pdf1, a palmitoyl protein thioesterase 1 ortholog in ... · containing 0.1% 5-ﬂuoroorotic acid...

EUKARYOTIC CELL, Apr. 2004, p. 302–310 Vol. 3, No. 21535-9778/04/$08.00�0 DOI: 10.1128/EC.3.2.302–310.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

pdf1, a Palmitoyl Protein Thioesterase 1 Ortholog inSchizosaccharomyces pombe: a Yeast Model of

Infantile Batten DiseaseSteve 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

Infantile Batten disease is a severe neurodegenerative storage disorder caused by mutations in the humanPPT1 (palmitoyl protein thioesterase 1) gene, which encodes a lysosomal hydrolase that removes fatty acidsfrom lipid-modified proteins. PPT1 has orthologs in many species, including lower organisms and plants, butnot in Saccharomyces cerevisiae. The fission yeast Schizosaccharomyces pombe contains a previously uncharac-terized open reading frame (SPBC530.12c) that encodes the S. pombe Ppt1p ortholog fused in frame to a secondenzyme that is highly similar to a previously cloned mouse dolichol pyrophosphatase (Dolpp1p). In the presentstudy, 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 complemen-tation by plasmids bearing mutations in various regions of the pdf1 sequence. Strains bearing a deletion of theentire pdf1 open reading frame are nonviable and are rescued by a pdf1 expression plasmid. Inactivatingmutations in the Dolpp1p domain do not rescue the lethality, whereas mutations in the Ppt1p domain resultin cells that are viable but abnormally sensitive to sodium orthovanadate and elevated extracellular pH. Thelatter 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-deficientphenotype is complemented by the human PPT1 gene. These results indicate that the function of PPT1 has beenwidely conserved throughout evolution and that S. pombe may serve as a genetically tractable model for thestudy of human infantile Batten disease.

The neuronal ceroid lipofusinoses (NCLs, also known col-lectively as Batten disease) are a group of progressive inheritedneurodegenerative disorders of children characterized by theaccumulation of autofluorescent inclusion bodies in the brainand other tissues (19, 31). These disorders are characterized bydeclining mental abilities, severe intractable seizures, blind-ness, loss of motor skills, and premature death. Traditionally,NCLs have been divided into subtypes based on the age ofonset and pathology. The three major forms (infantile, lateinfantile, and juvenile onset) are caused by autosomal recessivemutations 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 thio-esterase 1 (Ppt1p) (53). Ppt1p catalyzes the removal of thefatty acid palmitate from cysteine residues in posttranslation-ally lipid-modified proteins (29). In mammals, Ppt1p is a clas-sical globular monomeric enzyme, which possesses an �/� hy-drolase fold typical of lipases and a classical catalytic triadconsisting of serine, aspartic acid, and histidine residues (4).

As is true of many lysosomal storage disorders, the mecha-nism whereby the underlying enzyme deficiency leads to organpathology is poorly understood. The storage material (granularosmiophilic deposits) accumulating in affected Ppt1p-deficient

brain and other peripheral tissues is heterogeneous and com-plex, largely defying attempts at characterization. However,dolichol pyrophosphoryl oligosaccharides (DolPP-OS) havebeen found to accumulate to high levels in the brain tissue ofNCL patients, including those with infantile NCL (13–16, 23).This has led to the hypothesis that abnormal dolichol metab-olism may be involved in the pathogenesis of Batten disease.

Schizosaccharomyces pombe is a single-celled free living ar-chiascomycete fungus sharing many features associated withmore-complex eukaryotic cells. Because the genome of S.pombe can be manipulated experimentally, it has served as anexcellent model organism for many studies, particularly thoseinvolving cell cycle control, mitosis and meiosis (9), and DNArepair and recombination (20). S. pombe is also increasinglyused for investigating the functions of human disease genes.About 50 genes (of a total complement of approximately4,800) are orthologous to known human disease genes, includ-ing those underlying various metabolic, cardiac, renal, andneurological diseases and cancer. Most of these genes are alsofound in Saccharomyces cerevisiae (another common fungalmodel). However, two known disease-associated genes(SPAC630.13c and SPBC530.12c) are found only in S. pombeand not in S. cerevisiae (55). One of the genes, SPBC530.12c,encodes the S. pombe ortholog of the human PPT1 gene. Theother encodes a gene associated with tuberous sclerosis(TSC2).

Interestingly, examination of SPBC530.12c reveals that it isa fusion gene consisting of the S. pombe PPT1 ortholog fol-lowed by a single in-frame open reading frame (ORF), which

* Corresponding author. Mailing address: Hamon Center for Ther-apeutic Oncology Research, University of Texas Southwestern Medi-cal Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8593. Phone:(214) 648-4911. Fax: (214) 648-4940. E-mail: [email protected].

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is an ortholog of DOLPP1, a gene encoding dolichol pyrophos-phate (DolPP) phosphatase-1 (Dolpp1p), an enzyme also im-plicated in dolichol metabolism. Dolpp1p is a multimembrane-spanning resident endoplasmic reticulum (ER) protein thatcatalyzes the conversion of DolPP to dolichol phosphate and ispostulated to play a role in the recycling of dolichol utilized inthe synthesis of the DolPP-OS. DolPP-OS are intermediates inthe asparagine-linked glycosylation of proteins (43). Dolpp1pnull mutant alleles in S. cerevisiae produce normal dolichol anddolichol-linked oligosaccharide intermediates at a reducedlevel (20%) compared to wild-type yeast. The presence of aputative fusion protein containing both PPT1 and DOLPP1orthologs in S. pombe suggests coordinate regulation of thesynthesis 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 conse-quences of inactivating mutations in the Ppt1p and Dolpp1pdomains through plasmid complementation assays. We findthat pdf1 ablation causes lethality and that Dolpp1p alone canrescue the lethal phenotype. However, cells containing nofunctional copy of Ppt1p are abnormally sensitive to sodiumorthovanadate and elevated external pH, which are pheno-types associated with vacuolar dysfunction in yeast. By immu-noblotting yeast extracts with antibodies that recognize thecarboxyl terminus of Pdf1p, we provide evidence that Pdf1p isproduced as a precursor protein that is cleaved to two separatepolypeptides, probably as a result of a kex-related protease,Krp1. A human PPT1 cDNA is able to complement the phe-notypes of Ppt1p deficiency, demonstrating that S. pombe pro-vides a new genetic model for the study of Batten disease.

