Glycosylphosphatidylinositol-anchored Proteins AreRequired for the Transport of Detergent-resistantMicrodomain-associated Membrane ProteinsTat2p and Fur4p*

Received for publication, April 28, 2005, and in revised form, November 30, 2005 Published, JBC Papers in Press, December 15, 2005, DOI 10.1074/jbc.M504684200

Michiyo Okamoto, Takehiko Yoko-o, Mariko Umemura, Ken-ichi Nakayama1, and Yoshifumi Jigami2

From the Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST) , TsukubaCentral 6, Tsukuba, Ibaraki 305-8566, Japan

In eukaryotic cells many cell surface proteins are attached to themembrane via the glycosylphosphatidylinositol (GPI) moiety. Inyeast, GPI also plays important roles in the production of manno-protein in the cell wall. We previously isolated gwt1 mutants andfound that GWT1 is required for inositol acylation in the GPI bio-synthetic pathway. In this study we isolated a new gwt1 mutantallele, gwt1-10, that shows not only high temperature sensitivity butalso low temperature sensitivity. The gwt1-10 cells show impairedacyltransferase activity and attachment of GPI to proteins even atthe permissive temperature. We identified TAT2, which encodes ahigh affinity tryptophan permease, as a multicopy suppressor ofcold sensitivity in gwt1-10 cells. The gwt1-10 cells were also defec-tive in the import of tryptophan, anda lackof tryptophancaused lowtemperature sensitivity. Microscopic observation revealed thatTat2p is not transported to the plasmamembrane but is retained inthe endoplasmic reticulum in gwt1-10 cells grown under trypto-phan-poor conditions.We found thatTat2pwasnot associatedwithdetergent-resistant membranes (DRMs), which are required for therecruitment of Tat2p to the plasmamembrane. A similar result wasobtained for Fur4p, a uracil permease localized in the DRMs of theplasma membrane. These results indicate that GPI-anchored pro-teins are required for the recruitment of membrane proteins Tat2pandFur4p to the plasmamembrane viaDRMs, suggesting that somemembrane proteins are redistributed in the cell in response to envi-ronmental and nutritional conditions due to an association withDRMs that is dependent on GPI-anchored proteins.

Glycosylphosphatidylinositol (GPI)3 anchoring is a mechanism bywhich proteins are attached to the cell surface in all eukaryotic cells (1,2). GPI-anchored proteins have various physiological roles contributing

to transmembrane signaling, cell surface protection, cell adhesion, andcell wall synthesis (3, 4). In yeast, GPI-anchored proteins are majorcomponents of the cell wall, and they are essential for cell wall integrity(5, 6) and cell viability (7–9).The GPI anchor is synthesized from phosphatidylinositol (PI)

through multiple steps in the endoplasmic reticulum (ER). It is thentransferred to the C terminus of proteins bearing a GPI attachmentsignal sequence. The GPI anchor has a conserved core structure, NH2-CH2-CH2-PO4-6Man�1,2-Man�1,6-Man�1,4-GlcN�1,6Ins-PO4-lipid(2, 10), which can be modified by the addition of an acyl chain to theinositol and a ethanolamine phosphate to the mannose residues (11–14). The lipid moiety of the GPI anchor is also subject to modification,such as variation of the fatty acids, ceramide remodeling, or alkyl chainattachment (2). In yeast andmammalian cells, inositol acylation, mostlypalmitoylation, is the third step in theGPI anchor biosynthetic pathway,generating glucosaminyl-acyl-PI (GlcN-(acyl)PI) from glucosaminyl-PI.We previously reported thatGwt1p is required for this step in yeast (15).In mammalian cells a palmitoyl chain is added to glucosaminyl-PI byPIG-W, and acylation of inositol is critical for the attachment of GPI toproteins (16). In mammalian cells, GPI-anchored proteins are concen-trated in sphingolipid- and cholesterol-rich domains (17, 18). A distinc-tive feature of these domains is their resistance to extraction with deter-gent, typically Triton X-100 (TX-100). Therefore, they are referred to asdetergent-resistant membranes (DRMs). Some DRM-associated pro-teins are sensitive to TX-100 but resistant to other detergents such asCHAPS (19). These sphingolipid- and cholesterol-rich domains, alsoreferred to as lipid microdomains or lipid rafts, play key roles in signaltransduction and membrane trafficking (17, 20–24).In Saccharomyces cerevisiae, DRMs are composed of sphingolipids

