Copyright � 2006 by the Genetics Society of AmericaDOI: 10.1534/genetics.106.064709

Genetic Evidence for Phospholipid-Mediated Regulation of theRab GDP-Dissociation Inhibitor in Fission Yeast

Yan Ma,* Takayoshi Kuno,* Ayako Kita,† Toshiya Nabata,* Satoshi Uno* and Reiko Sugiura†,1

*Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine,Kobe, Hyogo 650-0017, Japan and †Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences,

Kinki University, Higashi-Osaka, Osaka 577-8502, Japan

Manuscript received August 9, 2006Accepted for publication September 7, 2006

ABSTRACT

We have previously identified mutant alleles of genes encoding two Rab proteins, Ypt3 and Ryh1,through a genetic screen using the immunosuppressant drug FK506 in fission yeast. In the same screen,we isolated gdi1-i11, a mutant allele of the essential gdi11 gene encoding Rab GDP-dissociation inhibitor.In gdi1-i11, a conserved Gly267 was substituted by Asp. The Gdi1G267D protein failed to extract Rabs frommembrane and Rabs were depleted from the cytosolic fraction in the gdi1-i11 mutant cells. Consistently, theGdi1G267D protein was found mostly in the membrane fraction, whereas wild-type Gdi1 was found in both thecytosolic and the membrane fraction. Notably, overexpression of spo201, encoding a phosphatidylcholine/phosphatidylinositol transfer protein, rescued gdi1-i11 mutation, but not ypt3-i5 or ryh1-i6. The gdi1-i11and spo20-KC104 mutations are synthetically lethal, and the wild-type Gdi1 failed to extract Rabs from themembrane in the spo20-KC104 mutant. The phosphatidylinositol-transfer activity of Spo20 is dispensablefor the suppression of the gdi1-i11 mutation, suggesting that the phosphatidylcholine-transfer activity isimportant for the suppression. Furthermore, knockout of the pct11 gene encoding a choline phosphatecytidyltransferase rescued the gdi1-i11 mutation. Together, our findings suggest that Spo20 modulatesGdi1 function via regulation of phospholipid metabolism of the membranes.

IN all eukaryotic cells, Rab family small GTPases(Rabs) form the largest branch of the small GTPase

superfamily (Takai et al. 2001). In mammals, Rabsdefine a family of almost 70 proteins that play criticalroles in the trafficking of vesicles that mediate transportbetween compartments of the exocytic and endocyticpathways (Pfeffer 2001, 2005). Like Ras, Rabs act asmolecular switches, cycling between an active GTP-boundstate and an inactive GDP-bound state. Thus, transportvesicles bear Rabs with bound GTP; concomitant withor after membrane fusion, Rabs are converted intotheir GDP-bound states. In this manner, target mem-branes acquire vesicle-derived Rabs in their GDP-boundconformations (Pfeffer et al. 1995).

We have previously identified two Rab family smallGTPases, Ypt3/Its5 and Ryh1/Its6, in the fission yeastSchizosaccharomyces pombe through a genetic screen usingthe immunosuppressant drug FK506, a specific inhibi-tor of calcineurin (Cheng et al. 2002; He et al. 2006). Inthe same genetic screen, we also isolated a mutant of theapm11 gene that encodes a homolog of the mammalianm1A subunit of the clathrin-associated adaptor protein-1 complex and that is implicated in the Golgi/endo-

some function (Kita et al. 2004). This prompted us tofurther screen immunosuppressant-sensitive mutants toidentify molecules involved in membrane trafficking.

Here, we report the identification and characteriza-tion of its11-1/gdi1-i11, a new addition to the series ofimmunosuppressant- and temperature-sensitive mu-tants. The gdi11 gene encodes a protein that is highlysimilar to the mammalian Rab GDP-dissociation in-hibitor (GDI) (Sasaki et al. 1990) and to Saccharomycescerevisiae GDI1/SEC19 (Garrett et al. 1994). GDI is aRab-specific regulator that plays a key role in therecycling of Rabs (Ullrich et al. 1993). GDI binds andreleases Rabs in the presence of GDP but not GTP,which is thought to be a key biochemical function ofGDI in retrieving Rabs from target membranes. GDI canretrieve a broad spectrum of Rabs from the membranefollowing bilayer fusion. In the cytosol, GDI binds Rabin the GDP-bound form. The heterodimeric complex ofGDI and Rab–GDP serves as a cytosolic reservoir fordelivering Rab to the newly formed vesicles, where itbecomes activated to the GTP-bound form (Luan et al.1999). Thus, GDI plays a key role in the Rab recycling byretrieving prenylated Rabs from their fusion targets inmembranes, maintaining Rabs as a soluble cytosoliccomplex and delivering Rabs to specific membrane-bound compartments for vesicle formation. In mam-mals, the importance of GDI function is reflected in thefindings that mutations in the gene encoding the

1Corresponding author: Laboratory of Molecular Pharmacogenomics,School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1,Higashi-Osaka, Osaka 577-8502, Japan.E-mail: sugiurar@phar.kindai.ac.jp

Genetics 174: 1259–1271 (November 2006)

human GDI result in X-linked nonspecific mental retar-dation (D’Adamo et al. 1998) and that Gdi1-deficientmice show impairment in associative memory and so-cial behavior (D’Adamo et al. 2002). In S. cerevisiae, GDI1is an essential gene, and depletion of Gdi1p in vivoleads to loss of the soluble pool of Sec4p and inhibi-tion of protein transport at multiple stages of the se-cretory pathway (Garrett et al. 1994).

The structural and mutational analysis of GDI hasbeen focused on the regions called sequence conservedregions (SCRs). SCR1 and SCR3B, forming a compactstructural unit at the apex of GDI, were reported to beimplicated for Rab binding in vitro and in vivo (Schalk

et al. 1996; Luan et al. 1999). In addition to Rab binding,domain II, which consists of part of SCR2 and SCR3A,is required for both Rab extraction and Rab loading(Gilbert and Burd 2001). In a study by Luan et al.(2000), the double mutation in SCR3B and SCR3A(R248A-R226A or R248A-Y227A) caused a defect in theassociation with the membrane.

