affected by vanadate, whereas cells harboring inactivating mutations in Ppt1p are sensitive to vanadate in a concentration-dependent manner (sectors 2 and 3). This sensitivity was reversed when the cells were transformed with a second plasmid containing the wild-type Ppt1p domain and an inactive Dolpp1p domain (Fig. 5A, center column). Inactivating mutations in the Ppt1p domain were also associated with an abnormal sensitivity to elevated extracellular pH, as shown in Fig. 5B. At pH 5.5, cells complemented with either the wild type or plasmids bearing mutations in the Ppt1p domain grow normally. However, when the external pH was elevated above pH 6.0, the haploid cells lacking functional Ppt1p did not grow. This pH sensitivity was reversed when the cells were transformed with a second plasmid containing a wild-type Ppt1p domain and an inactive Dolpp1p domain (Fig. 5B, center column). Transformation of cells with an empty plasmid vector had no effect on the phenotype (Fig. 5A and B, third column). These results strongly suggest that Ppt1p activity is required for normal vacuolar function in S. pombe.

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FIG. 5. Sensitivity of PPT-deficient cells to sodium orthovanadate and to extracellular pH. (A) Normally inviable pdf1::his3+ haploids rescued by wild-type pAAL-pdf1+ (1), mutated pAAL-pdf1(S106A) (2), or pAAL-pdf1(D226A) plasmids with mutations in the Ppt1p domain (3) were exposed to various concentrations of sodium orthovanadate for 4 days at 30°C. Mutations in the Ppt1p domain conferred sensitivity to sodium orthovanadate in a concentration-dependent manner. The sensitivity was reversed when the cells were transformed with a second plasmid, pAAU-pdf1(H478A), containing wild-type PPT and an inactivating mutation in the Dolpp1p domain (sectors 4 to 6). Empty vector controls are shown in sectors 7 to 9. (B) Yeast cells were incubated at 30°C for 4 days on EMM adjusted to the indicated pH. Ppt1p-inactivating mutations conferred an abnormal sensitivity to high pH to the cells. The sensitivity to higher pH wasreversed when the cells were transformed with a second plasmid containing a wild-type PPT domain and an inactivating point mutation in the Dolpp1p domain (sectors 4 to 6). This suggests that the pH sensitivity is due solely to the absence of wild-type Ppt1p activity in the cells. Note that even normal S. pombe cells were unable to grow at pH 7.0. Empty vector controls are shown in sectors 7 to 9. (C) The human PPT1 gene activity was able to complement the phenotypes of PPT-deficient mutants. A second plasmid containing a human PPT1 cDNA (pAAU-hPPT1) was transformed into the cells. Transformants were streaked onto plates containing either 4 mM sodium orthovanadate or medium buffered to pH 6.5.

We also tested for a number of other phenotypes, especially those associated with defects in cell wall integrity, as is seen in Dolpp1p (CWH8) mutants in S. cerevisiae (10, 51). Ppt1p-deficient cells grew normally in the presence of calcofluor white (up to 1 mg/ml), sorbitol or glucose (2 M), potassium chloride (0.9 M), or glycerol (as a sole carbon source) (data not shown). In addition, staining with the dye FM4-64, which reveals defects in endocytosis in S. pombe vps mutants (48), was entirely normal when observed after 30 min of incubation and up to 16 h after incubation (data not shown). The addition of vanadate (4 mM) or upward adjustment of the extracellular pH to 6.5 for 3 h prior to staining had no effect. Evaluation of wild-type and Ppt1p-deficient cells by electron microscopy revealed no significant differences in vacuolar morphology, and no clear accumulations of granular osmophilic deposits were noted (data not shown). In addition, heat sensitivity (as assessed by growth at 37°C) and cold sensitivity (growth at 23°C) were indistinguishable from those of the wild type.

Functional equivalence of human and S. pombe Ppt1p domains. The presence of vacuolar phenotypes in the cells lacking Ppt1p function allowed us to demonstrate whether the human PPT1 ortholog would substitute for the S. pombe gene to complement these phenotypes. As shown in Fig. 5C, a human PPT1 cDNA reestablished S. pombe cell viability in the presence of vanadate and at elevated extracellular pH. These results indicate that S. pombe can be used as a model organism for the study of Batten disease.

