Báo cáo khoa học: ERS1 encodes a functional homologue of the human lysosomal cystine transporter pptx

ERS1 encodes a functional homologue of the human lysosomal cystine transporter Xiao-Dong Gao 1 , Ji Wang 2 , Sabine Keppler-Ross 2 and Neta Dean 2 1 Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan 2 Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, USA Cystinosis is a lysosomal storage disease whose hall- mark is the accumulation of cystine in the lysosome [1,2]. During the normal degradation of proteins in the lysosome, cystine, the disulfide-linked form of cysteine, is not reduced in the oxidating environment of the lysosome but must be transported to the cytoplasm where it is reduced. Cystine is very insoluble and a defect in its efflux causes it to form crystals. Cystine is normally transported from the lysosome to the cyto- plasm by cystinosin, a lysosomal membrane protein encoded by the CTNS gene. Cystinosin is a proton symporter, coupling cystine transport with protons generated by the vacuolar (H + )-ATPase [3]. Thus cys- tine transport removes both cystine to the reducing environment of the cytoplasm, along with protons whose luminal accumulation may have an overly acidi- fying effect on the lysosome. A number of different mutations have been charac- terized from cystinosis patients, almost all of which map to the CTNS gene [3]. Several forms of this auto- somal recessive disease occur, which differ in both severity and age of onset. The most severe form is infantile nephropathic cystinosis. These individuals are normal at birth, but within several months develop progressively severe nephratic disorders that culminate in renal failure by the age of 10 years. In this form of the disease, small molecules fail to be re-absorbed in the renal tubules, resulting in excessive urinary loss of vital components. This generalized renal tubule dis- order, known as Fanconi syndrome, leads to growth Keywords cystinosin; ERS1; GTR1; MEH1; vacuole Correspondence N. Dean, Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794- 5215, USA Fax: +1 631 632 8575 Tel: +1 631 632 9309 E-mail: Neta.Dean@stonybrook.edu (Received 5 November 2004, revised 11 March 2005, accepted 18 March 2005) doi:10.1111/j.1742-4658.2005.04670.x Cystinosis is a lysosomal storage disease caused by an accumulation of insoluble cystine in the lumen of the lysosome. CTNS encodes the lyso- somal cystine transporter, mutations in which manifest as a range of disorders and are the most common cause of inherited renal Fanconi syndrome. Cystinosin, the CTNS product, is highly conserved among mam- mals. Here we show that the yeast Ers1 protein and cystinosin are func- tional orthologues, despite sharing only limited sequence homology. Ers1 is a vacuolar protein whose loss of function results in growth sensitivity to hygromycin B. This phenotype can be complemented by the human CTNS gene but not by mutant ctns alleles that were previously identified in cysti- nosis patients. A genetic screen for multicopy suppressors of an ers1D yeast strain identified a novel gene, MEH1, which is implicated in regulating Ers1 function. Meh1 localizes to the vacuolar membrane and loss of MEH1 results in a defect in vacuolar acidification, suggesting that the vac- uolar environment is critical for normal ERS1 function. This genetic sys- tem has also led us to identify Gtr1 as an Meh1 interacting protein. Like Meh1 and Ers1, Gtr1 associates with vacuolar membranes in an Meh1- dependent manner. These results demonstrate the utility of yeast as a model system for the study of CTNS and vacuolar function. Abbreviations ER, Endoplasmic reticulum; GFP, green fluorescent protein; HA, haemagglutinin; hygB, hygromycin B. FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS 2497 retardation, hypothyroidism, photophobias, and neuro- logical dysfunctions if untreated. These individuals appear to be null mutants with a complete loss of CTNS function. A less severe form is juvenile cystino- sis. In addition to having much milder renal problems, these individuals also suffer ocular disorders, such as light sensitivity or retinal blindness caused by cystine crystal deposits in the cornea of the eyes. This ocular, non-nephropathic form of cystinosis characterizes the mildest form of the disease in individuals whose onset occurs as adults. The mutations in these individuals suggests that these milder forms represent partial loss- of-function CTNS mutations [4]. The only proven therapeutic agent that exists for the treatment of cysti- nosis is cysteamine, a membrane permeable reagent that reduces cystine to produce cysteine and cysteam- ine:cystine, whose exit from the lysosome occurs via a lysine transporter [5,6]. Cysteamine is limited in its applications because its efficacy as a therapeutic requires early functional diagnosis and because it is difficult to administer [7,8]. The human CTNS gene product is highly conserved amongst all mammals but shows more limited similar- ity to the Saccaromyces cerevisiae Ers1protein (28% identical ⁄ 46% similar). ERS1 was isolated as a gene that when over expressed can suppress