In this study of fission yeast, we identified a novelmutant allele of the gdi11 gene (gdi1-i11), containing anamino acid change at codon 267 (Gly to Asp), which islocated outside the SCRs. The Gdi1G267D protein failed toextract Rabs from the membrane, yet maintained theability to bind Rabs. To further delineate Gdi1 function,we screened for dosage-dependent suppressors of thegdi1-i11 mutant and isolated the spo201 gene. spo201 isan essential gene, which encodes a phosphatidylcho-line/phosphatidylinositol transfer protein (PITP) ofthe Sec14 family (Nakase et al. 2001). Further study in-dicated that the gdi1-i11 and spo20-KC104 mutationsare synthetically lethal. Consistently, the wild-type Gdi1

failed to extract Rabs from the membrane in the spo20-KC104 mutant, indicating that Spo20 is necessary forGdi1 to efficiently extract Rabs. We also provide evidencesuggesting that the phosphatidylcholine-transfer activ-ity, but not the phosphatidylinositol-transfer activity, isthe mechanism of suppression of the gdi1-i11 mutationby Spo20. To the best of our knowledge, this articleprovides the first evidence suggesting that PITP modu-lates Gdi1 function via regulation of lipid metabolism.

MATERIALS AND METHODS

Strains, media, and genetic and molecular biology meth-ods: Strains used in this study are listed in Table 1. The com-plete medium YPD and the minimal medium EMM have beendescribed previously (Toda et al. 1996). Standard genetic andrecombinant-DNA methods (Moreno et al. 1991) were usedexcept where noted. FK506 was provided by Fujisawa Pharma-ceutical (Osaka, Japan).

Isolation of the its11-1/gdi1-i11 mutant and cloning of thegdi11 gene: The its11-1/gdi1-i11 mutant was isolated in ascreen of cells that had been mutagenized with nitrosoguani-dine as described previously (Zhang et al. 2000). To cloneits111, the its11-1 mutant (KP1892) was grown at 27� andtransformed with an S. pombe genomic DNA library con-structed in the vector pDB248 (Beach et al. 1982). Leu1

transformants were replica plated onto YPD plates at 36� andthe plasmid DNA was recovered from transformants thatshowed plasmid-dependent rescue. These plasmids comple-mented both the immunosuppressant sensitivity and thetemperature sensitivity of the its11-1 mutant. By DNA sequenc-ing, the suppressing plasmids were identified to contain thegdi11 gene (SPAC22H10.12c).

To investigate the relationship between the cloned gdi11

gene and its11-1 mutant, linkage analysis was performed asfollows. The entire gdi11 gene was subcloned into the pUC-derived plasmid containing the S. cerevisiae LEU2 gene and

TABLE 1

S. pombe strains used in this study

Strain Genotype Reference

HM123 h� leu1-32 Our stockKP162 h� leu1-32 ypt3-i5 Our stockHM528 h1 his2 Our stockKP928 h1 his2 leu1-32 ura4-D18 Our stockKP1248 h� leu1-32 ura4-294 Our stockKP1259 h1 leu1-32 ura4-294 nmt1GFP-ypt3Tura41 Our stockKP1892 h� leu1-32 gdi1-i11 This studyKP1893 h1 his2 leu1-32 gdi1-i11 This studyKP2035 h� leu1-32 ura4-294 nmt1GFP-syb1Tura41 Our stockKP2439 h� leu1-32 ura4-294 gdi1 nmt1GFP-syb1Tura41 This studyKP2454 h1 his2 leu1-32 ura4-D18 gdi1-i11 This studyKP2580 h� leu1-32 ura4-D18 ryh1Tura41 Our stockKP2591 h�/h1 leu1-32/leu1-32 ura4-D18/ura4-D18 his2/1

ade6-M210/ade6-M216 gdi11/gdi1Tura41

This study

KP2678 h� leu1-32 ura4-D18 spo20-KC104 Nakase et al.(2001)

KP2881 h1 leu1-32 ura4 spo20-KC104 nmt1GFP-ypt3Tura41 This studyKP2902 h� leu1-32 pct1Tura41 This studyKP2913 h� leu1-32 ura4-D18 gdi1-i11 pct1Tura41 This study

1260 Y. Ma et al.

integrated by homologous recombination into the genome ofthe wild-type strain HM123. The integrant was mated with theits11-1 mutant. The resulting diploid was sporulated, andtetrads were dissected. A total of 30 tetrads were dissected. Inall cases, only parental ditype tetrads were found, indicatingallelism between the gdi11 gene and the its11-1 mutation (datanot shown).

Cloning of the spo201 gene: To screen for dosage-depen-dent suppressors of its11-1, the its11-1 mutant (KP1892) wastransformed with an S. pombe genomic DNA library, and Leu1

transformants were replica plated onto YPD plates at 30�. BySouthern blot analysis, the suppressing plasmids fell into twoclasses, with one class containing the gdi11 gene, and the otherclass containing the spo201 gene (SPBC3H8.10). No othergene was isolated in this screening.

Tagging of the gdi11 gene: The gdi11 gene was amplified byPCR with genomic DNA of wild-type cells as a template. Thesense primer was 59-CGC GGA TCC ATG GAT GAG GAA TATGAT GTT ATA GTC C-39, and the antisense primer was 59-CGCGGA TCC TTA TTG ATC TTC CAT TTT GGG-39. Theamplified product containing the gdi11 gene was digestedwith BamHI, and the resulting fragment was subcloned intoBlueScriptSK (1) (Stratagene. La Jolla, CA).

For ectopic expression of proteins, we used the thiamine-repressible nmt1 promoter (Maundrell 1993). Expressionwas repressed by the addition of 4 mm thiamine to EMM. Toexpress GST–Gdi1, Gdi1 was tagged at its N terminus with GST.GST-Gdi1G267D was made in the same way except that thegenomic DNA was from gdi1-i11 mutant cells. Genes eithertagged or untagged were subcloned into the pREP1 vector toexpress the gene (Maundrell 1993).