and ergosterol, which is slightly different from cholesterol, and theycontain GPI-anchored proteins similar to those found in mammaliancells (25, 26). Although the physiological functions of GPI-anchoredproteins in DRMs of yeast cells are still unknown, lipid microdomainsare known to deliver a GPI-anchored protein, Gas1p, from the ER to theplasma membrane (25). The recent results obtained in sec18 cells, inwhich ER-to-Golgi transport is blocked in a secretory pathway, suggestthat the formation of DRMs and their association with certain proteins,including GPI-anchored proteins, are initiated in the ER (25, 27). Pre-sumably such domains contain ceramide as their sphingolipids becauseGPI-anchored proteins cannot associate with DRMs in the absence ofceramide (25) and because ceramide is required for the specific trans-port of GPI-anchored proteins from the ER to the Golgi (28–30). Cer-amide may help drive the incorporation of GPI-anchored proteins intoER-derived vesicles that are specific to the GPI-anchored proteins byparticipating in the formation of DRM structure in the ER.

* This study was supported by grants from New Energy and Industrial Technology Devel-opment Organization of Japan. The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Present address: The Health Technology Research Center, National Institute ofAdvanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Taka-matsu, Kagawa 761-0395, Japan.

2 To whom correspondence should be addressed: Research Center for Glycoscience,National Institute of Advanced Industrial Science and Technology (AIST), TsukubaCentral 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Tel.: 81-29-861-6160; Fax:81-29-861-6161; E-mail: jigami.yoshi@aist.go.jp.

3 The abbreviations used are: GPI, glycosylphosphatidylinositol; PI, phosphatidylinositol;GlcN-(acyl)PI, glucosaminyl(acyl)-PI; ER, endoplasmic reticulum; CoA, coenzyme A;DRMs, detergent-resistant membranes; TX-100, Triton X-100; CHAPS, 3-[(3-cholami-dopropyl)dimethylammonio]-1-propanesulfonate; SC, synthetic complete; mRFP,monomeric red fluorescent protein; GFP, green fluorescent protein; HA, hemaggluti-nin; ConA, concanavalin A; Ts�, high temperature sensitivity; Cs�, cold sensitivity;5-FU, 5-fluorouracil.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 7, pp. 4013–4023, February 17, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Besides GPI-anchored proteins, many plasma membrane transport-ers, such as Pma1p, Tat2p, Fur4p, and Can1p, have been reported to beassociated with DRMs, which are required for the precise delivery ofthese proteins to the plasma membrane (25, 27, 31–33). Tat2p is a highaffinity tryptophan permease whose cellular location is affected by theexternal tryptophan concentration. At high external tryptophan con-centrations, Tat2p membrane proteins are sorted to the early endo-somes and subsequently to the vacuole through the late endosomes,whereas at low tryptophan concentrations they are sorted to the plasmamembrane (31). In erg6 cells, which are defective in the synthesis ofergosterol, Tat2p cannot associate with DRMs and is inappropriatelypolyubiquitinated and mislocalized to the vacuole even at low trypto-phan concentrations (31). These results indicate that ubiquitination andassociation with DRMsmay be involved in the regulation of Tat2p sort-ing and that sterol may participate in this mechanism at the post-Golgistage.In this report we found that gwt1-10 cells show reduced tryptophan

uptake similar to erg6 cells and that this defect is caused by a failure ofTat2p to be recruited to the plasma membrane due to a lack of associ-ation with DRMs. Moreover, we show that GPI-anchored proteins mayplay an important role in the transport of membrane proteins thatchange their cellular localization in response to nutritional conditions.

EXPERIMENTAL PROCEDURES

Yeast Strains and Media—The strains used in this study are listed inTable 1. Except for the strains derived from RH401-7C, RH401-7D, andsec18-1, the mutant strains were all derivatives of W303. Cells weregrown in YPAD or synthetic complete (SC) medium (34). Geneticin-resistant yeast colonies were selected on YPAD plates containing 200�g/ml Geneticin (G418).