MATERIALS AND METHODS

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

Identification and subcloning of pdf1�. The SPBC530.12c gene of S. pombewas identified as containing sequences corresponding to a potential human PPT1orthologue through a BLAST search of GenBank with the human PPT1 aminoacid sequence (NP_000301) as the query. S. pombe cosmid 530 (GenBank ac-cession number AL023634), which contains the SPBC530.12c gene, was obtainedfrom the Sanger Center (http://www.sanger.ac.uk). PCR was carried out with Pfupolymerase (Stratagene) and cosmid 530 DNA as a template DNA with primers5�-TAAGAATTCGACTCAAGACAACAATCACT-3� and 5�-CTTGGATCCCCTTGGGTAATTTTCTGAAC-3�. The amplified product was digested withEcoRI and BamHI (underlined in the forward and reverse primers, respectively)and subcloned into the plasmid vector pGEM-T-Easy (Promega) to createpGPD1. The entire ORF was sequenced on both strands and corresponds exactlyto nucleotides 37983 through 39794 of AL023634.

Construction of the pdf1 disruption. Disruption of pdf1 was performed byremoval of a large portion of the ORF (Fig. 1, corresponding to amino acids 69through 565) of pGPD1 and insertion of a his3� gene. Briefly, pGPD1 wasdigested with BbsI and BlpI to remove most of the ORF, and the remaining3.5-kb fragment (containing vector and flanking sequence) was rendered bluntended. The his3� gene from pJB1 (a gift from Luis Rokeach, University ofMontreal) (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 containingthe his3� gene was rendered blunt ended and ligated to the 3.5-kb fragment ofpGPD. The resultant plasmid (pGPDH1) was digested with NotI, and the 2.6-kbinsert was transformed into a wild-type diploid strain SC2478. His� diploids wereselected and designated SC41. Replacement of the wild-type allele by the dis-rupted gene was confirmed by Southern analysis with a randomly primed labeledprobe generated by PCR amplification of a 300-bp portion of pGPD with primers5�-CCCTTGGGTAATTTTCTGAAC-3� and 5�-GTCTTCAAATTCGTTGTCACC-3�. The correct targeting of the deletion cassette into the genomic locus wasalso 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 thehis3� cassette (data not shown). To determine the effect of the gene disruptionon spore viability, SC41 cells were plated on malt extract plates to inducesporulation. After 36 to 48 h, the presence of asci was confirmed under the lightmicroscope, and tetrad analysis was performed by using a micromanipulator asdescribed previously (1).

Expression plasmids. The ORF of pdf1� was amplified by PCR with pGPD1as a template, and the amplified product was subcloned into three plasmidvectors—pREP1, pAAU (a gift from Steven Marcus, M. D. Anderson CancerCenter), and pAAL. The pAAL vector was created by replacing the ura4�

marker with LEU2� in pAAU by digestion with HindIII and ligation. Pointmutations were introduced in the pdf1� ORF by using the QuikChange site-directed mutagenesis kit (Stratagene). Oligonucleotide sequences used in theconstruction of point mutations are available upon request. The truncationmutation (�373 to 602) was constructed by PCR with pGPD1 as the template.

Isolation of haploid pdf1� strains complemented by pdf1�. SC41 cells weretransformed with pAAU-pdf1�, and His� and Ura� transformants were selectedon medium lacking adenine, histidine, and uracil. Transformants were induced tosporulate on malt extract plates, the ascus walls were digested with glusulase(Sigma), and the individual spores were germinated on plates lacking histidineand uracil. The resulting colonies were examined as His� Ade� haploids only ifthey were also Ura�, indicating plasmid rescue of the disruption phenotype. Lossof the wild-type allele was confirmed by Southern analysis. The haploid strainswere designated SC41R, SC41U, or SC41L, for strains complemented bypREP1-pdf1�, pAAU-pdf1�, and pAAL-pdf1�, respectively.

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

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

Affinity-purified polyclonal antibodies. Three New Zealand White rabbitswere each immunized with 300 �g of a synthetic peptide corresponding to amino

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

Strain Genotype Source

SC247 h� his3-D1 ade6-M210 ura4-D18 leu1-32 L. RokeachSC248 h� his3-D1 ade6-M216 ura4-D18 leu1-32 L. RokeachSC2478 h�/h� his3-D1/his3-D1 ade6-M210/ade6-M216 ura4-D18/ura4-D18 leu1-32/leu1-32 This studySC41 h�/h� pdf1�::his3�/pdf1� his3-D1/his3-D1 ade6-M210/ade6-M216 ura4-D18/ura4-D18 leu1-32/leu1-32 This studySC41R h� pdf1�::his3� his3-D1 ade6-M210 ura4-D18 leu1-32 pREP1 [pdf1�-LEU2�] This studySC41U h� pdf1�::his3� his3-D1 ade6-M210 ura4-D18 leu1-32 pAAU [pdf1�-ura4�] This studySC41L h� pdf1�::his3� his3-D1 ade6-M210 ura4-D18 leu1-32 pAAL [pdf1�-LEU2�] This study

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acid residues 583 to 603 of Pdf1p that was coupled to keyhole limpet hemocyaninby using methods described previously (43). The antigen was injected intrader-mally in Freund’s complete adjuvant, and rabbits were boosted three times at3-week intervals with 300 �g of peptide in Freund’s incomplete adjuvant. Animmunoglobulin G (IgG) fraction was prepared from preimmune and immuneserum by specific binding to protein A-Sepharose CL-4B (Pharmacia). The IgGwas affinity purified by specific binding to a column consisting of the peptidecross-linked to SulfoLink coupling gel (Pierce).