Gene deletion: A one-step gene disruption by homologousrecombination was performed (Rothstein 1983). Thegdi1Tura41 disruption was constructed as follows. The BamHIfragment containing the gdi11 gene was subcloned into theBamHI site of BlueScriptSK (1). Then, a BamHI fragmentcontaining the ura41 gene was inserted into the BglII site of theprevious construct. The fragment containing the disruptedgdi11 gene was transformed into diploid cells. Stable inte-grants were selected on medium lacking uracil, and disruptionof the gene was checked by genomic Southern hybridizationand tetrad analysis. The pct11 gene (SPCC1827.02c) was dis-rupted by the insertion of the ura41 gene at the HincII site,using a one-step gene disruption by homologous recombina-tion as described above.

Microscopy: Methods in light microscopy, such as fluores-cence microscopy and differential interference contrast micros-copy, were performed as described (Kita et al. 2004). FM4-64labeling and staining of vacuoles with Lucifer Yellow (LY) wereperformed as described (Kita et al. 2004). Septa were visualizedby adding 1 ml of 1 mg/ml Calcofluor (Sigma, St. Louis) to a 10-to 20-ml aliquot of cell suspension. Conventional electron mi-croscopy was performed as described (Kita et al. 2004).

Assays and miscellaneous methods: Tetrad analysis (Zhang

et al. 2000), expression and detection of GFP-Pho1 (Cheng et al.2002), and cell extract preparation and immunoblot analysis(Sio et al. 2005) were performed as previously described.Quantification of the immunoblot data was performed byImageJ software (http://rsb.info.nih.gov/ij/). Antibody to fis-sion yeast Cdc4 protein was prepared by immunizing the rabbitwith purified Cdc4 protein and was used for the detection ofendogenous Cdc4 as a loading control.

RESULTS

Isolation of the its11-1 mutant: To identify proteinsthat function in membrane trafficking, we searched for

mutants that are sensitive to the immunosuppressivedrug FK506 and isolated the its11-1 mutant (for immu-nosuppressant and temperature sensitive). As shown inFigure 1A, its11-1 mutants grew equally as well as thewild-type cells at 27�. However, its11-1 mutant cells couldnot grow at 36� nor could they grow on YPD containingFK506 at the permissive temperature, whereas wild-typecells grew normally (Figure 1A). As predicted, no dou-ble mutant was obtained at any temperature by thegenetic cross between its11-1 and calcineurin deletion(Dppb1), indicating that its11-1 and Dppb1 are synthet-ically lethal (data not shown).

The its11-1/gdi1-i11 is an allele of the gdi11 gene thatencodes a homolog of mammalian GDP-dissociationinhibitor: The its111 gene was cloned by complementa-tion of the temperature-sensitive growth defect of theits11-1 mutant (Figure 1A, YPD36�, 1gdi11). The its111

gene also complemented the immunosuppressant sen-sitivity of the its11-1 mutant (Figure 1A, YPD27� 1FK506,1gdi11). Nucleotide sequencing of the cloned DNAfragment revealed that its111 is identical to the gdi11

gene (SPAC22H10.12c), which encodes a protein of 440amino acids that is highly similar to human a-GDI (249/440, 56.59% identity) and S. cerevisiae Gdi1p (272/440,61.82% identity) (Figure 1B). Linkage analysis was per-formed (see materials and methods) and resultsindicated the allelism between the gdi11 gene and theits11-1 mutation. We therefore renamed its11-1 as gdi1-i11. The its11-1 mutant is the only allele of gdi11 that wasidentified in the screen of mutants that show sensitivityto the immunosuppressant.

To identify the mutation in the gdi1-i11 allele, ge-nomic DNA from the gdi1-i11 mutant was isolated, andthe full-length coding region of the gdi1-i11 gene wassequenced. The G-to-A nucleotide substitution caused ahighly conserved glycine to be altered to an aspartic acidresidue at amino acid position 267 (Figure 1B, arrow).We therefore refer to the protein product of the gdi1-i11gene as Gdi1G267D. It should be noted that Gly267Aspis a novel mutation located outside the SCRs, known tobe required for many aspects of GDI function (Figure1C).

Gdi1 is essential for cell viability: To determine theeffect of complete loss of gdi11 function, the gdi11 genewas knocked out in a wild-type diploid strain by homo-logous recombination using the ura41 gene as a marker(Rothstein 1983). Southern blotting of the genomicDNA of one such transformant confirmed that the wild-type gdi11 gene had been replaced by the derivativecontaining the ura41 insertion (data not shown). Tetradanalysis indicated that each ascus consisted of two viableand two inviable spores and that all viable spores failedto grow in EMM lacking uracil (data not shown).Microscopic observation of nonviable meiotic progenyshowed that these spores germinated but ceased growthsoon thereafter. Therefore, the gdi11 gene is essentialfor vegetative cell growth and viability.

Gdi1 and Spo20 in S. pombe 1261

Rabs failed to distribute in the cytosolic fraction inthe gdi1-i11 mutant cells: To determine which activity ofRab GDI is affected in the gdi1-i11 mutant, we examinedthe distribution of several Rabs by performing immu-noblot analyses of the cytosolic and membrane fractionsobtained from wild-type and the gdi1-i11 mutant cells. Inwild-type cells, Ypt3, Ryh1, and Ypt2 predominantlylocalized to the membrane fraction and were also foundin the cytosolic fraction [Figure 2, wild type (wt)]. Ingdi1-i11 mutant cells, the distribution of the three Rabschanged from cytosol to membrane (Figure 2, gdi1-i11).The distribution of Cdc4 as a loading control remainedunchanged (Figure 2, Cdc4). Thus, the gdi1-i11 muta-tion affects the activity of Gdi1 in extracting Rabs fromthe membrane.