Gene Disruption and Gene Tagging—Deletions of BUL1 and ERG6were carried out by a PCR-based method using disruption cassettesderived from the plasmid pFA6a-HIS3MX6or pFA6a-kanMX6 (35) andthe following primers: for deletion of BUL1, BUL1-F (5�-CGAAAAG-AGACTGTTCGTGTGTGTCAACAGGTATATCGTACGCTAAC-GGATCCCCGGGTTAATTAA-3�) and BUL1-R (5�-ATCTATATC-TATAAGAAAAGTAACGAGAATTTTTTCTAATGTTTTTGAAT-TCGAGCTCGTTTAAAC-3�); and for deletion of ERG6, ERG6-F (5�-TTAAAAAAACAAGAATAAAATAATAATATAGTAGGCAGCA-TAAGCGGATCCCCGGGTTAATTAA-3�) and ERG6-R (5�-TATA-TCGTGCGCTTTATTTGAATCTTATTGATCTAGTGAATGAAT-TCGAGCTCGTTTAAAC-3�). Deletion of TAT2 was carried out byusing pKU41 (kindly provided by K. Umebayashi, National Institute ofGenetics) as described previously (31). C-terminal green fluorescentprotein (GFP)-tagging ofHxt1pwas carried out by a PCR-basedmethod

TABLE 1Yeast strains used in this studypMO10, YCpTAT2-mRFP; pMO12, YCpTAT2-HA; pMO13, YCp50-GFP-HDEL; pMO14, YCpGFP-HDEL; pMO24, YEpFUR4-GFP.

Strain Genotype Harboring plasmid SourceW303-1A MATa ade2-1 his3-11 ura3-1 leu2-3,112 trp1-1 can1-100 Dr. RothsteinW303D MATa/� ade2-1/ade2-1 his3-11/his3-11 ura3-1/ura3-1 Dr. Rothstein

leu2-3,112/leu2-3,112 trp1-1/trp1-1 can1-100/can1-100WDG2 MATa/� GWT1/gwt1::his5� Ref. 36gwt1-10 MAT� gwt1�::his5� URA3::gwt1-10 This studygwt1-16 MATa gwt1�:: his5� TRP1::gwt1-16 Ref. 15gwt1-20 MATa gwt1�:: his5� TRP1::gwt1-20 Ref. 15KE121 MATa gwt1�:: his5� URA3::gwt1-20 This studyMFY1 MATa gpi7�:: his5� Ref. 49RH401-7D MAT� URA3 leu2 his4 Ref. 9RH401-7C MAT� gaa1-1 URA3 leu2 Ref. 9YMO20 MATa bul1�::his5� This studyYMO21 MATa bul1�::his5� gwt1�::his5� URA3::gwt1-10 This studyYMO15-1 MATa gwt1�:: his5� YEp351-GWT1 (2�, LEU2) This studyYMO15-2 MATa gwt1�:: his5� pRS315-GWT1 (CEN, LEU2) This studyYMO18-1 MAT� gwt1�::his5� URA3::gwt1-10 YEp351 (2�, LEU2) This studyYMO18-2 MAT� gwt1�::his5� URA3::gwt1-10 YEp13-GWT1 (2�, LEU2) This studyYMO18-3 MAT� gwt1�::his5� URA3::gwt1-10 YEp13-TAT2 This studyYMO18-4 MAT� gwt1�::his5� URA3::gwt1-10 YEp351-UBP5 This studyYMO18-5 MAT� gwt1�::his5� URA3::gwt1-10 YEp351-UBP11 This studyYMO18-6 MAT� gwt1�::his5� URA3::gwt1-10 YEp351-EAF3 This studyYMO18-7 MAT� gwt1�::his5� URA3::gwt1-10 YEp351-SSB1 This studyYMO18-8 MAT� gwt1�::his5� URA3::gwt1-10 YEp351-YDR266C This studyYMO24-1 MATa tat2�::ADE2 pMO12, pRS314 (CEN, TRP1) This studyYMO24-2 MATa tat2�::ADE2 pMO10, pMO14 This studyYMO25-1 MATa gwt1�::his5� tat2�::ADE2 URA3::gwt1-10 pMO12, pRS314 This studyYMO25-2 MATa gwt1�::his5� tat2�::ADE2 URA3::gwt1-10 pMO10, pMO14 This studyYMO28-1 MATa gpi7�::his5� tat2�::ADE2 pMO10, pMO14 This studyYMO29-1 MATa gwt1�::his5� tat2�::ADE2 URA3::gwt1-20 pMO10, pMO14 This studyYMO31-1 MATa erg6�::kanMX6 tat2�::ADE2 pMO10, pMO14 This studyYMO32-1 MATa erg6�::kanMX6 gwt1�::his5� tat2�::ADE2 URA3::gwt1-10 pMO10, pMO14 This studyYMO34-1 MATa fur4�::his5� pRS316 (CEN, URA3) This studyYMO35-1 MATa pRS316 This studyYMO35-2 MATa pRS316, pMO24 This studyYMO36-1 MAT� gwt1�::his5� URA3::gwt1-10 pMO24 This studyYMO37 MATa HXT1::GFP::TRP1 This studyYMO38 MATa gwt1�::his5� URA3::gwt1-10 HXT1::GFP::TRP1 This studyYMO41 MATa sec18-1 leu2 URA3 trp1 his3 pMO12, pRS314 This studyYMO45-1 MAT� ura3 leu2 his4 pMO10, pMO13 This studyYMO45-2 MAT� ura3 leu2 his4 pMO12 This studyYMO45-3 MAT� ura3 leu2 his4 pMO24, pRS316 This studyYMO46-1 MAT� gaa1-1 ura3 leu2 pMO10, pMO13 This studyYMO46-2 MAT� gaa1-1 ura3 leu2 pMO12 This studyYMO46-3 MAT� gaa1-1 ura3 leu2 pMO24, pRS316 This studyMFY1-1 MATa gpi7�::his5� pRS316 This study