Protein extraction and immunoblotting. Cells were grown to mid-log phase inEMM with appropriate amino acids. The harvested cells were washed twice withdistilled water, and cell lysates were prepared by glass bead lysis in homogeni-zation buffer (20 mM HEPES [pH 7.0], 50 mM potassium acetate, 5 mM mag-

nesium acetate, 100 mM sorbitol) with the addition of 1 mM dithiothreitol anda cocktail of protease inhibitors (2 �g [each] of leupeptin, pepstatin, and apro-tinin/ml and 1 mM phenylmethylsulfonyl fluoride) (28). Lysates were cleared bycentrifugation at 500 � g for 1 min at 4°C, and resulting supernatants werecentrifuged at 20,000 � g for 30 min at 4°C in a Beckman Optima TLX ultra-centrifuge. The supernatant fraction was reserved and the membrane pellet waswashed once with homogenization buffer and resuspended in 8 M urea forimmunoblotting. Samples were analyzed by electrophoresis in sodium dodecylsulfate (SDS)–12.5% polyacrylamide gel electrophoresis (PAGE) gels and im-munoblotting essentially as described previously (43). Filters were blocked for1 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

FIG. 1. Amino acid sequence alignment (A) and domain structure (B) of proteins encoded by S. pombe pdf1, human PPT1, and mouseDOLPP1. Gaps in the sequences are indicated by dashes. Identical amino acid residues are shaded dark gray; similar residues are light gray. Thelinker region contains two distinct clusters (�) of basic amino acids, which are putative cleavage sites of serine endopeptidase Krp1. Amino acidsthat participate in catalysis and that were mutated to catalytically inactive residues for the purposes of this study are indicated (✶). An acidic linkerregion of 76 amino acids between the Ppt1p and Dolpp1p domains is indicated by a shaded bar.

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rabbit antipeptide polyclonal antibody (0.1 �g/ml). The filters were washed withPBS-T and incubated for 45 min in a solution containing horseradish peroxidase-conjugated secondary antibody (sheep anti-rabbit IgG; Amersham) diluted1:2,500 in PBS-T. The filters were washed and developed with ECL chemilumi-nescence reagents (Amersham).

RESULTSCloning of the fission yeast pdf1 gene. A BLAST search of

the S. pombe genome database with the human PPT1 aminoacid sequence yielded a gene (SPBC530.12c) that encodes a603-amino-acid-residue protein that contains a Ppt1p domain(31% identical to human Ppt1p), a linker region, and a domainthat shares 33% sequence identity to mouse Dolpp1p (Fig. 1).Residues of human Ppt1p and mouse Dolpp1p known to par-ticipate in catalysis (i.e., catalytic triad residues Ser115,Asp233, and His289 of human PPT1) (4) and the consensuslipid-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 tocleavage recognition sites for a kex-related processing pro-tease, Krp1 (40).

Disruption of fission yeast pdf1 is lethal. To investigate thefunction of pdf1, we constructed and examined the phenotypesof S. pombe strains in which pdf1 was deleted. The pdf1 gene is1,812 bp in length and contains no predicted intron sequences(Fig. 2A). One copy of the pdf1 ORF (comprising amino acids69 to 565) was replaced with the his3� selectable marker in adiploid S. pombe strain, and chromosomal deletion of the genewas confirmed by Southern analysis (Fig. 2B), which yields a2.2-kb band in the wild type (lane 1), 2.2- and 1.8-kb bands inheterozygous diploids (lane 2), and a single band of 5.5 kb inthe disruption strain rescued by a plasmid (pREP1-pdf1�) inthe haploid state (lane 3). Correct targeting of the deletioncassette into the genomic locus was also confirmed by PCR asdescribed in Materials and Methods (data not shown). Sporu-lation and tetrad analysis of independently derived heterozy-gous diploid strains (� pdf1::his3�/pdf1�) resulted in only twoviable spores, showing a 2:0 segregation ratio (Fig. 2C). All ofthe viable spores were his� and pdf1�, indicating that pdf1 is anessential gene in S. pombe.

Dolpp1p domain of Pdf1p is essential for viability whereasPpt1p domain is dispensable. To gain a further insight into therole of two domains of pdf1, we analyzed a pdf1� strain com-plemented by various plasmids as shown in Fig. 3. Of note, theanalysis was complicated by an inability to express the S. pombeDolpp1p domain alone in the absence of upstream sequences,presumably due to the need for a signal sequence or othersequences needed for proper Dolpp1p membrane insertionand folding.

A structure-function analysis was performed by using a plas-mid shuffling technique. First, we isolated a haploid strainSC41U 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 containingthe plasmid were viable. The SC41U cells were transformedwith a second plasmid (pAAL) containing a series of mutantconstructs or S. cerevisiae DOLPP1 or human PPT1 cDNAs.Those plasmids included specific point mutants and deletionand nonsense mutants within the pdf1 coding sequences on theplasmid. The growth of each transformant in the presence and

absence of 5-FOA was scored (Fig. 3). As a result, we observedthat plasmids bearing inactivating mutations in the Ppt1p do-main (18) (S106A and D226A) that contained an intactDolpp1p domain complemented the lethal phenotype (Fig. 3,sectors 3 and 4), whereas plasmids truncated before theDolpp1p domain or bearing a mutation in a highly conservedresidue in the phosphate binding pocket of Dolpp1p (47)(H478A) did not rescue the phenotype (Fig. 3, sectors 2, 5, and6). Therefore, it is clear that loss of the Dolpp1p domain isresponsible for the lethal phenotype. Interestingly, neither hu-man Ppt1p nor S. cerevisiae Dolpp1p expression plasmids alonecomplemented the lethal phenotype.

Phenotype of Ppt1p-deficient mutants. We observed thatwhile pdf1� mutants rescued by functional Dolpp1p alone

FIG. 2. Disruption of pdf1�. (A) Restriction map and schematic ofthe pdf1� locus. The direction and location of the pdf1� ORF areindicated. The position of the probe used for Southern blot analysis isindicated as a shaded box. The location of the his3� gene in thepdf1�::his3� disruption construct is shown. An additional HindIII siteis located within the his3� gene. (B) Southern blot analysis of thechromosomal pdf1� deletion. Genomic DNA was digested with Hin-dIII, resolved on a 1% agarose gel, and probed with the 0.3-kb frag-ment 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 analysisof the pdf1� gene disruption. A 2:0 segregation ratio resulted frommicrodissection of heterozygous diploid (pdf1�::his3�/pdf1�) cells af-ter sporulation. The spores that formed colonies in any tetrad werehis� and hence pdf1�.