The Gdi1G267D protein failed to extract Rabs from themembrane, yet maintained the ability to bind Rabs:Glycine267 of the Gdi1 protein is highly conserved fromfission yeast to humans. This prompted us to investigatethe functional significance of the Gdi1G267D mutation. Toinvestigate whether Gdi1G267D protein is affected in itsability to bind Rabs, we expressed GST, GST-taggedGdi1, or GST-tagged Gdi1G267D protein in GFP-Ypt3 in-tegrated cells and performed GST pull-down experi-ments. Expression of GST-Gdi1 fully suppressed thephenotypes of the gdi1-i11 mutation, indicating that thefusion protein is functional (data not shown). As shownin Figure 3A, GST-Gdi1G267D interacted with GFP-Ypt3 as

Figure 2.—Rabs failed to distribute in the cytosolic fractionin the gdi1-i11 mutant cells. Wild-type cells and gdi1-i11 mu-tant cells transformed with each of the indicated GFP-Rabwere grown in EMM containing 4 mm thiamine for 12 hr. Cellswere lysed in the absence of detergent and the resulting ex-tracts were centrifuged at 100,000 3 g for 70 min to gener-ate membrane and cytosolic fractions. Equal aliquots weretreated with antibodies to GFP. Endogenous Cdc4 was usedas a loading control and was immunoblotted using anti-Cdc4antibodies. C, cytosolic fraction; M, membrane fraction.

Figure 1.—Mutation in the its111/gdi11 gene causes immu-nosuppressant- and temperature-sensitive phenotypes. (A)The immunosuppressant and temperature sensitivities ofthe its11-1/gdi1-i11 mutant cells. Cells transformed with themulticopy vector pDB248 or the vector containing the gdi11

gene were streaked onto each plate containing YPD andYPD plus 0.5 mg/ml FK506, and then incubated for 4 daysat 27� and for 3 days at 36�, respectively. (B) Alignment of pro-tein sequences of S. pombe Gdi1 with related proteins from hu-man and S. cerevisiae. Sequence alignment was performedusing the Clustal W program. Asterisks indicate identicalamino acids. Arrow indicates the mutation site in the gly-cine267 of Gdi1, which, when mutated to aspartic acid, re-sulted in immunosuppressant- and temperature-sensitivefunction in Gdi1. (C) Schematic of the SCRs and where thegdi1-i11 mutation resides. Star indicates the mutation site.

1262 Y. Ma et al.

efficiently as GST-Gdi1. GST-Gdi1G267D also interactedwith GFP-Ryh1 (data not shown). The results suggestthat Gdi1G267D protein is not affected in its ability to bindRabs, consistent with our finding that Gly267 is locatedoutside SCR1 and SCR3B.

Furthermore, to investigate the ability of the Gdi1G267D

protein to extract Rabs from membranes, we expressedthe GST-fused wild-type gdi11 gene, the gdi1G267D gene,or the control GST vector in strains chromosomallyexpressing nmt1-GFP-Ypt3 or nmt1-GFP-Ryh1 and per-

formed the fractionation experiments. Fractions fromthe cells were probed with the anti-GST antibody todetect Gdi1 and with the anti-GFP antibody to detectYpt3 and Ryh1. As shown in Figure 3, B and D, over-expression of wild-type Gdi1 resulted in a significantincrease in the amount of Ypt3 and Ryh1 detected in thecytosolic fraction (1Gdi1), whereas overexpression ofthe Gdi1G267D protein failed to alter the distribution ofthese Rabs between the cytosolic and membrane frac-tions (1Gdi1G267D). Notably, the Gdi1G267D protein was

Figure 3.—The characterization of the Gdi1G267D protein. (A) Coprecipitation of Ypt3 with GST-Gdi1G267D. GFP-Ypt3-integrated cells expressing pREP1-GST (control), pREP1-GST-Gdi1, or pREP1-GST-Gdi1 G267D were grown in EMM medium inthe absence of thiamine for 20 hr. The proteins were extracted in the presence of detergent. GST-Gdi1, GST-Gdi1G267D, andGST were precipitated by glutathione beads, washed extensively, subjected to SDS–PAGE, and immunoblotted using anti-GFPor anti-GST antibodies. (B) Distribution of Ypt3 and Ryh1 between membrane and cytosolic fractions after wild-type Gdi1 andGdi1G267D overexpression. GFP-Ypt3- and GFP-Ryh1-integrated cells harboring pREP1-GST-Gdi1 or pREP1-GST-Gdi1G267D weregrown in EMM containing 4 mm thiamine for 12 hr. The cells were collected and the fractionation experiment was performedas described in Figure 2. Equal aliquots were treated with anti-GFP antibodies to detect Ypt3 and Ryh1 and with anti-GST anti-bodies to detect Gdi1. C, cytosolic fraction; M, membrane fraction. (C) Distribution of overexpressed wild-type Gdi1 or Gdi1G267D

in cytosolic and membrane fractions in wild-type cells. Wild-type cells harboring pREP1-GST-Gdi1 or pREP1-GST-Gdi1G267D weregrown in EMM containing 4 mm thiamine for 20 hr. The cells were collected and the fractionation experiment was performedas described in Figure 2. (D) Quantifications of the distribution of Rabs and GDIs (coexpressed with GFP-Ypt3) between cytosolicand membrane fractions. The panels in Figure 3B were quantitated using ImageJ software.

Gdi1 and Spo20 in S. pombe 1263

detected in the membrane fraction, while it was almostnegligible in the cytosolic fraction (Figure 3, B andD, 1Gdi1G267D), whereas wild-type Gdi1 was found inboth the cytosolic and the membrane fraction (Figure 3,B and D, 1Gdi1). To exclude the possibility that theoverexpression of Rabs might affect the distribution ofGdi1, we expressed the wild-type Gdi1 or Gdi1G267D alonein wild-type cells and examined the distribution. Again,the Gdi1G267D protein was not detected in the cytosolicfraction in wild-type cells (Figure 3C).

The gdi1-i11 mutant cells are defective for secretion:The function and genetic interactions between two Rabsand Gdi1 as described above prompted us to examinethe role of Gdi1 in the secretory pathway, as these twoRabs are involved in Golgi/endosome membrane traf-ficking. For this, we examined the ability of gdi1-i11mutant cells to secrete S. pombe leader (SPL)-GFP,because this fusion protein is secreted through thesame pathway as Pho1 acid phosphatase (Cheng et al.2002).