the secretion of resident endoplasmic reticulum (ER) proteins in erd1D mutants [9]. Yeast strains lacking ERD1 display a number of pleiotropic Golgi defects, including the secretion of ER proteins that are normally retrieved from the Golgi and the underglycosylation of proteins normally modified in the Golgi [10]. Neither the pre- cise function of ERD1 nor the mechanism of ERS1- mediated suppression of erd1D is known. Although ERS1 is related in sequence to CTNS, there are some notable differences in their gene prod- ucts. Both genes encode membrane proteins that are predicted to contain seven membrane-spanning domains, reminiscent of G-protein-coupled receptors. However, cystinosin differs from Ers1p in that it con- tains an extended N-terminal domain of 121 amino acids predicted to face the lumen [11]. Furthermore, unlike CTNS mutants, loss of ERS1 in yeast leads to no detectable growth phenotypes. These differences in both protein sequence and mutant phenotype have raised the question of whether or not these two pro- teins perform similar functions. Here we show that CTNS and ERS1 are indeed orthologous genes. The Ers1 protein localizes to the endosomes and yeast vacuole, an organelle that is functionally equivalent to the mammalian lysosome. Although ers1D mutant strains are not defective in growth, we identify a drug phenotype, hygromycin B (hygB) sensitivity, which can be reversed by the human CTNS gene but not by mutant CTNS alleles identified in cystinosis patients. A screen for genes that when overexpressed can sup- press the drug sensitivity of ers1D strains has led to the identification of MEH1, a novel gene product that is implicated in the regulation of vacuolar function. We also identify Gtr1, a conserved GTPase whose interaction with Meh1 is required for Gtr1 vacuolar localization. These results demonstrate that yeast serves as a useful model for the study of CTNS. Results The mammalian CTNS lysosomal H + -driven transporter encodes a functional homologue of ERS1 Yeast strains lacking ERS1 exhibit no detectable growth phenotypes but are sensitive to the aminoglyco- side, hygB (Fig. 1). The ERS1 gene was deleted by replacement with TRP1. Tetrad dissection of over Fig. 1. CTNS encodes a functional homologue of ERS1. Isogenic wild type (SEY6210) and ers1D cells (XGY51) expressing either the human CTNS cDNA or the ERS1 gene under the control of the ERS1 promoter, were streaked on YPAD plates in the presence or absence of 50 lgÆmL )1 hygB and grown for 2 days at 30 °C. ERS1 and CTNS are functional homologues X D. Gao et al. 2498 FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS 20ERS1 ⁄ ers1D::TRP1 diploids demonstrated complete cosegregation of hygB sensitivity and tryptophan pro- totrophy (data not shown). Further, hygB sensitivity can be completely complemented by expression of the normal ERS1 gene (Fig. 1), demonstrating this drug phenotype is a direct consequence of the ERS1 dele- tion. HygB sensitivity has been described for many yeast mutants, including those with defects in cell wall biosynthesis, glycosylation, ion transport, and vacuolar function (e.g. [12–15]). ers1D strains exhibit no detect- able cell wall or glycosylation phenotypes (data not shown). Although the basis for this complex drug sen- sitivity is not understood, the identification of a pheno- type associated with loss of ERS1 function provided a system to analyse the relationship between CTNS and ERS1. To ask if CTNS is a functional homologue of ERS1, we took advantage of the hygB sensitive phenotype to examine whether or not it can be complemented by CTNS. The human CTNS gene contains 11 introns. To analyse complementation of ers1D in yeast, the human CTNS cDNA was cloned into a yeast expres- sion vector that places CTNS expression under the control of the ERS1 promoter (see Experimental pro- cedures). Under these conditions, the human CTNS gene completely complemented the hygB sensitive growth phenotype of an ers1D strain, in a manner equivalent to its complementation by ERS1, demon- strating that cystinosin functions in yeast (Fig. 1). CTNS mutants fail to complement ers1D strains To further confirm the functional conservation between ERS1 and CTNS, a series of mutant CTNS genes was constructed, and their functional activities were monitored by the complementation of hygB sensi- tive growth phenotype of ers1D cells. Mutations were introduced into CTNS that correspond to well-charac- terized mutations that have been repeatedly isolated from cystinosin patients. First, two missense mutations were introduced that had been identified in patients with infantile nephropathic cystinosis [4]. The glycine at position 308 was replaced by an arginine (ctns- G308R) and the leucine at position 338 was replaced by a proline (ctns-L338P). These amino acids, G308 and L338, lie within regions of cystinosin that are among the most highly conserved, within the sixth (G308) or seventh (L338) membrane spanning domains (see Fig. 2A). These membrane spanning domains have been postulated to be important for cystinosin function [4]. Two additional mutations were generated that encode