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were viable, the growth rate of these cells was somewhat slowerthan that of the haploid cells containing the wild-type pdf1�

gene (Fig. 4, left panel) This growth retardation phenotype wasreversed by a second plasmid encoding a wild-type Ppt1p do-main (Fig. 4, right panel) and an inactivated Dolpp1p domain,suggesting that the growth retardation was caused by the lackof Ppt1p function. Therefore, we have further examined thephenotype of these strains. Of note, Ppt1p enzymatic activity inthese strains could not be measured due to interfering activi-ties or substances in the yeast that raised background signals inthe assay.

Ppt1p-deficient mutants are sensitive to vanadate and ele-vated extracellular pH. As Ppt1p is a lysosomal enzyme inmammalian cells and as the fungal vacuole is the equivalentorganelle of the mammalian lysosome, we thought that itwould be of interest to see whether deficiency of Ppt1p func-

tion would lead to phenotypes normally associated with vacu-olar mutants in fungi, such as sensitivity to sodium orthovana-date or external pH (3, 21, 24, 25, 49). As shown in Fig. 5A,cells harboring a wild-type pdf1 gene were not affected byvanadate, whereas cells harboring inactivating mutations inPpt1p are sensitive to vanadate in a concentration-dependentmanner (sectors 2 and 3). This sensitivity was reversed whenthe cells were transformed with a second plasmid containingthe wild-type Ppt1p domain and an inactive Dolpp1p domain(Fig. 5A, center column). Inactivating mutations in the Ppt1pdomain were also associated with an abnormal sensitivity toelevated extracellular pH, as shown in Fig. 5B. At pH 5.5, cellscomplemented with either the wild type or plasmids bearingmutations in the Ppt1p domain grow normally. However, whenthe external pH was elevated above pH 6.0, the haploid cellslacking functional Ppt1p did not grow. This pH sensitivity wasreversed when the cells were transformed with a second plas-mid containing a wild-type Ppt1p domain and an inactiveDolpp1p domain (Fig. 5B, center column). Transformation ofcells with an empty plasmid vector had no effect on the phe-notype (Fig. 5A and B, third column). These results stronglysuggest that Ppt1p activity is required for normal vacuolarfunction in S. pombe.

We also tested for a number of other phenotypes, especiallythose associated with defects in cell wall integrity, as is seen inDolpp1p (CWH8) mutants in S. cerevisiae (10, 51). Ppt1p-deficient cells grew normally in the presence of calcofluorwhite (up to 1 mg/ml), sorbitol or glucose (2 M), potassiumchloride (0.9 M), or glycerol (as a sole carbon source) (data notshown). In addition, staining with the dye FM4-64, which re-veals defects in endocytosis in S. pombe vps mutants (48), wasentirely normal when observed after 30 min of incubation andup to 16 h after incubation (data not shown). The addition ofvanadate (4 mM) or upward adjustment of the extracellular pHto 6.5 for 3 h prior to staining had no effect. Evaluation ofwild-type and Ppt1p-deficient cells by electron microscopy re-vealed no significant differences in vacuolar morphology, andno clear accumulations of granular osmophilic deposits werenoted (data not shown). In addition, heat sensitivity (as as-sessed 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 do-mains. The presence of vacuolar phenotypes in the cells lack-ing Ppt1p function allowed us to demonstrate whether thehuman PPT1 ortholog would substitute for the S. pombe geneto complement these phenotypes. As shown in Fig. 5C, a hu-man PPT1 cDNA reestablished S. pombe cell viability in thepresence of vanadate and at elevated extracellular pH. Theseresults indicate that S. pombe can be used as a model organismfor the study of Batten disease.

Posttranslational processing of Pdf1p. To determinewhether the two functional domains of Pdf1p remain associ-ated as a single polypeptide or whether they are subject toposttranslational proteolytic processing, we made an antibodythat recognizes the carboxyl terminus of Pdf1p and used it toexamine the molecular masses of proteins produced from plas-mid-derived expression of Pdf1p (Fig. 6). Membrane fractionswere prepared from the yeast, and solubilized samples weresubjected to SDS-PAGE and transferred onto nitrocellulosemembranes for immunoblotting. In Fig. 6A, lane 1 (wild type),

FIG. 3. Plasmid shuffling by counterselection on 5-FOA revealsthat the Dolpp1p domain is essential for viability. The ability to growin the presence of 5-FOA indicates complementation of pdf1� by theindicated plasmid in SC41U haploid cells. The schematic (1 to 8)indicates the amino acid sequences encoded by the complementingplasmid with inactivating point mutations, deletions, or nonsense mu-tations 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 werestreaked to EMM plates supplemented only with adenine (�5�FOA)or adenine, uracil, and 5-FOA (�5�FOA). The growth of each trans-formant on the plates was scored. �, growth; �, no growth.

FIG. 4. Growth retardation of cells harboring point mutants in thePpt1p domain. (Left panel) pdf1�::his3� haploids were rescued byplasmids containing wild-type pdf1� or plasmids bearing mutations inactive site residues of the Ppt1p domain (S106A and D226A). (Rightpanel) Rescue of growth retardation by a second plasmid containing awild-type Ppt1p domain and catalytically inactive Dolpp1p domain.Serial 10-fold dilutions were spotted onto plates containing the rele-vant amino acid supplement.


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one major band at 31 kDa and two minor bands at 101 and 33kDa are seen. The major band corresponds precisely to thepredicted molecular mass of the Dolpp1p domain (in the ab-sence of the linker region), whereas the upper minor band at101 kDa corresponds to the full-length unprocessed Pdf1p.The Pdf1p linker region contains two putative cleavage sitesfor 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 fromto the processed form. However, mutation of the dibasic motifat the internal site (R354A) (Fig. 6B and C) essentially abol-ished cleavage (Fig. 6A, lane 3). Mutation of the dibasic motifsat both the primary and internal sites also prevented the pro-cessing of Pdf1p and increased the level of precursor Pdf1p(Fig. 6A, lane 4). These results suggest that Pdf1p is proteo-lytically cleaved to form distinct Ppt1p and Dolpp1p domainsand that Arg354 is crucial for posttranslational processing.