As shown in Figure 4, at the permissive temperaturethere was no marked decrease in the amount of secretedSPL-GFP in the growth medium (gdi1-i11, lane 1).However, when shifted to 33� for 4 hr, the secretion ofSPL-GFP was almost abolished in the gdi1-i11 mutant(gdi1-i11, lane 2), whereas no remarkable change wasseen in wild-type cells (wt, lane 2). These results clearlydemonstrate a defect in the secretory pathway associ-ated with the gdi1-i11 mutant. On the other hand, therewas no dramatic change of secreted SPL-GFP when thegdi1-i11 mutant was treated with FK506 for 8 hr (gdi1-i11, lane 4). This suggests that calcineurin is not directlyinvolved in the regulation of the secretory pathway. Ascalcineurin plays a key role in maintaining cell wallintegrity by regulating the enzymes involved in cell wallsynthesis, impairment of both Gdi1 and calcineurin, thetwo key players in cell wall synthesis, might explain thesynthetic lethal interaction.

Fluid-phase uptake of Lucifer Yellow is impaired ingdi1-i11 mutant cells: To assess the effect of the gdi1-i11 mutation on the endocytic pathway, we used themembrane-impermeable fluorescence dye LY to moni-tor the fluid-phase endocytosis (Riezman 1985). Wild-type cells and gdi1-i11 mutant cells were incubated in LYat permissive temperature. At the indicated periods,aliquots were washed extensively to remove excess LYand the cells were viewed under Nomarski differentialinterference contrast and fluorescence microscope. LYclearly labeled the vacuoles of wild-type cells after a30-min incubation (Figure 4B, wt, 30 and 60 min). How-ever, the gdi1-i11 mutant cells showed only weak stainingof the cell wall (Figure 4B, gdi1-i11, 15 and 30 min) andsmall internal structures (Figure 4B, gdi1-i11, 60 min),indicating that fluid-phase endocytosis is significantlyimpaired in gdi1-i11 mutant cells even at the permissivetemperature. It should be noted that, in ypt3-i5 and ryh1-i6 mutant cells, no defect was detected in endocytosis

(data not shown), indicating that Gdi1 has other Rabpartner(s) functioning in the endocytic pathway.

Abnormal findings in gdi1-i11 mutant cells as ob-served in electron micrographs: As noted, gdi1-i11 mu-tant cells exhibited various defects in membranetrafficking and this prompted us to perform electronmicroscopy analysis. Wild-type cells and gdi1-i11 mutantcells were grown at the permissive temperature of 27�,and then each culture was divided into two portions.One portion was maintained at 27�, while the remainingportion was shifted to 33� for 4 hr. Cells were then fixedand examined by electron microscopy. At 27�, themorphology of gdi1-i11 mutant cells was almost in-distinguishable from wild-type cells, except that frag-mented vacuoles were often observed (data not shown).Upon temperature upshift to 33� for 4 hr, the appear-ance of gdi1-i11 mutant cells was clearly distinct fromthat of wild-type cells (Figure 4C). First, the accumula-tion of putative large post-Golgi vesicles ranging from100 to 150 nm in size (intensely stained after perman-ganate fixation) was observed around the Golgi struc-tures [Figure 4D(a)]. These large vesicles were oftenobserved in gdi1-i11 mutant cells (.50%), whereas theywere rarely observed in the wild-type cells. Second, theabnormal membranous structure, which may be aBerkeley body that represents abnormal Golgi mem-brane, was also observed [Figure 4D(b)]. Third, theabnormal electron-dense membranous structures thatwere �500 nm in diameter were observed and wereclosely connected to the Golgi structures [Figure4D(d)]. These structures were negligible in wild-typecells. Abnormal membranous structures related to theGolgi structures were observed in gdi1-i11 mutant cells(�3%), whereas they were seldom observed in the wild-type cells. Fourth, almost all the gdi1-i11 mutant cellsshowed fragmented vacuoles [Figure 4D(c)], whereasthe wild-type cells showed larger vacuoles (data notshown). As observed in the electron micrographs, itshould be noted that the abnormal findings in gdi1-i11mutant cells are similar to those that we previouslyreported in ypt3-i5 mutant cells (Cheng et al. 2002).Thus, these findings are consistent with our data thatYpt3 accumulated in membrane fraction in gdi1-i11mutant (Figure 2) and our finding that Gdi1 plays animportant role in recycling of Ypt3.

Gdi1 shows genetic interactions with the Sec14-likePITP Spo20: To identify proteins that function togetherwith Gdi1, or that bypass the requirement for Gdi1, weperformed a screen for gene dosage-dependent sup-pressors that could rescue the growth defect of the gdi1-i11 mutants at the nonpermissive temperature of 30�.This led to the isolation of several clones containing thespo201 gene. As shown in Figure 5A, the overexpressionof the spo201 gene suppressed the growth defect of thegdi1-i11 mutants at 30�, but not at 33�. The spo201 genefully suppressed the immunosuppressant sensitivity ofthe gdi1-i11 mutants.

1264 Y. Ma et al.

Figure 4.—Defects in membrane traffic in the gdi1-i11 mutant cells. (A) Defective secretion of SPL-GFP to the growth mediumin gdi1-i11 mutants. Wild-type cells and gdi1-i11 mutant cells expressing SPL-GFP were cultured at 27� or 33� for 4 hr or at 27� withor without the addition of FK506 for 8 hr. Proteins were separated by SDS–PAGE and analyzed by Western blotting using anti-GFPantibody. Extra: the trichloroacetic-acid precipitate of supernatant of 5 3 106 cells; Intra: cell extract of 1 3 106 cells. (B) Time-course analysis of LY internalization. Wild-type (wt) and gdi1-i11 mutant cells were incubated in the medium containing Luciferyellow (5 mg/ml) and the washed cells were viewed under a fluorescence microscope (LY) and a differential interference contrastmicroscope. Bar, 10 mm. (C) Electron microscopic analysis of gdi1-i11 mutant cells grown at 33� for 4 hr. (D) The boxed regions(a, b, and c) in C are enlarged. (a) Putative post-Golgi vesicles around Golgi structures. Bar, 1 mm. (b) An abnormal membranousstructure of a Berkeley body. Bar, 0.5 mm. (c) Fragmented vacuoles in gdi1-i11 mutant. Bar, 1 mm. (d) Abnormal electron-densemembranous structures closely connected to the Golgi structure. Bar, 1 mm.