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using an integrative cassette derived from the plasmid pFA6a-GFP(S65-T)-TRP1 and primers HXT1-F (5�-ACCTAATGCATGATGACCAA-CCATTTTACAAGAGTTTGTTTAGCAGGAAACGGATCCCCG-GGTTAATTAA-3�) and HXT1-R (5�- TAAATACTGTATAAG-TCATTAAAATATGCATATTGAGCTTGTTTAGTTTAGAATTC-GAGCTCGTTTAAAC-3�).

Construction of Plasmids—The plasmid to produce the monomericred fluorescent protein (mRFP)-tagged Tat2p was constructed as fol-lows. The SpeI-SpeI fragment containing the open reading frame ofmRFP was prepared from pBlues-mRFP (kindly provided by T. Sumita,National Institute of Advanced Industrial Science and Technology) andsubcloned into the XbaI site of pKU42 (31) containing the TAT2 openreading frame to generate pMO9 (pUCTAT2-mRFP). The PstI-HindIIIfragment containingTAT2-mRFPwas cloned into the PstI-EcoT22I siteof pRS315 (CEN, LEU2) to generate pMO10 (YCpTAT2-mRFP). Theplasmid for the expression of TAT2 fused with three copies of thehemagglutinin (HA) epitope was derived from pKU46 (31). The EcoRI-PstI fragment containing TAT2-3HA was blunt-ended using a DNAblunting kit (Takara, Shiga, Japan) and then inserted into the SmaI siteof pRS315 to generate pMO12 (YCpTAT2-3HA).The plasmid to produce the GFP-HDEL fusion was constructed as

follows. The Kar2p signal-peptide sequence (the first 135 nucleotides oftheKAR2 gene), which bears a SalI site upstream of the initiation codonof KAR2 and a ClaI site at the C terminus, was amplified by PCR andinserted into pBluescript SK�. Next, the GFP gene modified to encodea C-terminal HDEL tetrapeptide, which bears a ClaI site at the N termi-nus and an XbaI site at the C terminus, was amplified by PCR andinserted into this plasmid. The SalI-XbaI fragment containing KAR2-GFP-HDELwas cloned into the YCp50 expression vector (CEN,URA3),which contains the TDH3 (which encodes glyceraldehyde-3-phosphatedehydrogenase) promoter and the actin terminator, to generatepMO13. The AatII-SphI fragment containing the TDH3 promoter andKAR2-GFP-HDEL was blunt-ended and then inserted in the SmaI siteof pRS314 (CEN, TRP1) to generate pMO14 (YCpGFP-HDEL).