DISCUSSION

In the present study, we demonstrated that an ortholog ofhuman PPT1 is present in the genome of S. pombe as a fusiongene with a partner, DOLPP1. The entire ORF of the pdf1gene is translated as a unit and subjected to posttranslationalproteolytic processing. Mutation at either one of two consen-sus sequences for the endopeptidase Krp1 inhibits processing,whereas mutation at both sites abolishes it. We found thatDolpp1p function is absolutely essential for viability in S.pombe. However, lack of Ppt1p function causes hypersensitiv-ity to elevated extracellular pH and to sodium orthovanadate.The human PPT1 cDNA complements these phenotypes, sug-gesting that the function of PPT1 has been conserved through-out evolution.

Abnormalities in lysosomal pH homeostasis in the juvenile-onset form of Batten disease were first uncovered through thestudy 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 muta-tions in CLN3, which encodes a multimembrane-spanning in-trinsic lysosomal membrane protein of unknown function (27).The orthologous S. cerevisiae gene BTN1 is an intrinsic vacu-olar protein. In contrast to Ppt1p-deficient S. pombe, BTN1�cells do not exhibit an overt phenotype referable to defectivevacuolar function (in over 100 experimental conditionsscreened) (37), with the exception that the cells are resistant toa primary amine growth inhibitor, D-(�)-threo-2-amino-1[p-nitrophenyl]-1, 3-propanediol (ANP). The ANP resistance of

reversed when the cells were transformed with a second plasmid con-taining a wild-type PPT domain and an inactivating point mutation inthe Dolpp1p domain (sectors 4 to 6). This suggests that the pH sen-sitivity is due solely to the absence of wild-type Ppt1p activity in thecells. Note that even normal S. pombe cells were unable to grow at pH7.0. Empty vector controls are shown in sectors 7 to 9. (C) The humanPPT1 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 werestreaked onto plates containing either 4 mM sodium orthovanadate ormedium buffered to pH 6.5.

FIG. 5. Sensitivity of PPT-deficient cells to sodium orthovanadateand to extracellular pH. (A) Normally inviable pdf1�::his3� haploidsrescued by wild-type pAAL-pdf1� (1), mutated pAAL-pdf1(S106A)(2), or pAAL-pdf1(D226A) plasmids with mutations in the Ppt1pdomain (3) were exposed to various concentrations of sodium or-thovanadate for 4 days at 30°C. Mutations in the Ppt1p domain con-ferred sensitivity to sodium orthovanadate in a concentration-depen-dent manner. The sensitivity was reversed when the cells weretransformed with a second plasmid, pAAU-pdf1(H478A), containingwild-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 tothe indicated pH. Ppt1p-inactivating mutations conferred an abnormalsensitivity to high pH to the cells. The sensitivity to higher pH was


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BTN1� cells was shown to be related to a subtly enhancedacidification of the external medium, and BTN1� strains havea slightly more acidic vacuolar pH in early phases of growth(37). The human CLN3 cDNA complements this phenotypeand substitutes functionally for BTN1 (7, 36, 39). Observationsof more robust phenotypes in Ppt1p versus Cln3p deficiencyappear to hold for yeast, mice (12, 22, 30), and humans (11).

The phenotypes we observed in Ppt1p loss-of-function mu-tations 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 previouslydescribed for the vma (vacuolar membrane ATPase) and classC and D vps (vacuolar protein sorting) mutants of S. cerevisiae(2, 21, 33, 34, 41, 56) and vps33 mutants of S. pombe. vmamutants carry mutations in subunits of the vacuolar H�-ATPase (an enzyme responsible for the acidification of vacu-olar contents and maintenance of intracellular pH), and class Cand class D vps mutants display defects in vacuole morphogen-esis (41, 42). These mutants are also sensitive to environmentalmanipulations such as external pH and sodium orthovanadate(3, 21, 24, 25, 49). Therefore, our data for S. pombe suggestthat Ppt1p functions in the vacuole just as it functions in theanalogous structure (lysosome) of mammalian cells.

The observed proteolytic processing of the Pdf1p fusionprotein of S. pombe is also entirely consistent with the knownsubcellular compartmentalization of Ppt1p and Dolpp1p inmammalian cells. In higher organisms, Ppt1p is a residentsoluble lysosomal enzyme that traffics through the classicalmannose 6-phosphate receptor pathway (17, 52), whereasDolpp1p is a resident ER protein (10, 43). Therefore, our dataare consistent with a model that, for S. pombe, the Pdf1p fusionprotein is directed into the secretory pathway by virtue of thePpt1p signal sequence, with the appearance of the Ppt1p do-main on the lumenal side, followed by cotranslational incor-poration of Dolpp1p into the ER membrane. The fusion pro-tein would then become available for processing by Krp1 (or arelated enzyme), and the Ppt1p domain would be free fortransport to the vacuole.

Whether Pdf1p is indeed a physiological substrate for Krp1premains to be determined experimentally. Deletion of Krp1 inS. pombe is lethal (40), and as deletion of Dolpp1p is alsolethal, one may speculate that lack of processing of Dolpp1p byKrp1 is responsible for the lethality in Krp1 null strains. Ofnote, Dolpp1p processing mutants created in our study wereviable, but the mutants appeared to be leaky because smallamounts of processed Ppt1p were detectable upon longer ex-posures.

The translation of Pdf1p as a single polypeptide suggeststhat there may be an advantage in coordinately regulatinglevels of Ppt1p and Dolpp1p in S. pombe. In mammalian cells,a number of lysosomal enzymes are coordinately upregulatedin a compensatory fashion under conditions of lysosomal dys-function (6, 26, 50). Dolpp1p is postulated to participate in therecycling of dolichols following transfer of the oligosaccharidechain from DolPP-OS to nascent proteins in the biosyntheticpathway (45). As DolPP-OS accumulate to some degree in anumber of lysosomal storage diseases (reviewed in reference54), it is plausible that the initial steps of DolPP-OS degrada-tion occur in the lysosome and that Dolpp1p, though not alysosomal enzyme per se, is upregulated in response to theneed to recycle dolichol under conditions of increased lysoso-mal metabolic demand.