Gdi1 and Spo20 in S. pombe 1265

We also investigated whether the overexpression ofthe spo201 gene could suppress the growth defects ofypt3-i5 and ryh1-i6 mutants. As shown in Figure 5B,overexpression of the spo201 gene failed to suppress thetemperature sensitivity and immunosuppressant sensi-tivity of these Rab mutants. Thus, Spo20 overexpressionsuppressed phenotypes of gdi1-i11 mutants, but notthose of ypt3-i5 and ryh1-i6 mutants, indicating thatSpo20 specifically suppressed the growth defects of gdi1-i11 mutants.

In fission yeast, the spo201 gene encodes Sec14-likePITP (Nakase et al. 2001), which thus far contains fivemembers, including spo201. Then, we examined theability of each of these gene members to suppress thetemperature sensitivity and immunosuppressant sen-sitivity of the gdi1-i11 mutants. The results clearlyshowed that overexpression of Spo20, but not of otherSec14 family members, suppressed the phenotypes ofthe gdi1-i11 mutants (Figure 5C), indicating that thesuppression of the phenotypes of the gdi1-i11

Figure 5.—The isolation of spo201 as a specific dosage-dependent suppressor of the gdi1-i11 mutant cells. (A and B) Overex-pression of Spo20-suppressed phenotypes of the gdi1-i11 mutant, but not those of the ypt3-i5 or ryh1-i6 mutant. The indicated cellstransformed with the control vector or spo201 were spotted onto each plate as indicated. Cells were spotted in serial 10-fold di-lutions starting with OD660 ¼ 0.3 of log-phase cells (5 ml). (C) Overexpression of Spo20, but not of other Sec14 family members,suppressed the phenotypes of the gdi1-i11 mutant. The gdi1-i11 mutants expressing control vector, SPBC23B6.04, SPBC3H8.02,SPBC365.01, SPBC589.09, and spo201 were spotted onto the indicated plates and then incubated for 4 days at 27� and 30�, re-spectively. (D) The gdi1-i11 mutant is synthetically lethal with the spo20-KC104 mutant. Tetrad analysis used progeny derived fromcrossing KP1893 (h1 his2 leu1-32 gdi1-i11) with KP2678 (h� leu1-32 ura4-D18 spo20-KC104). Colonies grown on YPD at 27 � werereplica plated onto various plates as indicated. Colonies that failed to grow on YPD at 33� and at 36� were predicted to be gdi1-i11(gdi1), and those that grew on YPD at 33� and grew very slowly at 36� were predicted to be spo20-KC104 (spo20). The rest of thecolonies that grew at either of the temperatures were predicted to be wild-type cells (wt). Spores that failed to grow were predictedto be gdi1-i11spo20-KC104 double mutants (D).

1266 Y. Ma et al.

mutant was specifically caused by the overexpression ofSpo20.

To further examine the genetic interaction betweenGdi1 and Spo20, we crossed the gdi1-i11 mutant with thespo20-KC104 mutant and performed tetrad analysis(Figure 5D). The spo20-KC104 mutant is a missenseallele of the essential spo201 gene that shows a temper-ature-sensitive growth defect and is defective in cell sepa-ration at the restrictive temperature (Nakase et al. 2001).The results showed that no double mutant was obtained,indicating that gdi1-i11 and spo20-KC104 mutations aresynthetically lethal and therefore that Spo20 activity isessential for the viability of gdi1-i11 mutants. Together,these data are indicative of a highly specific functionalrelationship between Gdi1 and Spo20.

Spo20 suppressed the defective membrane traf-ficking of the gdi1-i11 mutant cells: To determine ifoverexpression of Spo20 could rescue the membrane-trafficking defects of the gdi1-i11 mutants, first weexamined the effect of Spo20 overexpression on theabnormal localization of GFP-fused Syb1, the synapto-brevin in fission yeast (Edamatsu and Toyoshima

2003). As the secretory vesicle SNARE, Syb1 would beexpected to cycle between the cell surface and theendocytic pathway. In gdi1-i11 mutants harboring vec-tor, GFP-Syb1 failed to localize to the cell ends andaccumulated as large dots in the cytosol as comparedwith wild-type cells (Figure 6A). While in gdi1-i11 mu-tants harboring spo201, GFP-Syb1 was visible at the cellends and the accumulation of the large dots in thecytosol was greatly improved (Figure 6A, 1spo201). Thisresult indicated that Spo20 overexpression suppressedthe defective GFP-Syb1 localization of gdi1-i11 mutant.

Second, we examined the effect of Spo20 overexpres-sion on vacuole fusion, as fragmented vacuoles wereobserved in the electron micrographs [Figure 4D(c)].FM4-64 is a vital fluorescent dye that is internalized inliving cells and then accumulates at the vacuoles (Vida

and Emr 1995). When the cells were labeled with FM4-64 for 60 min, tiny vacuoles were observed in the gdi1-i11mutants (Figure 6B, �H2O, 1vector), while big vacu-oles were observed in wild-type cells (Figure 6B, �H2O,1gdi11). In a study by Bone et al. (1998), hypotonicstress causes a dramatic fusion of vacuoles in S. pombe.When cells were collected, washed, and resuspendedin water for 60 min, the wild-type cells had evidentlylarge vacuoles that resulted from vacuole fusion (Figure6B, 1H2O, 1gdi11), while vacuoles remained smalland numerous in gdi1-i11 mutants (Figure 6B, 1H2O,1vector). In contrast, gdi1-i11 mutants transformedwith spo201 contained large vacuoles Figure 6B, 1H2O,1spo201), indicating that overexpression of Spo20 par-tially but clearly suppressed the defective vacuole fusionof gdi1-i11 mutants.

Finally, we examined the effect of Spo20 overexpres-sion on defective cytokinesis. Cells grown to midlogphase at 27� in liquid YPD medium were subjected to a

temperature upshift or to the medium containing FK506at 27�. Upon shifting to 33� for 6 hr, the frequencyof septated cells in gdi1-i11 mutant cells significantlyincreased to 46% (Figure 6, left panels in C and D,1vector), whereas the septation index of wild-type cellsremained unchanged (Figure 6, left panels in C and D,1gdi11). Strikingly, the magnitude of the defect in gdi1-i11 mutant cells was even more pronounced after theaddition of FK506 for 6 hr, because nearly 66% of gdi1-i11 mutant cells were septated (Figure 6, right panels inC and D, 1vector) as compared with 34% of wild-typecells that were septated (Figure 6, right panels in Cand D, 1gdi11). As shown in Figure 6, C and D, Spo20overexpression partially suppressed the defect in cyto-kinesis at the restrictive temperature of 33�. Notably,Spo20 overexpression almost completely suppressed thedefective cytokinesis caused by FK506 treatment.