The plasmid used to produce the Fur4p-GFP was constructed as fol-lows. The promoter and open reading frame region of FUR4, which hasthe SpeI site just before the stop codon at the C terminus, was amplifiedby PCR and inserted into YEp351 (2�, LEU2) containing a CMK1 ter-minator. Next, the XbaI-XbaI fragment containing GFP was preparedfrom pUC119-GFP and inserted into the SpeI site to generate pMO24(YEpFUR4-GFP).

Isolation of gwt1-10 Mutant Cells—Temperature-sensitive alleles ofGWT1were screened by PCRmutagenesis as described previously (15).The multiple missense mutations were separated by replacing thehomologous fragments of wild-type GWT1, and a gwt1-10 allele con-taining a single mutation that generates temperature sensitivity wasidentified. The gwt1-10 allele was subcloned into integration vectorpRS306 and then integrated into the ura3 locus on the chromosome ofWDG2 cells (36). Ura� transformants were selected, and gwt1-10 cellswere identified by segregating with His�, Ura�, and temperature sensi-tivity selection after the tetrad analysis.

[3H]Inositol Labeling of Proteins—Cells were grown to the early log-arithmic phase at 24 °C and then incubated at 24 or 16 °C for 12 h. Cellswere washed and resuspended in SC inositol-free medium containing0.67% yeast nitrogen base without inositol and amino acids (Bio101,Carlsbad, CA) and supplemented with 5% glucose and nutrient supple-ments, incubated at 24 °C or 16 °C for 30 min, mixed with 0.74 MBq ofmyo-[1,2-3H]inositol (1.11 TBq/mmol; American Radiolabeled Chemi-cals Inc., St. Louis,MO), and incubated at the indicated temperature for2 h. Proteins and lipids labeled with [3H]inositol were isolated as

described previously (15). Lipids were separated by thin-layer chroma-tography (TLC) in 65:25:4 (v/v) CHCl3/CH3OH/H2O, and proteinswere separated by SDS-PAGE (10% acrylamide gel). Labeled proteinswere detected after TLC and electrophoresis using a Molecular ImagerFX (Bio-Rad).

Biosynthesis of GPI Precursors in Vitro—ER-enriched membraneswere prepared from the cells grown in YPAD medium at 24 °C asdescribed previously (37). Membrane proteins (300 �g) were incubatedin TM buffer (50mMTris-HCl, pH 7.5, and 2mMMgCl2) containing 21�g/ml tunicamycin, 10�Mnikkomycin, and 0.5mMdithiothreitol in thepresence of 1 mM coenzyme A (CoA), and 1mMATP. The reaction wasinitiated by adding 3.7 kBq of UDP-[14C]GlcNAc (10.7 GBq/mmol;PerkinElmer Life Sciences) and then incubated for 1 h at 24 or 16 °C.The reaction was stopped by adding 1 ml of 1:1 (v/v) CHCl3/CH3OH toyield a final mixture of 10:10:3 (v/v) CHCl3/CH3OH/H2O. Radiolabeledlipids were separated by centrifugation, and the supernatants werepooled. The pellet was re-extracted with 10:10:3 (v/v) CHCl3/CH3OH/H2O. The confined lipid extracts were dried and desalted by n-butylalcohol extraction. Radiolabeled lipids were separated byTLC in 10:10:3(v/v) CHCl3/CH3OH/H2O and detected by autoradiography with aMolecular Imager FX.