Studies in lower organisms, particularly yeast, have providedsurprising insights into human biology, even in the area ofhuman neurodegenerative disease (46). The two yeast modelsof Batten disease have provided evidence for abnormal vacu-olar function due to lack of the relevant disease proteins andhave begun to implicate abnormalities in pH homeostasis inthe pathogenesis of these disorders, with implications for ther-

A

208

124

101

55

352921

1 2 3 4

WT

R34

4AR

354A

R34

4,35

4A

kDa

Pdf1p

Dolpp1p

FIG. 6. Posttranslational processing of Pdf1p at dibasic cleavagesites. (A) Membrane fractions of cell lysates were prepared, separatedby SDS-PAGE, and transferred to nitrocellulose for immunoblottingwith an antibody that recognizes the carboxyl terminus of Pdf1p. Cellswere expressing either wild-type Pdf1p or Pdf1p that contained pointmutations in the linker domain (R344A, R354A, or both mutations).Substitution of alanine for arginine at position 354 blocks the proteo-lytic cleavage, suggesting that arginine 354 is crucial for the posttrans-lational processing. (B) Schematic of Pdf1p cleavage sites. Two clustersof dibasic motifs, KR344 and RKR354, are indicated. (C) Comparison ofknown 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 aminoacids between the two sites is smaller in Pdf1 (10 versus 20). Inaddition, the arginine in the primary site of Krp1 is replaced by leucinein Ppt1p.


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apy. Future work in these genetically tractable organisms maytherefore provide further insights into the pathogenesis of Bat-ten Disease.

ACKNOWLEDGMENTS

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.

REFERENCES

1. Alfa, C., P. Fantes, J. Hyams, M. McLeod, and E. Warbrick (ed.). 1993.Experiments with fission yeast: a laboratory course manual. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.

2. Bankaitis, V. A., L. M. Johnson, and S. D. Emr. 1986. Isolation of yeastmutants defective in protein targeting to the vacuole. Proc. Natl. Acad. Sci.USA 83:9075–9079.

3. Banta, L. M., J. S. Robinson, D. J. Klionsky, and S. D. Emr. 1988. Organelleassembly in yeast: characterization of yeast mutants defective in vacuolarbiogenesis and protein sorting. J. Cell Biol. 107:1369–1383.

4. Bellizzi, J. J., III, J. Widom, C. Kemp, J. Y. Lu, A. K. Das, S. L. Hofmann,and J. Clardy. 2000. The crystal structure of palmitoyl protein thioesterase 1and the molecular basis of infantile neuronal ceroid lipofuscinosis. Proc.Natl. Acad. Sci. USA 97:4573–4578.

5. Burke, J. D., and K. L. Gould. 1994. Molecular cloning and characterizationof the Schizosaccharomyces pombe his3 gene for use as a selectable marker.Mol. Gen. Genet. 242:169–176.

6. Cataldo, A. M., P. A. Paskevich, E. Kominami, and R. A. Nixon. 1991.Lysosomal hydrolases of different classes are abnormally distributed inbrains of patients with Alzheimer disease. Proc. Natl. Acad. Sci. USA 88:10998–11002.

7. Chattopadhyay, S., N. E. Muzaffar, F. Sherman, and D. A. Pearce. 2000. Theyeast model for batten disease: mutations in BTN1, BTN2, and HSP30 alterpH homeostasis. J. Bacteriol. 182:6418–6423.

8. Davey, J., K. Davis, Y. Imai, M. Yamamoto, and G. Matthews. 1994. Isolationand characterization of krp, a dibasic endopeptidase required for cell via-bility in the fission yeast Schizosaccharomyces pombe. EMBO J. 13:5910–5921.

9. Davis, L., and G. R. Smith. 2001. Meiotic recombination and chromosomesegregation in Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. USA98:8395–8402.

10. Fernandez, F., J. S. Rush, D. A. Toke, G. S. Han, J. E. Quinn, G. M. Carman,J. Y. Choi, D. R. Voelker, M. Aebi, and C. J. Waechter. 2001. The CWH8gene encodes a dolichyl pyrophosphate phosphatase with a luminally ori-ented active site in the endoplasmic reticulum of Saccharomyces cerevisiae.J. Biol. Chem. 276:41455–41464.

11. Goebel, H. H. 1995. The neuronal ceroid-lipofuscinoses. J. Child Neurol.10:424–437.

12. Gupta, P., A. A. Soyombo, A. Atashband, K. E. Wisniewski, J. M. Shelton,J. A. Richardson, R. E. Hammer, and S. L. Hofmann. 2001. Disruption ofPPT1 or PPT2 causes neuronal ceroid lipofuscinosis in knockout mice. Proc.Natl. Acad. Sci. USA 98:13566–13571.

13. Hall, N., and A. Patrick. 1988. Accumulation of dolichol-linked oligosaccha-rides. Am. J. Med. Genet. 5:221–232.

14. Hall, N. A., and A. D. Patrick. 1987. Accumulation of phosphorylated doli-chol in several tissues in ceroid-lipofuscinosis (Batten disease). Clin. Chim.Acta 170:323–330.

15. Hall, N. A., and A. D. Patrick. 1985. Dolichol and phosphorylated dolicholcontent of tissues in ceroid-lipofuscinosis. J. Inherit. Metab. Dis. 8:178–183.

16. Hall, N. A., J. E. Thomas-Oates, A. Dell, M. Haltia, B. D. Lake, and A. D.Patrick. 1992. Stored dolichyl pyrophosphoryl oligosaccharides in Battendisease. Am. J. Med. Genet. 42:580–585.

17. Hellsten, E., J. Vesa, V. M. Olkkonen, A. Jalanko, and L. Peltonen. 1996.Human palmitoyl protein thioesterase: evidence for lysosomal targeting ofthe enzyme and disturbed cellular routing in infantile neuronal ceroid lipo-fuscinosis. EMBO J. 15:5240–5245.