Phosphatidylinositol-transfer activity is dispensablefor the suppression of the phenotypes of gdi1-i11 mu-tants: To provide insight into the mechanism by whichSpo20 overexpression suppresses the phenotypes ofgdi1-i11 mutants, we examined whether Spo20 influen-ces the ability of Gdi1 to extract Rab from the mem-brane. The spo20-KC104 mutant cells integrated withGFP-Ypt3, harboring control vector or pREP1-GST-Gdi1,were cultured in EMM containing 4 mm thiamine for 8hr, and then each culture was divided into two portions.One portion was maintained at 27�, while the remainingportion was shifted to 36� for 4 hr. The cells werecollected and the fractionation experiment was per-formed. Notably, in spo20-KC104 mutant cells, Gdi1failed to extract Ypt3 from membrane when shifted to36�, although it maintains the ability at the permissivetemperature of 27� (Figure 7A). In wild-type cells, Gdi1maintains its ability to extract Ypt3 from membraneeven at 36� (data not shown). Thus, Spo20 is necessaryfor facilitating Gdi1 to extract Rab from membrane.

In a study by Phillips et al. (1999) cytosol fromSec14K66A- or Sec14K239A-expressing strains exhibiteda reduced phosphatidylinositol-transfer activity, andSec14K66AK239A-expressing strains exhibited no detectablephosphatidylinositol-transfer activity, but normal phos-phatidylcholine-transfer activity. To examine whetherphosphatidylinositol-transfer activity is necessary for sup-pression of the gdi1-i11 mutant, we created three mu-tants, namely Spo20K61A, Spo20K234A, and Spo20K61AK234A,on the basis of an analogy to budding yeast Sec14. Asshown in Figure 7B, all three mutant versions of Spo20suppressed FK506- and temperature-sensitive pheno-types of the gdi1-i11 mutant, and the suppression was thesame as that of wild-type Spo20. The result suggests thatphosphatidylinositol-transfer activity is dispensable forthe suppression of the phenotypes of gdi1-i11 mutants.

Deletion of the pct11 gene encoding a homolog ofcholine phosphate cytidyltransferase suppressed thephenotypes of gdi1-i11 mutants: Sec14p, budding yeasthomolog of Spo20, plays a key role in regulating inositol

Gdi1 and Spo20 in S. pombe 1267

Figure 6.—Spo20 suppressed the defective phenotypes associated with gdi1-i11 mutants. (A) Spo20 partially suppressed thedefective localization of GFP-Syb1 in the gdi1-i11 mutant cells. The gdi1-i11 mutant cells expressing chromosome-borne GFP-Syb1 were transformed with the control vector, gdi11, or spo201 and were examined under the fluorescence microscope. (B)Spo20 suppressed the defective vacuole fusion in the gdi1-i11 mutant cells. The gdi1-i11 mutant cells transformed with eachof the indicated plasmids were grown in YPD medium at 27�. Cells were harvested, labeled with FM4-64 fluorescent dye for60 min (see materials and methods), and then resuspended in water and examined by fluorescence microscopy. Photographswere taken after 60 min. Bar, 10 mm. (C) Spo20 suppressed the defective cytokinesis in the gdi1-i11 mutant cells. The gdi1-i11mutant cells expressing control vector, spo201, or gdi11 were shifted to the restrictive temperature (33�) for 6 hr, or FK506was added for 6 hr and incubated at 27� and then stained with Calcofluor to visualize cell wall and septum. Bar, 10 mm. (D) Per-centage of cells forming a division septum at each time point in the gdi1-i11 mutant cells expressing control vector, spo201, or gdi11

after the shift to 33� (left) or the addition of FK506 at 27� (right). Values are the average of three independent experiments with500 cells counted for each time point. Standard deviations between experiments were ,10%.

1268 Y. Ma et al.

and choline phospholipid metabolism (Huijbregts

et al. 2000; Li et al. 2000). The phosphatidylcholine-and phosphatidylinositol-bound forms of Sec14p areproposed to independently regulate inositol and cho-line phospholipid metabolism (Kearns et al. 1998).Phosphatidylcholine-bound Sec14p is hypothesized todownregulate flux through the diacylglycerol-utilizingcitidine 59-diphosphocholine (CDP-choline) pathway forphosphatidylcholine biosynthesis via inhibiting the ac-tivity of the PCT1 gene encoding choline phosphatecytidyltransferase, a rate-limiting enzyme. Assuming thatoverexpression of Spo20 may suppress the phenotypesof the gdi1-i11 mutant by inhibiting Pct1 activity, weexamined whether the pct1 deletion can rescue thephenotypes of gdi1-i11 mutants. As expected, the growthdefect of the gdi1-i11 mutant was rescued by pct1 dele-tion (Figure 7C) to the same extent as that of Spo20overexpression (Figure 5A), thus providing further evi-dence that Spo20 may affect Gdi1 function via modu-lating phosphatidylcholine metabolism.

DISCUSSION

Our study highlights the functional significance ofthe highly conserved Gly267 located outside the SCRs ofGdi1. More importantly, we discovered a genetic andfunctional interaction between Gdi1 and genes encod-ing proteins involved in phospholipid metabolism.

Highly conserved Gly267 is important for Gdi1 func-tion in vivo: Structural and mutational analyses of Gdi1have focused on the SCRs. SCR1 and 3B were impli-cated for Rab binding in vitro and in vivo (Schalk et al.1996; Luan et al. 1999). In S. cerevisiae, two mutants werereported to be defective in extracting Rabs from mem-brane. One mutant contained multiple mutations lo-cated in domain II, which consists of part of SCR2and SCR3A (Gilbert and Burd 2001), and the othermutant contained mutations in both SCR3B and SCR3A(Luan et al. 2000). However, the gdi1-i11 single sub-stitution Gly267Asp mutation is located outside theSCRs and the overexpression of Gdi1G267D protein failedto increase the cytosolic pool of Rabs, thus indicating adefect in extracting Rabs from membranes. However,the Gdi1G267D protein maintains the ability to bind Rabs.