truncated forms of cystinosin. The first of these is ctns-ND121, which lacks the first 121 amino acids at the NH 2 -terminus of cystinosin (Fig. 2A). This domain is present in cystinosin but completely absent in Ers1. This region is also the least conserved among CTNS orthologues from other species, including birds, worms, flies, mosquitos, and rats (Fig. 2A), so it was of interest to examine the functional consequence of its deletion. The second mutant is ctns-CD82, which lacks the last 82 amino acids at the COOH-terminus of cys- tinosin. This C-terminal domain of CTNS encompasses the sixth and seventh predicted transmembrane regions of cystinosin that have been implicated as functionally important [4]. This C-terminal deletion is analogous to several deletion mutations found in severe infantile nephropathic cystinosis patients [4]. These mutant CTNS alleles were expressed in an ers1D strain and assayed quantitatively for complemen- tation of its hygB sensitive growth phenotype. Neither ctns-G308R nor ctns-L338P alleles can complement the hygB sensitivity of an ers1D strain at levels observed by the wild type CTNS (Fig. 2B). Both the G308R and the L338P mutations were almost 100-fold less efficient for ers1D complementation than the wild type CTNS. Thus, these missense mutations in CTNS mimic their affect in humans when expressed in yeast. The most severe mutant phenotype was seen in the ctns-CD82 allele, which completely failed to comple- ment ers1D. In contrast, the ctns-ND121 allele had no effect and complemented ers1D in a manner equivalent to the wild type CTNS gene (Fig. 2B). These results suggest that the sixth and seventh transmembrane domains in both CTNS and ERS1 are probably essen- tial for protein function, while the N-terminal 121 amino acid domain of cystinosin is dispensable. A trivial explanation for the failure of these mutant ctns alleles to complement ers1D is that these muta- tions grossly perturb protein structure and lead to its instability. To determine if the lack of complementa- tion by mutant ctns alleles was due to reduced levels of cystinosin protein, these mutant alleles were tagged with sequences encoding the haemagglutinin (HA) epi- tope to compare levels of protein expression. Neither Ers1 nor cystinosin could be detected by Western blot analysis of whole cell extracts, suggesting that neither of these proteins normally accumulate to high steady- state levels. As described below, these HA-tagged pro- teins could be readily detected after enrichment from vacuolar membrane fractions, sedimented by centrifu- gation at 16 000 g (see Experimental procedures). Equivalent amounts of protein from these fractions were separated by SDS ⁄ PAGE and immunoblotted with antibodies against HA. Ers1 has a predicted molecular mass of  30 kDa and contains one recogni- tion site for N-linked glycosylation, in the lumenal X D. Gao et al. ERS1 and CTNS are functional homologues FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS 2499 domain between the sixth and seventh membrane span- ning regions. Ers1 migrated with molecular weight markers in this range as a slightly heterogeneous smear, as expected for a glycosylated protein (Fig. 2C). Cystinosin is predicted to have a molecular mass of 41.7 kDa and in addition, has seven predicted recog- nition sites for N-linked glycosylation. Most of the cystinosin migrated as a large heterogeneous smear, A BC Fig. 2. Mutant forms of CTNS fail to complement ers1D. (A) Schematic diagram of the predicted topology of cystinosin. Magenta and blue dots represent those amino acids that are invariant in an alignment of representative cystinosin-related proteins from humans (AAH32850.1), birds (Gallus gallus; XP_415851.1), flies (Drosophila melanogaster; AAM50956.1), mosquitoes (Anopheles gambiae XP_312994.1), worms (Caenorhabditis elegans NP_495704.1) and yeast (Saccharomyces cerevisiae YCR075C). Mutations that were introduced in this study are denoted in red. ND120 denotes a deletion of the N-terminal 120 amino acids. CD82 denotes a deletion of the C-terminal 82 amino acids, which remove the sixth and seventh membrane spanning domains. (B) The parental wild type (SEY6210) and ers1D (XGY51) strains expres- sing the indicated wild type or mutant alleles of human CTNS under the ERS1 promoter were assayed for complementation of the hygromy- cin B sensitive phenotype of ers1D. Exponentially growing cells were serially diluted (10-fold), spotted onto YPAD plates with or without 50 lgÆmL )1 hygB and grown for 2 days at 30 °C. (C) Whole cell lysates from cells expressing HA-tagged ERS1, CTNS, ctns-D121,or ctnsL338P were subjected to differential centrifugation as described in Experimental procedures and equivalent amounts of protein were separated by SDS ⁄ PAGE and immunoblotted with anti-HA. ERS1 and CTNS are functional homologues X D. Gao et al. 2500 FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS suggesting that the majority of cystinosin is glycosylated when expressed in yeast. An additional band corres- ponding to a molecular mass of  33 kDa was also seen, though its identity has not been further