18. Hofmann, S. L., A. Atashband, S. K. Cho, A. K. Das, P. Gupta, and J.-Y. Lu.2002. Neuronal ceroid lipofuscinoses caused by defects in soluble lysosomalenzymes (CLN1 and CLN2). Curr. Mol. Med. 2:423–437.

19. Hofmann, S. L., and L. Peltonen. 2001. The neuronal ceroid lipofuscinoses,p. 3877–3894. In C. R. Scriver, A. L. Beaudet, W. S. Sly, B. Childs, and B.Vogelstein (ed.), The metabolic and molecular bases of inherited disease,vol. 8. McGraw-Hill, New York, N.Y.

20. Humphrey, T. 2000. DNA damage and cell cycle control in Schizosaccharo-myces pombe. Mutat. Res. 451:211–226.

21. Iwaki, T., F. Osawa, M. Onishi, T. Koga, Y. Fujita, A. Hosomi, N. Tanaka,Y. Fukui, and K. Takegawa. 2003. Characterization of vps33�, a gene re-quired for vacuolar biogenesis and protein sorting in Schizosaccharomycespombe. Yeast 20:845–855.

22. Katz, M. L., H. Shibuya, P. C. Liu, S. Kaur, C. L. Gao, and G. S. Johnson.

1999. A mouse gene knockout model for juvenile ceroid-lipofuscinosis (bat-ten disease). J. Neurosci. Res. 57:551–556.

23. Keller, R. K., D. Armstrong, F. C. Crum, and N. Koppang. 1984. Dolicholand dolichyl phosphate levels in brain tissue from English setters with ceroidlipofuscinosis. J. Neurochem. 42:1040–1047.

24. Kitamoto, K., K. Yoshizawa, Y. Ohsumi, and Y. Anraku. 1988. Mutants ofSaccharomyces cerevisiae with defective vacuolar function. J. Bacteriol. 170:2687–2691.

25. Klionsky, D. J., P. K. Herman, and S. D. Emr. 1990. The fungal vacuole:composition, function, and biogenesis. Microbiol. Rev. 54:266–292.

26. Kopacek, J., S. Sakaguchi, K. Shigematsu, N. Nishida, R. Atarashi, R.Nakaoke, R. Moriuchi, M. Niwa, and S. Katamine. 2000. Upregulation of thegenes encoding lysosomal hydrolases, a perforin-like protein, and peroxi-dases in the brains of mice affected with an experimental prion disease.J. Virol. 74:411–417.

27. Lerner, T. J., R. N. Boustany, J. W. Anderson, K. D. D’Arigo, K. Schlumpf,A. J. Buckler, J. F. Gusella, J. L. Haines, G. Kremmidiotis, I. L. Lensink,G. R. Sutherland, D. F. Callen, P. E. M. Taschner, N. deVos, G.-J. B. vanOmmen, M. H. Breuning, N. A. Doggett, L. J. Meincke, Z.-Y. Liu, L. A.Goodwin, J. G. Tesmer, H. M. Mitchison, A. M. O’Rawe, P. B. Munroe, I. E.Jarvela, R. M. Gardiner, and S. E. Mole. 1995. Isolation of a novel geneunderlying Batten disease, CLN3 (The International Batten Disease Con-sortium). Cell 82:949–957.

28. Liang, D. T., and S. L. Forsburg. 2001. Characterization of Schizosaccharo-myces pombe mcm7(�) and cdc23(�) (MCM10) and interactions with rep-lication checkpoints. Genetics 159:471–486.

29. Lu, J. Y., L. A. Verkruyse, and S. L. Hofmann. 1996. Lipid thioesters derivedfrom acylated proteins accumulate in infantile neuronal ceroid lipofuscino-sis: correction of the defect in lymphoblasts by recombinant palmitoyl-pro-tein thioesterase. Proc. Natl. Acad. Sci. USA 93:10046–10050.

30. Mitchison, H. M., D. J. Bernard, N. D. Greene, J. D. Cooper, M. A. Junaid,R. K. Pullarkat, N. de Vos, M. H. Breuning, J. W. Owens, W. C. Mobley,R. M. Gardiner, B. D. Lake, P. E. Taschner, and R. L. Nussbaum. 1999.Targeted disruption of the Cln3 gene provides a mouse model for Battendisease. Neurobiol. Dis. 6:321–334.

31. Mitchison, H. M., and S. E. Mole. 2001. Neurodegenerative disease: theneuronal ceroid lipofuscinoses (Batten disease). Curr. Opin. Neurol. 14:795–803.

32. Moreno, S., A. Klar, and P. Nurse. 1991. Molecular genetic analysis of fissionyeast Schizosaccharomyces pombe. Methods Enzymol. 194:795–823.

33. Nelson, H., and N. Nelson. 1990. Disruption of genes encoding subunits ofyeast vacuolar H(�)-ATPase causes conditional lethality. Proc. Natl. Acad.Sci. USA 87:3503–3507.

34. Oluwatosin, Y. E., and P. M. Kane. 1998. Mutations in the yeast KEX2 genecause a Vma(-)-like phenotype: a possible role for the Kex2 endoprotease invacuolar acidification. Mol. Cell. Biol. 18:1534–1543.

35. Pearce, D. A. 2000. Localization and processing of CLN3, the protein asso-ciated to Batten disease: where is it and what does it do? J. Neurosci. Res.59:19–23.

36. Pearce, D. A., C. J. Carr, B. Das, and F. Sherman. 1999. Phenotypic reversalof the btn1 defects in yeast by chloroquine: a yeast model for Batten disease.Proc. Natl. Acad. Sci. USA 96:11341–11345.

37. Pearce, D. A., T. Ferea, S. A. Nosel, B. Das, and F. Sherman. 1999. Actionof BTN1, the yeast orthologue of the gene mutated in Batten disease. Nat.Genet. 22:55–58.