Tryptophan Uptake Assay—The rate of import of radiolabeled tryp-tophan was measured as described previously (38). Cells were grown tothe early logarithmic phase in YPADmedium at 28 °C and divided intotwo aliquots. One aliquot was used for an assay at 28 °C, and the otherwas incubated for an additional 8 h at 16 °C and then used for an assay at16 °C. Cells were resuspended to an optical density at 600 nm (A600) of0.7 in a solution containing 10 mM sodium citrate, pH 4.5, 20 mM

(NH4)2SO4, and 2% glucose. The uptake assay was started bymixing 297�l of cell culture with 3 �l of radiolabeled tryptophan solution (1:100dilution of 1.11 Bq of [5-3H]tryptophan (1.18 TBq/mmol); AmershamBiosciences) at 28 or 16 °C. Aliquots (0.5 ml) of import reaction mix-tures were withdrawn at 0, 5, 10, 20, and 45min. Cells were filtered ontoa Whatman GF/C filter and washed twice with 2 ml of cold water. Thebound radioactivity was quantified using a liquid scintillation counterand corrected for differences in cell density.

Isolation of DRMs and Western Immunoblotting—Cells were grownto the logarithmic phase at 25 °C in tryptophan-free or uracil-free SCmedium. DRMs for the Tat2p analysis were isolated essentially asdescribed by Umebayashi and Nakano (31) with a slight modification.After incubation with 20 mM CHAPS (Sigma) for 30 min on ice, thelysates were subjected to Optiprep density gradient floatation by cen-trifuging for 5.5 h at 37,000 rpm in a SW55 Ti rotor (Beckman Instru-ments) at 4 °C. After centrifugation, nine fractions of equal volumewerecollected starting from the top. Each fraction was mixed with samplebuffer, incubated at 37 °C for 10 min, and resolved by 10% SDS-PAGE.Proteins were transferred to a polyvinylidene fluoride membrane forWestern blotting (Millipore, Billerica, MA). DRMs for the Fur4p anal-ysis were isolated as described by Dupre and Haguenauer-Tsapis (27).Western blotting was carried out using anti-HA monoclonal antibody16B12 (Berkeley Antibody Co., Berkeley, CA), anti-GFP monoclonalantibody JL-8 (BD Biosciences Clontech, Palo Alto, CA), anti-Pma1pmonoclonal antibody MCA-40B7 (Molecular Probes, Eugene, OR), oranti-vacuolar alkali phosphatase monoclonal antibody 1D3-A10(Molecular Probes) followed by horseradish peroxidase-conjugatedgoat anti-mouse IgG (Cell Signaling Technology, Beverly,MA). An ECLPlus kit (Amersham Biosciences) was used to visualize the immunore-active proteins.

Metabolic Labeling and Immunoprecipitation—Cells were grown tologarithmic phase at 24 °C in SDmediumwith low SO4

2� concentration

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(0.17% yeast nitrogen base without amino acids and ammonium sulfate,0.5% casamino acids, 5% glucose, nutrient supplements, and 200 �M

ammonium sulfate). Cells were centrifuged and resuspended in SD-SO4

2� medium (0.17% yeast nitrogen base without amino acids andammonium sulfate, 0.5% casamino acids, 2% glucose, nutrient supple-ments) lacking tryptophan at 10 A600 eq/ml. Cells were incubated at 24or 37 °C for 10 min and then labeled with 925 kBq of [35S]methionine/cysteine (37 TBq/mmol; Amersham Biosciences) per A600 equivalent ofcells for 40 min at each temperature. Radiolabeled proteins were sub-jected to CHAPS extraction and density gradient floatation as describedabove (see “Isolation of DRMs and Western Immunoblotting”). Frac-tions 2 and 3 were separated as insoluble fractions, and fractions 7 and 8were soluble fractions. For immunoprecipitation of Tat2-3HAp andGas1p, each fraction was divided in two halves, one of which was usedfor immunoprecipitation of Tat2-3HAp and the other for immunopre-cipitation of Gas1p. After adding SDS (final 1%), the samples for immu-noprecipitation of Tat2-3HAp were incubated at 37 °C for 10 min, andthose of Gas1p were incubated at 95 °C for 5 min. The samples werediluted in IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM

EDTA, and 1% TX-100) and precleared with Sepharose CL-4B (Sigma)at 4 °C for 30 min. After removal of Sepharose CL-4B by centrifugation,the supernatants were incubated overnight at 4 °Cwith 30�l of anti-HAantibody-bound affinity matrix (Roche Applied Science) or 1:150 rabbitanti-Gas1p antibody (kindly provided by K. Hata, Eisai co., Ltd.) andprotein G-agarose (Roche Applied Science). Immunoprecipitates werewashed three times with IP buffer and once with 0.2 mM Tris-HCl, pH7.5, and then resuspended in SDS sample buffer. After incubating at37 °C for 10 min (for Tat2-3HAp) or 95 °C for 5 min (for Gas1p), thesamples were separated by SDS-PAGE and analyzed using a MolecularImager FX.