investi- gated. Importantly, strains expressing ctns mutant alle- les expressed altered proteins at levels comparable to the wild type (Fig. 2C and data not shown). The only mutation that markedly reduced steady state levels of cystinosin was the deletion of the N-terminal 121 amino acids. This domain is predicted to face the lumen and contains all of the sites for N-linked glycan addition. Consistent with this prediction, cystinosin- ND120 runs as a single band of about 28 kDa. This protein accumulated at levels lower than the wild type cystinosin, but these levels are apparently sufficient as this mutant allele fully complemented ers1D (Fig. 2B). Both ctns-L338P (Fig. 2C) and ctns-G308R (not shown) expressed protein that comigrated with the wild type although the cystinosin-L338P protein appeared to have a larger proportion of fully glycosyl- ated forms than the wild type. As each of these mutant proteins accumulated to levels that are comparable to the wild type cystinosin, these results suggest that the failure to complement ers1D is not due to reduced pro- tein levels. Ers1 protein localizes in the endosomes and vacuole Cystinosin is a resident lysosomal protein. If cystinosin and Ers1 perform similar functions, a strong prediction is that Ers1 resides in the vacuole, the yeast counter- part of the lysosome. To determine if this is the case, we analysed the intracellular localization of Ers1 in living cells by examining a green fluorescent protein (GFP)–Ers1 fusion protein (see Experimental proce- dures). While we were unable to detect GFP–Ers1when expressed from the ERS1 promoter, when driven by the GAL1 promoter GFP–Ers1 localized in the vacuole and in a punctate pattern reminiscent of endosomes in yeast (Fig. 3). To confirm that these puncta represent components of the endocytic pathway, the localization of GFP–Ers1 was compared to that of FM4-64, a fluorescent dye that is a marker for the endocytic com- partments. At very short times after addition, FM4-64 is first localized on the plasma membrane. With increasing times of incubation, FM4-64 is found in endosomes and finally in the vacuole [16]. When cells expressing GFP–Ers1were stained with Fm4-64 and viewed after 10 min of incubation, Ers1 largely colo- calized with FM4-64 fluorescence (Fig. 3), suggesting that Ers1 is primarily found in endosomes and in the vacuole. To rule out the possibility of mislocalization of GFP–Ers1due to its over expression by the GAL1 pro- moter, the localization of Ers1 expressed at physiologi- cal levels was examined by subcellular fractionation. Yeast strains were constructed that express a low copy plasmid-borne HA-tagged allele of ERS1 and used to prepare whole cell lysates. Subcellular organelles were separated by differences in their densities using differ- ential centrifugation (see Experimental procedures). Using this method, we found that the vast majority of Ers1 sedimented with vacuolar membranes at 16 000 g (P16), cofractionating with the 100-kDa subunit of the vacuolar-ATPase, Vph1 (Fig. 3B) and away from other Golgi markers that sediment in the 100 000 g pellet (P100) (data not shown). The faint 31 kDa band comi- grating with Ers1 in Fig. 3B is probably not Ers1 but rather a nonspecific membrane localized protein that cross reacts with the anti-HA Ig as it appears in lysates from strains not expressing Ers1–HA (data not shown). The simplest interpretation of these results is that Ers1 does indeed localize in the endosomes and vacuole, a result that provides additional evidence for its functional conservation with cystinosin. Identification of MEH1 as a high copy suppressor of ers1D To identify genes involved in regulating ERS1, and hence CTNS functions, we carried out a screen for genes that when overexpressed, suppres the hygB sensi- tivity of an ERS1 deletion mutant. The ers1D strain was transformed with a yeast genomic DNA library in a high copy vector. About 10 000 ers1D transformants, representing at least a fourfold excess of the entire yeast genome were screened for growth resistance to hygB (see Experimental procedures). Isolation and sequence analysis of plasmids conferring this growth resistance led to the identification of ERS1 itself as well as nine other genes. At sufficiently high concentra- tions ( 100–200 lgÆmL )1 ), wild type yeast are sensi- tive to hygB, and several genes have been identified that confer hygB resistance at these high concentra- tions. To rule out the possibility that the genes we identified are nonspecific high copy suppressers of hygB sensitivity, each of these genes was further ana- lysed for the ability to suppress the hygB sensitivity of a wild type yeast strain on media containing elevated concentrations of hygB (100 lgÆmL )1 ) at which wild type cells fail to grow. Indeed we found that over- expression of five of these genes, including PRP3, SAT4, HAL5, SKN7 and PDR5, suppress the hygB sensitivity of wild type cells (data not shown). Thus four remaining genes were identified as high copy X D. Gao et al. ERS1 and CTNS are functional homologues FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS 2501 suppressors