38. Pearce, D. A., and F. Sherman. 1997. BTN1, a yeast gene corresponding tothe human gene responsible for Batten’s disease, is not essential for viability,mitochondrial function, or degradation of mitochondrial ATP synthase.Yeast 13:691–697.

39. Pearce, D. A., and F. Sherman. 1998. A yeast model for the study of Battendisease. Proc. Natl. Acad. Sci. USA 95:6915–6918.

40. Powner, D., and J. Davey. 1998. Activation of the kexin from Schizosaccha-romyces pombe requires internal cleavage of its initially cleaved prose-quence. Mol. Cell. Biol. 18:400–408.

41. Robinson, J. S., D. J. Klionsky, L. M. Banta, and S. D. Emr. 1988. Proteinsorting in Saccharomyces cerevisiae: isolation of mutants defective in thedelivery and processing of multiple vacuolar hydrolases. Mol. Cell. Biol.8:4936–4948.

42. Rothman, J. H., and T. H. Stevens. 1986. Protein sorting in yeast: mutantsdefective in vacuole biogenesis mislocalize vacuolar proteins into the latesecretory pathway. Cell 47:1041–1051.

43. Rush, J. S., S. K. Cho, S. Jiang, S. L. Hofmann, and C. J. Waechter. 2002.Identification and characterization of a cDNA encoding a dolichyl pyrophos-phate phosphatase located in the endoplasmic reticulum of mammalian cells.J. Biol. Chem. 277:45226–45234.

44. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

45. Schenk, B., F. Fernandez, and C. J. Waechter. 2001. The ins(ide) and out-(side) of dolichyl phosphate biosynthesis and recycling in the endoplasmicreticulum. Glycobiology 11:61R–70R.

46. Sherman, M. Y., and P. J. Muchowski. 2003. Making yeast tremble: yeast


on Novem


.org/D

ownloaded from

http://ec.asm.org/

models as tools to study neurodegenerative disorders. Neuromolecular Med.4:133–146.

47. Studdert, V. P., and R. W. Mitten. 1991. Clinical features of ceroid lipofus-cinosis in border collie dogs. Aust. Vet. J. 68:137–140.

48. Takegawa, K., D. B. DeWald, and S. D. Emr. 1995. Schizosaccharomycespombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for nor-mal cell growth and vacuole morphology. J. Cell Sci. 108(Pt 12):3745–3756.

49. Takeshige, K., M. Baba, S. Tsuboi, T. Noda, and Y. Ohsumi. 1992. Autoph-agy in yeast demonstrated with proteinase-deficient mutants and conditionsfor its induction. J. Cell Biol. 119:301–311.

50. Tyynela, J., I. Sohar, D. E. Sleat, R. M. Gin, R. J. Donnelly, M. Baumann, M.Haltia, and P. Lobel. 2000. A mutation in the ovine cathepsin D gene causesa congenital lysosomal storage disease with profound neurodegeneration.EMBO J. 19:2786–2792.

51. van Berkel, M. A., M. Rieger, S. te Heesen, A. F. Ram, H. van den Ende, M.Aebi, and F. M. Klis. 1999. The Saccharomyces cerevisiae CWH8 gene isrequired for full levels of dolichol-linked oligosaccharides in the endoplasmicreticulum and for efficient N-glycosylation. Glycobiology 9:243–253.

52. Verkruyse, L. A., and S. L. Hofmann. 1996. Lysosomal targeting of palmitoyl-protein thioesterase. J. Biol. Chem. 271:15831–15836.

53. Vesa, J., E. Hellsten, L. A. Verkruyse, L. A. Camp, J. Rapola, P. Santavuori,S. L. Hofmann, and L. Peltonen. 1995. Mutations in the palmitoyl proteinthioesterase gene causing infantile neuronal ceroid lipofuscinosis. Nature376:584–587.

54. Wolfe, L. S., S. Gauthier, M. Haltia, and J. Palo. 1988. Dolichol and dolichylphosphate in the neuronal ceroid-lipofuscinoses and other diseases. Am. J.Med. Genet. Suppl. 5:233–242.

55. Wood, V., R. Gwilliam, M. A. Rajandream, M. Lyne, R. Lyne, A. Stewart, J.Sgouros, N. Peat, J. Hayles, S. Baker, D. Basham, S. Bowman, K. Brooks, D.Brown, S. Brown, T. Chillingworth, C. Churcher, M. Collins, R. Connor, A.Cronin, P. Davis, T. Feltwell, A. Fraser, S. Gentles, A. Goble, N. Hamlin, D.Harris, J. Hidalgo, G. Hodgson, S. Holroyd, T. Hornsby, S. Howarth, E. J.Huckle, S. Hunt, K. Jagels, K. James, L. Jones, M. Jones, S. Leather, S.McDonald, J. McLean, P. Mooney, S. Moule, K. Mungall, L. Murphy, D.Niblett, C. Odell, K. Oliver, S. O’Neil, D. Pearson, M. A. Quail, E. Rabbi-nowitsch, K. Rutherford, S. Rutter, D. Saunders, K. Seeger, S. Sharp, J.Skelton, M. Simmonds, R. Squares, S. Squares, K. Stevens, K. Taylor, R. G.Taylor, A. Tivey, S. Walsh, T. Warren, S. Whitehead, J. Woodward, G.Volckaert, R. Aert, J. Robben, B. Grymonprez, I. Weltjens, E. Vanstreels, M.Rieger, M. Schafer, S. Muller-Auer, C. Gabel, M. Fuchs, A. Dusterhoft, C.Fritzc, E. Holzer, D. Moestl, H. Hilbert, K. Borzym, I. Langer, A. Beck, H.Lehrach, R. Reinhardt, T. M. Pohl, P. Eger, W. Zimmermann, H. Wedler, R.Wambutt, B. Purnelle, A. Goffeau, E. Cadieu, S. Dreano, S. Gloux, et al.2002. The genome sequence of Schizosaccharomyces pombe. Nature 415:871–880.

56. Yoshida, S., and Y. Anraku. 2000. Characterization of staurosporine-sensi-tive mutants of Saccharomyces cerevisiae: vacuolar functions affect stauro-sporine sensitivity. Mol. Gen. Genet. 263:877–888.


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