Remarkably, the cytosolic Gdi1G267D protein dramati-cally decreased, yet maintained the ability to associatewith the membrane. This is the first report of an aminoacid change located outside SCRs affecting the intra-cellular distribution of Gdi1, and this might be a novelregulatory mechanism of Gdi1 function. It may not bemerely the charge of the aspartic acid residue thataltered the intracellular Gdi1 distribution; instead, themutation may cause a conformational change by in-troducing a bulkier group than the wild-type glycine.

Functional link between Gdi1 and Sec14-like phos-phatidylinositol transfer protein Spo20: Here, we

Figure 7.—Spo20 modulates Gdi1 function via regulationof phospholipid metabolism. (A) Spo20 affects extraction ofRab from membrane. The spo20-KC104 mutant cells inte-grated with GFP-Ypt3 harboring control vector or pREP1-GST-Gdi1 were cultured in EMM containing 4 mm thiaminefor 8 hr, and then each culture was divided into two portions.One portion was maintained at 27�, while the remaining por-tion was shifted to 36� for 4 hr. The cells were collected andthe fractionation experiment was performed as described inFigure 2. Endogenous Cdc4 was used as a loading control andwas immunoblotted using anti-Cdc4 antiserum. C, cytosolicfraction; M, membrane fraction. (B) phosphatidylinositol-transfer activity of Spo20 is dispensable for the suppressionof the gdi1-i11 mutant phenotype. The gdi1-i11 mutants ex-pressing the control vector, Spo20K60AK234A, Spo20K60A, Spo20-K234A, or wild-type Spo20 were spotted onto the plates asindicated and then incubated for 4 days at 27� and 30�, re-spectively. (C) The growth defect of the gdi1-i11 mutant waspartially rescued by pct1 deletion. Wild-type, gdi1-i11 mutant,Dpct1, and gdi1-i11Dpct1 cells were spotted onto each plate asindicated and then incubated for 4 days at 27� or 30� and for 3days at 33�, respectively.

Gdi1 and Spo20 in S. pombe 1269

identified Spo20 as a dosage-dependent suppressor ofthe gdi1-i11 mutation. Overexpression of Spo20 alsosuppressed the defects in membrane trafficking, in-cluding abnormal Syb1 localization, defective vacuolefusion, and defective cytokinesis.

How does the overexpression of Spo20 suppress thephenotypes of gdi1-i11 mutants? Spo20, similar to S.cerevisiae Sec14p, regulates the Golgi secretory functionin fission yeast (Nakase et al. 2001). Thus, it might bethat Spo20 suppressed phenotypes of gdi1-i11 by pro-moting the membrane trafficking from Golgi. However,Spo20 overexpression suppressed the growth defectsof gdi1-i11 mutants, but not those of ypt3-i5 or ryh1-i6mutants that demonstrate defects in Golgi membranetrafficking, indicating that the suppression by Spo20 ishighly specific to gdi1-i11 mutants and not caused by itseffects on Golgi trafficking.

To address the role of Gdi1 and its regulation bySpo20, we examined whether the phosphatidylinositol-transfer activity of Spo20 is required for suppression ofgdi1-i11. Surprisingly, our results showed that phospha-tidylinositol-transfer activity is dispensable for the sup-pression of gdi1-i11 phenotypes, suggesting that themechanisms of suppression include phosphatidylcho-line-transfer activity of Spo20. Consistently, the ability tosuppress gdi1-i11 is unique to Spo20 that was reported toexhibit both phosphatidylinositol- and phosphatidyl-choline-transfer activity (Nakase et al. 2001) and is notshared by other PITP-encoding genes, which were re-ported to exhibit only phosphatidylinositol-transfer ac-tivity in budding yeast.

To further investigate whether Spo20 modulates Gdi1function, we examined Rab membrane extraction byGdi1 in spo20-KC104 mutant cells. The wild-type Gdi1was able to extract Rabs from the membrane in the wild-type background, but failed to extract them in the spo20-KC104 mutant at the restrictive temperature. Thus, thecombination of mutations in spo20-KC104 and gdi1-i11,by causing severe reduction of Rab extraction frommembranes, would disrupt membrane trafficking me-diated by Rabs. This might explain the synthetic lethalitybetween spo20-KC104 and gdi1-i11. According to thestudy by Fovick and Novick (2000), a synthetic lethalitycan be interpreted as indicating that both genes func-tion in the same essential pathway if the mutation causespartial loss-of-function in cases where single null muta-tions are lethal. As single null mutations in gdi11 andspo201 are lethal, the synthetic lethal interaction be-tween gdi1-i11 and spo20-KC104 indicates that thesegenes function in the same essential pathway.

As mentioned above, phosphatidylcholine-transfer ac-tivity of Spo20 may be important in that it modulates thelipid composition of the membrane structure, and thisin turn affects the ability of Gdi1 to extract Rab. Consis-tent with these findings, deletion of the pct11 gene thatencodes a homolog of choline phosphate cytidyltransfer-ase, a rate-limiting enzyme in the diacylglycerol-utilizing

CDP-choline pathway for phosphatidylcholine biosynthe-sis, rescued the gdi1-i11 mutation to the same extent asthat of Spo20 overexpression. This further strengthensour hypothesis that Spo20 exerts its effect on the gdi1-i11mutant through regulation of phospholipid metabolism.

We thank Takashi Toda, Mitsuhiro Yanagida, Chikashi Shimoda,Kaoru Takegawa, and the National Bio-Resource Project for providingstrains and plasmids; Susie O. Sio for critical reading of themanuscript; Hideyuki Mukai for helpful suggestions; and FujisawaJapan for gifts of FK506. This work was supported by the 21st CenturyCenter of Excellence Program, the Asahi Glass Foundation, theUehara Memorial Foundation, and research grants from the Ministryof Education, Culture, Sports, Science and Technology of Japan.

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Communicating editor: P. Russell

Gdi1 and Spo20 in S. pombe 1271