of the ers1D phenotype. Of these four genes, YKR007W is the strongest suppressor, whose overexpression reversed the hygB sensitive phenotype of ers1D as efficiently as ERS1 itself (Fig. 4A). We henceforth refer to this gene as MEH1 (Multicopy sup- pressor of ERS1 Hygromycin sensitivity) and describe its further characterization below. MEH1 displays genetic interactions with ERS1 and localizes to the vacuole MEH1 is predicted to encode a 20.2-kDa protein that is highly conserved among fungi, but its function is unknown. As an initial investigation of MEH1,we analysed its null phenotype. A deletion of MEH1 results in a slow growth phenotype, although these meh1D cells are viable. A deletion of MEH1 also results in hypersensitivity to hygB (Fig. 4B) and tet- rad dissection of meh1D::S.p. his3 + heterozygous diploids demonstrated complete linkage between the meh1 deletion and hygB sensitivity (data not shown). Similar to the ers1D phenotype, meh1D strains do not display any apparent cell wall or glycosylation defects (data not shown). To obtain further evidence for the functional relatedness of ERS1 and MEH1, we ana- lysed their genetic interactions. We found that meh1D hygB sensitivity can be suppressed by overexpression of ERS1 (Fig. 4B). As was seen for the complementa- tion of ers1D by the wild type ERS1 gene, suppres- sion of meh1D by ERS1 was most efficient when ERS1 is expressed from its own promoter (data not shown). A B GFP-Ers1p FM4-64 DICMerge Fig. 3. Ers1p localizes in the vacuole and endosome-like compartments. (A) Cells expressing GFP-ERS1 (XGY50) were stained with FM4-64 and analysed by fluorescence microscopy as described in Experimental procedures. GFP–Ers1 is shown in green, FM4-64 is shown in red and their colocaliza- tion (merge) is in yellow. Also shown are cells imaged by Nomarski optics. (B) Whole cell lysates from cells expressing HA-tagged Ers1(pRs305ERS1p-ERS1-HA) were subjec- ted to differential centrifugation and equival- ent amounts of protein from each fraction were separated by SDS ⁄ PAGE and immuno- blotted with anti-HA or anti V-ATPase Igs as described in Experimental procedures. ERS1 and CTNS are functional homologues X D. Gao et al. 2502 FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS MEH1 is predicted to encode a hydrophilic protein with no obvious transmembrane spanning domains, but it contains an N-terminal recognition sequence for the attachment of a myristate. To determine its subcel- lular localization, yeast strains were constructed that expressed an MEH1 allele that was GFP-tagged at the C terminus. Fluorescence analysis of this Meh1–GFP fusion suggested that it tightly localized to the vacuo- lar membrane (Fig. 5B). This result was confirmed by the determining the localization in these cells of the vacuole lumen fluorescent marker, CMAC. While Meh1–GFP and CMAC colocalize to the same com- partment, Meh1 is found at the membrane, while CMAC is within the lumen (Fig. 4C). A similar local- ization pattern was observed by using a Meh1 HA-tagged protein (data not shown). Unlike Ers1, which localizes to the endosomes as well as the vacu- ole, Meh1 appears to be largely confined to the vacuo- lar membrane. Nonetheless, taking together both the genetic and subcellular localization data, these results provide good evidence for a functional relationship between Ers1 and Meh1. Meh1 is required for vacuolar acidification To determine if loss of MEH1 plays a role in regula- ting vacuolar function, we examined the acidity of the vacuole indirectly using LysoSensor green. LysoSensor green is a pH-sensitive fluorescent probe that accumu- lates in the membranes of acidic organelles. In wild type yeast, LysoSensor green labels the vacuolar mem- branes and this staining is greatly diminished in A B C Fig. 4. MEH1, a multisuppressor of ers1D, encodes a vacuolar protein. (A) ers1D (XGY51) cells expressing MEH1 (YKR007W) or ERS1 in YEp213 were streaked on an YPAD plate containing 50 lgÆmL )1 hygB. (B) Isogenic wild type (SEY6210) or meh1D cells (XGY53) with or without a high copy plasmid containing the MEH1 or ERS1 gene were serially diluted (10-fold), spotted onto YPAD plates with or without 50 lgÆmL )1 hygB and grown for 2 days at 30 °C. (C) Yeast cells expressing a GFP-tagged MEH1 allele (XGY52) were stained with the vacuolar probe, CMAC, and analysed by fluorescence microscopy as described in Experimental procedures. Also shown are cells imaged by Nomarski optics. X D. Gao et al. ERS1 and CTNS are functional homologues FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS 2503 mutant strains that are defective in the vacuolar (H+) ATPase (V-ATPase) that pumps protons into the lumen ([17] and Fig. 5). We qualitatively measured vacuolar acidification by a visual assay of LysoSensor green intensity by fluorescence microscopy. For com- parative purposes, we also assayed LysoSensor green staining of a vma1D strain, which lacks the 118-kDa subunit of the V-ATPase (Fig. 5A) and is defective in vacuolar acidification. Compared to the ers1D strain (data not shown) or to the isogenic parental wild type strain, in the meh1D strain