Posttranslational processing of Pdf1p. To determine whether the two functional domains of Pdf1p remain associated as a single polypeptide or whether they are subject to posttranslational proteolytic processing, we made an antibody that recognizes the carboxyl terminus of Pdf1p and used it to examine the molecular masses of proteins produced from plasmid-derived expression of Pdf1p (Fig. 6). Membrane fractions were prepared from the yeast, and solubilized samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting. In Fig. 6A, lane 1 (wild type), one major band at 31 kDa and two minor bands at 101 and 33 kDa are seen. The major band corresponds precisely to the predicted molecular mass of the Dolpp1p domain (in the absence of the linker region), whereas the upper minor band at 101 kDa corresponds to the full-length unprocessed Pdf1p. The Pdf1p linker region contains two putative cleavage sites for the kex-related endopeptidase designated Krp1p (5, 22). Mutation at one of the two putative Krp1p cleavage sites (R344A) did not prevent cleavage of the linker domain (Fig. 6A, lane 2) but did increase the ratio of the unprocessed from to the processed form. However, mutation of the dibasic motif at the internal site (R354A) (Fig. 6B and C) essentially abolished cleavage (Fig. 6A, lane 3). Mutation of the dibasic motifs at both the primary and internal sites also prevented the processing of Pdf1p and increased the level of precursor Pdf1p (Fig. 6A, lane 4). These results suggest that Pdf1p is proteolytically cleaved to form distinct Ppt1p and Dolpp1p domains and that Arg354 is crucial for posttranslational processing.

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FIG. 6. Posttranslational processing of Pdf1p at dibasic cleavage sites. (A) Membrane fractions of cell lysates were prepared, separated by SDS-PAGE, and transferred to nitrocellulose for immunoblotting with an antibody that recognizes the carboxyl terminus of Pdf1p. Cells were expressing either wild-type Pdf1p or Pdf1p that contained point mutations in the linker domain (R344A, R354A, or both mutations). Substitution of alanine for arginine at position 354 blocks the proteolytic cleavage, suggesting that arginine 354 is crucial for the posttranslational processing. (B) Schematic of Pdf1p cleavage sites. Two clusters of dibasic motifs, KR344 and RKR354, are indicated. (C) Comparison of known cleavage sites for Krp1p within Krp1p itself (autocatalytic sites) versus putative Krp1p cleavage sites within the Pdf1p linker region. Note that the order of internal and primary sites for Krp1p (8, 40) along the polypeptide is reversed and the number of intervening amino acids between the two sites is smaller in Pdf1 (10 versus 20). In addition, the arginine in the primary site of Krp1 is replaced by leucine in Ppt1p.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrated that an ortholog of human PPT1 is present in the genome of S. pombe as a fusion gene with a partner, DOLPP1. The entire ORF of the pdf1 gene is translated as a unit and subjected to posttranslational proteolytic processing. Mutation at either one of two consensus sequences for the endopeptidase Krp1 inhibits processing, whereas mutation at both sites abolishes it. We found that Dolpp1p function is absolutely essential for viability in S. pombe. However, lack of Ppt1p function causes hypersensitivity to elevated extracellular pH and to sodium orthovanadate. The human PPT1 cDNA complements these phenotypes, suggesting that the function of PPT1 has been conserved throughout evolution.

Abnormalities in lysosomal pH homeostasis in the juvenile-onset form of Batten disease were first uncovered through the study of a yeast model of the disease (35-39; D. A. Pearce, S. A. Nosel, and F. Sherman, Proc. 7th Int. Congr. NCLs, abstr. 034, 1998). Juvenile-onset Batten disease is caused by mutations in CLN3, which encodes a multimembrane-spanning intrinsic lysosomal membrane protein of unknown function (27). The orthologous S. cerevisiae gene BTN1 is an intrinsic vacuolar protein. In contrast to Ppt1p-deficient S. pombe, BTN1 cells do not exhibit an overt phenotype referable to defective vacuolar function (in over 100 experimental conditions screened) (37), with the exception that the cells are resistant to a primary amine growth inhibitor, D-(–)-threo-2-amino-1[p-nitrophenyl]-1, 3-propanediol (ANP). The ANP resistance of BTN1 cells was shown to be related to a subtly enhanced acidification of the external medium, and BTN1 strains have a slightly more acidic vacuolar pH in early phases of growth (37). The human CLN3 cDNA complements this phenotype and substitutes functionally for BTN1 (7, 36, 39). Observations of more robust phenotypes in Ppt1p versus Cln3p deficiency appear to hold for yeast, mice (12, 22, 30), and humans (11).