Fluorescence Microscopy—To obtain fluorescence images of Tat2p-mRFP or Fur4p-GFP fusion proteins, cells were grown to the logarith-mic phase at 25 °C in tryptophan-free or uracil-free SC medium. Thecellular localization of Tat2p was observed using a microscope (Olym-pus, Tokyo, Japan) equipped with a confocal laser scanning unit(Yokogawa Electric, Tokyo, Japan). Images were obtained with a CCDcamera (Andor Technology, Belfast, UK) or a High-gain AvalancheRushing Amorphous Photoconductor camera (Hitachi Kokusai Electricand NHK, Tokyo, Japan) and processed by IPLab software (Scanalytics,Fairfax, VA). The cellular localization of Fur4p was observed using afluorescence microscope (Olympus)

RESULTS

Isolation of gwt1-10 Cells That ShowCold Sensitivity—Wepreviouslyisolated three gwt1mutants, gwt1-16, gwt1-20, gwt1-28, that show hightemperature sensitivity (Ts�) at 37 °C, and we showed that GWT1encodes an inositol acyltransferase involved in the biosynthesis of theGPI anchor (15). To further investigate the physiological role of the GPIanchor in yeast, we attempted to isolate other gwt1mutants that cannotsurvive at low temperatures. Using in vitro error-prone PCR, we gener-ated one mutant, gwt1-10, that has a single amino acid substitutionconferring a growth defect at 16 and 37 °C. In gwt1-10 cells, the eighthlysine of Gwt1p is replacedwith glutamic acid (K8E). This lysine residueis located in an N-terminal region conserved amongmany species fromyeast to human (Fig. 1A). Unlike other gwt1mutants, the gwt1-10 cellsexhibited not only the Ts� phenotype but also cold sensitivity (Cs�) at16 °C (Fig. 1B). The gwt1-10 cells grow slower than wild-type cells evenat the permissive temperature (30 °C). They also show sensitivity toCalcofluorWhite, a drug that affects the cell wall architecture, and theirgrowth defect at 37 °C was suppressed by adding an osmotic stabilizer,

0.3 M KCl, to the medium (Fig. 1B). Moreover, gwt1-10 cells were proneto lysis at 37 °C (data not shown). These observations indicate that thismutant has defects in the cell wall similar to othermutants related to thebiosynthesis of the GPI anchor (6, 39).

gwt1-10 Cells Show Decreased Inositol Acyltransferase Activity—TheGWT1 gene is required for the acylation of inositol in glucosaminyl-PIto form GlcN-(acyl)PI, an intermediate in the biosynthesis of GPIanchors (15). We measured the activity of inositol acylation in gwt1-10cells in vitro to determine whether the K8E mutation affects the acyl-transferase activity. Membranes from wild-type and gwt1-10 cells wereincubated with UDP-[14C]GlcNAc at 24 and 16 °C, and radiolabeledlipids were extracted and separated by TLC.Membranes prepared fromwild-type cells synthesized the acylated product, GlcN-(acyl)PI, in thepresence of CoA and ATP at 24 and 16 °C (Fig. 2A). Although mem-branes from gwt1-10 cells also synthesized GlcN-(acyl)PI at both tem-peratures, the amount produced by gwt1-10 membranes was muchlower than that produced by wild-type membranes even at the permis-sive temperature (24 °C) (Fig. 2A). There was no significant differencebetween the acyltransferase activities at 16 and 24 °C in gwt1-10 cells. At37 °C, GlcN-(acyl)PI was not synthesized from gwt1-10 membranes(data not shown).Inositol acylation is essential for the transfer of GPI anchors to pro-