LysoSensor staining was diminished (Fig. 5A) although it was not absent, as it was in the vma1D strain. No obvious morphological abnormalities were seen when these different mutant cells were viewed by bright field microscopy, although meh1D strains appeared to be slightly swollen (Fig. 5B). The yeast V-ATPase is a large membrane associated complex of proteins containing at least 13 different subunits. In mutants lacking any of subunits, assembly of the complex is impaired [18]. As a further test for an affect of MEH1 on vacuolar acidification, we compared the steady state levels of the 60-kDa Vma2 protein in meh1D and wild type cells by western immu- noblotting, using anti-Vma2 antibodies. As expected, no Vma2 protein was detected in a vma2D mutant strain, and a 60-kDa protein corresponding to Vma2 was seen in wild type cells. A slightly diminished level of Vma2 (about twofold) was also observed in ers1D strains. In contrast to wild type cells, a significant decrease in Vma2p steady state levels was observed in meh1D strains (Fig. 6) suggesting that the 60-kDa V-ATPase subunit is unstable as a consequence of loss of MEH1 function. While the basis for this instability is unknown, these results are consistent with the decreased LysoSensor green staining in meh1D cells and provide further support for a role of Meh1 in regulating vacuolar, and hence ERS1 function. Myristylation of Meh1 is required for its vacuolar association The Meh1 protein does not contain any predicted membrane spanning domains and is quite hydrophilic, Fig. 5. Loss of MEH1 affects the vacuolar pH. (A) The isogenic parental strain (BY4741), meh1D,orvma1D were stained with the pH-sensitive fluorescent probe, LysoSensor Green and viewed by fluores- cence microscopy as described in Experi- mental procedures. (B) Brightfield view of the meh1D and vma1D cells imaged in (A). ERS1 and CTNS are functional homologues X D. Gao et al. 2504 FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS raising the question of how it localizes to the vacuolar membrane. Sequence analysis predicts that Meh1p contains a conserved N-terminal recognition sequence for myristyolation (MGAVLSC). Myristate is normally added to the consensus sequence at glycine-2 (G2) after removal of the initiator methionine. We wished to determine if this protein is myristoylated and if so, whether or not this lipid modification facilitates its interaction with the vacuolar membrane and is there- fore important for Meh1 function. To approach these questions, we created a mutant allele (meh1-ND5) that replaces the first five N-terminal amino acids with a methionine residue, and therefore produces an altered protein that is predicted to lack acylation. To enable detection of this altered protein, we also tagged the C terminus with the HA epitope. This plasmid-borne mutant allele was introduced into an meh1D strain and tested for complementation of the hygromycin B sensi- tive phenotype of meh1D. While an identical plasmid harbouring the wild type MEH1 gene complemented this phenotype, the mutant meh1ND5 failed to do so, suggesting these N-terminal five amino acids are essen- tial for MEH1 function (Fig. 7A). The failure to com- plement meh1D was not due to the absence of protein, since this mutant allele produced protein at levels com- parable to the wild type (e.g. Fig. 7B). To determine if the absence of this N-terminal region is important for Meh1 localization to the vacuole, we analysed its local- ization by subcellular fractionation. Subcellular organ- elles were separated by differences in their densities using differential centrifugation (see Experimental pro- cedures). Using this method, we found that while most of Meh1 sedimented with vacuolar membranes at 16 000 g (P16), though a proportion was found in the S14 fraction (Fig. 7B). This result is consistent with our observation that Meh1–GFP is associated with vacuolar membranes. It is notable that the majority of Meh1in the P16 fraction migrated as a smear, while Meh1 in the S16 fraction migrated as a sharp band, suggesting the possibility that the soluble portion of Meh1 lacks a myristate and is therefore not associated with the membrane. In sharp contrast, Meh1–ND5- HA, lacking the consensus myristoylation site, largely fractionated in the S16 fraction and away from the vacuolar membrane. Unlike most of the wild type Meh1 protein, this protein migrated as a sharp band. Taken together, these data demonstrate that the Fig. 6. Loss of MEH1 results in the instability of the 60-kDa V-ATPase subunit. Protein extracts ( 50 lg) prepared from equiv- alent amounts of wild type cells or those containing a deletion of VMA2, MEH1 or ERS1 were separated by 8% SDS ⁄ PAGE and immunoblotted with antibodies against Vma2p. AB Fig. 7. The N-terminal myristoylation consensus sequence is required for Meh1 function and vacuolar membrane association. (A) meh1ND5 fails to complement an meh1D strain. Wild type (SEY6210) or and meh1D mutant strain (XGY53) harbouring plasmids containing wild type MEH1 or the mutant meh1ND5 allele, encoding protein lacking the N-terminal myristoylation consensus