The phenotypes we observed in Ppt1p loss-of-function mutations in S. pombe (hypersensitivity to elevated extracellular pH and vanadate), while not seen in the yeast model of late-onset Batten disease, do overlap some of those previously described for the vma (vacuolar membrane ATPase) and class C and D vps (vacuolar protein sorting) mutants of S. cerevisiae (2, 21, 33, 34, 41, 56) and vps33 mutants of S. pombe. vma mutants carry mutations in subunits of the vacuolar H⁺-ATPase (an enzyme responsible for the acidification of vacuolar contents and maintenance of intracellular pH), and class C and class D vps mutants display defects in vacuole morphogenesis (41, 42). These mutants are also sensitive to environmental manipulations such as external pH and sodium orthovanadate (3, 21, 24, 25, 49). Therefore, our data for S. pombe suggest that Ppt1p functions in the vacuole just as it functions in the analogous structure (lysosome) of mammalian cells.

The observed proteolytic processing of the Pdf1p fusion protein of S. pombe is also entirely consistent with the known subcellular compartmentalization of Ppt1p and Dolpp1p in mammalian cells. In higher organisms, Ppt1p is a resident soluble lysosomal enzyme that traffics through the classical mannose 6-phosphate receptor pathway (17, 52), whereas Dolpp1p is a resident ER protein (10, 43). Therefore, our data are consistent with a model that, for S. pombe, the Pdf1p fusion protein is directed into the secretory pathway by virtue of the Ppt1p signal sequence, with the appearance of the Ppt1p domain on the lumenal side, followed by cotranslational incorporation of Dolpp1p into the ER membrane. The fusion protein would then become available for processing by Krp1 (or a related enzyme), and the Ppt1p domain would be free for transport to the vacuole.

Whether Pdf1p is indeed a physiological substrate for Krp1p remains to be determined experimentally. Deletion of Krp1 in S. pombe is lethal (40), and as deletion of Dolpp1p is also lethal, one may speculate that lack of processing of Dolpp1p by Krp1 is responsible for the lethality in Krp1 null strains. Of note, Dolpp1p processing mutants created in our study were viable, but the mutants appeared to be leaky because small amounts of processed Ppt1p were detectable upon longer exposures.

The translation of Pdf1p as a single polypeptide suggests that there may be an advantage in coordinately regulating levels of Ppt1p and Dolpp1p in S. pombe. In mammalian cells, a number of lysosomal enzymes are coordinately upregulated in a compensatory fashion under conditions of lysosomal dysfunction (6, 26, 50). Dolpp1p is postulated to participate in the recycling of dolichols following transfer of the oligosaccharide chain from DolPP-OS to nascent proteins in the biosynthetic pathway (45). As DolPP-OS accumulate to some degree in a number of lysosomal storage diseases (reviewed in reference 54), it is plausible that the initial steps of DolPP-OS degradation occur in the lysosome and that Dolpp1p, though not a lysosomal enzyme per se, is upregulated in response to the need to recycle dolichol under conditions of increased lysosomal metabolic demand.

Studies in lower organisms, particularly yeast, have provided surprising insights into human biology, even in the area of human neurodegenerative disease (46). The two yeast models of Batten disease have provided evidence for abnormal vacuolar function due to lack of the relevant disease proteins and have begun to implicate abnormalities in pH homeostasis in the pathogenesis of these disorders, with implications for therapy. Future work in these genetically tractable organisms may therefore provide further insights into the pathogenesis of Batten Disease.

 
We thank David Pearce for helpful discussions.

This work was supported by the National Institutes of Health (NS 35323) and the Robert A. Welch Foundation.

 
* Corresponding author. Mailing address: Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8593. Phone: (214) 648-4911. Fax: (214) 648-4940. E-mail: Sandra.Hofmann{at}UTSouthwestern.edu.

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Eukaryotic Cell, April 2004, p. 302-310, Vol. 3, No. 2
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