sequence, were plated on YPAD media containing 50 lgÆmL )1 hygromycin B. (B) The N-myristoylation consensus sequence is required for Meh1 membrane association. Extracts were prepared from the SEY6210 expressing pTiMEH1-HA 3 or pTI-meh1-ND5-HA 3 and subjected to sedimentation centrifugation, as described in Experimental procedures. Equivalent amounts of each fraction were separated by 10% SDS ⁄ PAGE and analysed by immuno- blotting with anti-HA Igs. X D. Gao et al. ERS1 and CTNS are functional homologues FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS 2505 N-terminal five amino acids are essential for function and localization to the vacuole, and provide evidence to support the idea that Meh1 is myristoylated. Thus, it is likely that Meh1 localizes to the vacuolar mem- brane through a myristate tail and probably functions on the cytosolic face of the vacuole. Meh1 recruits the small GTPase Gtr1 to the vacuolar membrane Proteomic analyses (http://bind.ca/); (http://dip.doe-mbi. ucla.edu/dip/); (http://biodata.mshri.on.ca/yeast_grid/) identify a highly conserved protein, the small Ras-rela- ted GTP binding protein, Gtr1, as a protein that Meh1 interacts with. To obtain further information about the function of Meh1, we examined whether or not Meh1 and Gtr1 interact with one another, under physiological conditions, using coimmunoprecipitation assays. To determine if Meh1 interacts with Gtr11, we used a coimmunoprecipitation assay. Yeast strains were constructed that coexpressed HA and myc tagged GTR1 and MEH1 genes. The chromosomal loci of GTR1 and MEH1 were replaced with the correspond- ing HA or myc-tagged alleles (see Experimental proce- dures). Extracts from each of these strains were prepared in buffer containing the nonionic detergent, digitonin, to maintain oligomeric interactions between membrane proteins, and these extracts were subjected to coimmunoprecipitation assays. Gtr1–myc protein was precipitated from these extracts with anti-myc antibody and the immunoprecipitates were fractionated by SDS ⁄ PAGE. The relative steady state levels of Gtr– myc and Meh1–HA in the same extracts used for the immunoprecipitations were determined by Western blot analysis of aliquots removed prior to immunopre- cipitation and were found to be similar (data not shown; Fig. 8A, lanes 2 and 3). Meh1–HA that copre- cipitated with Gtr1–myc was detected by immunoblot- ting with anti-HA Ig (Fig. 8A). The result of this experiment demonstrates that Meh1 coprecipitated with Gtr1–myc (Fig. 8A, lanes 6). This interaction is dependent on the coexpression of Meh1 with Gtr1 as Meh–HA did not coprecipitate in a control strain that does not coexpress Gtr1–myc (Fig. 8A, lane 5). We also find no evidence that Meh1 interacts with another vacuolar protein, Ers1, further demonstrating the spe- cificity of this interaction. If the interaction between Meh1 and Gtr1 is of bio- logical relevance, we would expect to find Gtr1 locali- zed in the vacuole, in an Meh1-dependent manner. To test this idea, we constructed yeast cells whose chro- mosomal GTR1 locus was replaced with a GFP-tagged allele, and examined the intracellular localization of Gtr1–GFP fusion proteins by fluorescence microscopy. By this analysis, we found that Gtr1–GFP localized in the vacuole, coincident to the pattern observed by the vacuolar marker, CMAC. However, unlike CMAC, which localizes in the lumen of the vacuole, Gtr1–GFP appears to localize to the vacuolar membrane (Fig. 8B), in a pattern similar to that of Meh1. To determine if the vacuolar association of Gtr1 is dependent on Meh1, we compared the fractionation behaviour of Gtr1–myc in a wild type or meh1D strain, by subcellular fraction. Fractions enriched for vacuolar membranes were prepared and separated using differ- ential centrifugation (see Experimental procedures). Consistent with our observation that Gtr1–GFP local- izes with vacuolar membranes, using this method, we found that in a wild type MEH1 background, Gtr1 sedimented in the P16 fraction, with very little protein observed in the S16 fraction (Fig. 8C). In striking con- trast, in extracts prepared from the meh1D cells that lack Meh1, the vast majority of Gtr1 appeared soluble, fractionating in the S16 supernatant. These results dem- onstrate that the membrane association of Gtr1 is dependent on Meh1 and suggest that Meh1 is required to recruit Gtr1 to the vacuolar membrane. Discussion In this study we have demonstrated that the S. cerevis- iae Ers1 and human cystinosin proteins are functional homologues. Like cystinosin, Ers1 is a vacuolar pro- tein whose loss of function results in hygB sensitivity. The human CTNS gene can complement the ers1D phenotype, reversing its hygB sensitivity. Moreover, the severity of CTNS mutants, as identified in different patients afflicted with varying forms of the disease, mimics the degree with which these CTNS mutant alle- les can complement ers1D. Importantly, the inability of these mutant ctns alleles to complement ers1D provides the proof of principle for the utility of yeast as model system for functional analyses of cystinosin. We have used this yeast system to identify novel yeast genes that regulate Ers1 and other vacuolar functions. Through a high copy suppressor screen we identify MEH1, a previously uncharacterized yeast gene that is required for maintaining vacuolar acidity. We also identify Gtr1, a small Ras-related GTPase, whose recruitment to the vacuolar membrane is dependent upon its interaction with Meh1. Although cystinosis has been described primarily as a kidney disease, mutations in CTNS affect a number of different organs. It is not understood how cystine accumulation in the lysosome causes cellular damage or why this accumulation specifically targets the ERS1 and CTNS are functional homologues X D. Gao et al. 2506 FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS [...]... These observations suggest that the accumulation of cystine in the vacuole of yeast does not lead to the types of cellular damage that is observed in mammalian cells Two explanations for these results can be envisaged First, a formal possibility that our studies have not ruled out is that, in yeast, Ers1 and cystinosin are involved in the transport of a molecule other than cystine, whose accumulation gives... measurements of cystine levels in the yeast vacuole have been hampered by technical difficulties (data not shown) A second explanation is that the mild ers1D phenotype may be more related to the effect of proton accumulation in the vacuole than to cystine accumulation Further 2507 ERS1 and CTNS are functional homologues investigation is required to clarify the basis for the ers1D hygB phenotype in yeast... 13314–13321 Dean N (1995) Yeast glycosylation mutants are sensitive to aminoglycosides Proc Natl Acad Sci USA 92, 1287– 1291 Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper SL & Fink GR (1999) The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast Proc Natl Acad Sci USA 96, 1480–1485 Madrid R, Gomez MJ, Ramos J & Rodriguez-Navarro A (1998) Ectopic potassium... by ERS1 and whether these relate to cystine transport from the vacuole Unlike CTNS in mammalian cells, deletion of ERS1 does not dramatically affect cellular growth properties FEBS Journal 272 (2005) 2497–2511 ª 2005 FEBS Further, ers1D mutants do not display any apparent abnormal vacuolar morphology and treatment of ers1D cells with cysteamine does not rescue the ers1D phenotype (data not shown) These... DNA sequence analysis Linearization of these integrative plasmids with A II within the LEU2 gene targets integration at the leu2-3 locus To tag the Erd1 protein, a HindIII ⁄ EcoRI fragment containing the ERD1 ORF lacking the stop codon was isolated by PCR and cloned into pSK–P ⁄ X HA3 [28], a derivative of Bluescript SK– (Stratagene, La Jolla, CA, USA) pSK:ERD1-HA3 encodes Erd1p containing an in-frame... consequences of CTNS mutations may also be due to secondary affects [3,19,20] For instance, CTNS encodes a proton-driven pump, so defects in cystine transport may affect lysosomal pH indirectly via an accumulation of protons or an affect on the lysosomal ATPase that pumps protons into the lysosome upon hydrolysis of cytosolic ATP These ideas bear on the question of what biological functions in yeast are regulated... highly related GTR1 homologue (GTR2) that may be redundant in function to GTR1 Further investigation is required to determine the precise mechanism of suppression of ers1D by MEH1, and the role of Meh1 and Gtr1 in regulating vacuolar function The results we have presented validate the utility of yeast as a model system for the functional analysis of cystinosin, both as a simple plate assay for the detection... identified MEH1 as a high copy suppressor of ers1D Meh1 is localized in the vacuole and this membrane association appears to be dependent on an N-terminal myristate modification The ability of Meh1 to associate with the vacuolar membrane is critical for its function because mutations in this putative myristoylation site fail to complement an meh1 deletion mutant We demonstrated that vacuolar acidification is... and 6) but the precise mechanism by which Meh1 affects vacuolar function remains unknown At least one vacuolar subunit, Vma2, is unstable in an meh1D mutant background (Fig 6) While an Meh1 homologue cannot be identified among mammals, we also identified the highly conserved Rasrelated GTPase Gtr1, as a protein that interacts with Meh1 A clue that these proteins physically interact came from the databases... WA, Charnas L, Markello TC, Bernardini I, Ishak KG & Dalakas MC (1992) Parenchymal organ cystine depletion with long-term cysteamine therapy Biochem Medical Metab Biol 48, 275–285 Gahl WA (2003) Early oral cysteamine therapy for nephropathic cystinosis Eur J Pediatr 162 (Suppl 1), S38–S41 Hardwick KG & Pelham HR (1990) ERS1 a seven transmembrane domain protein from Saccharomyces cerevisiae Nucleic Acids . ERS1 encodes a functional homologue of the human lysosomal cystine transporter Xiao-Dong Gao 1 , Ji Wang 2 , Sabine Keppler-Ross 2 and Neta Dean 2 1. found that the vast majority of Ers1 sedimented with vacuolar membranes at 16 000 g (P16), cofractionating with the 100-kDa subunit of the vacuolar-ATPase,