teins (16).We, therefore, examined whether the incorporation of radio-labeled inositol into proteins is impaired in gwt1-10 cells. Because alldetectable protein-bound inositols are present as the GPI-attachedform in yeast (40), cells were labeled withmyo-[1,2-3H]inositol at 24 or16 °C for 2 h. Labeled proteins were prepared and subjected to SDS-PAGE. The gwt1-10 cells showed a decrease in inositol labeling of pro-teins compared with the wild-type cells at both 24 and 16 °C, althoughno significant difference was observed between the quantities of inosi-tol-labeled proteins at each temperature (Fig. 2B). This result indicatedthat gwt1-10 cells are defective in the transfer of GPI anchors to proteinseven at the permissive temperature (24 °C).

FIGURE 1. gwt1-10 mutation and phenotype. A, alignment of the amino acidsequences of GWT1 orthologs of S. cerevisiae, Schizosaccharomyces pombe, Candida albi-cans, human, mouse, and rat. Black and gray boxes indicate identical and similar aminoacids, respectively. In gwt1-10 mutant cells, the eighth residue, lysine (K), which is con-served among many organisms, is replaced with glutamic acid (E). B, phenotypes ofvarious gwt1 alleles. The sites of mutation in each gwt1 mutant were as follows: gwt1-10(K8E), gwt1-16 (N330S, L362P, and V479A), and gwt1-20 (W63R and V64A). Cells weregrown at the indicated temperatures on YPAD, YPAD supplemented with 7 �g/ml Cal-cofluor White (CFW), or YPAD supplemented with 0.3 M KCl.

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Isolation of a Multicopy Suppressor Gene for Cold Sensitivity ofgwt1-10 Cells—Most mutants defective in the biosynthesis of GPIanchors show a loss of cell wall integrity (6, 39). This was also observedin gwt1-10 cells (Fig. 1B). First, we screened for genes that suppress theTs� phenotype at 37 °C in gwt1-10 cells, and we obtainedGFA1, EXG1,and RHO2, which are involved in the biosynthesis of the cell wall (datanot shown). Because these suppressor genes did not suppress the Cs�

phenotype at 16 °C, it is conceivable that the cold sensitivity of gwt1-10cells was not caused by a defect in the integrity of the cell wall.To investigate what causes the death of gwt1-10 cells at low temper-

ature, we isolated multicopy suppressors for the cold sensitivity of thecells at 16 °C. We obtained five suppressor genes, TAT2, UBP5, EAF3,SSB1, and YDR266C (Fig. 3A). Tat2p is a high affinity tryptophan per-mease (41), and Ubp5p is a putative ubiquitin-specific protease whosefunction remains unknown (42). Eaf3p is a component of the NuA4histone acetyltransferase complex (43), and Ssb1p is a pair of Hsp70molecular chaperones that associate with ribosomes to fold nascentpolypeptide chains (44). Ydr266cp is a protein of unknown function thatcontains a RING finger domain. These genes did not suppress thegrowth defect of gwt1-10 cells at 37 °C (Fig. 3A).

TheCold Sensitivity of gwt1-10Cells Is Caused by an ImpairedUptakeof Tryptophan—Amulticopy suppressor ofTAT2was themost effectiveat suppressing the cold sensitivity of gwt1-10 cells (data not shown).Therefore, we focused on TAT2 to understand why gwt1-10 cells have aCs� phenotype. First, we measured the uptake of a radiolabeled trypto-phan into gwt1-10 cells (Fig. 3B). The relative rate of the uptake waslower than that in wild-type cells even at the permissive temperature(28 °C); the uptake by gwt1-10 cells at 45 min was �62% that of thewild-type cells. The difference in the uptake of tryptophan betweengwt1-10 and wild-type cells was much greater at 16 °C than at 28 °C; theuptake by gwt1-10 cells at 45 min was 30% that of the wild-type cells.These results suggest that the growth defect of gwt1-10 cells at 16 °Cmay be caused by impaired tryptophan uptake.

Recently, it was reported that the localization of Tat2p is regulated bythe external tryptophan concentration. At low concentrations of tryp-tophan, Tat2p is sorted to the plasma membrane to import tryptophaninto the cells, whereas at high concentrations of tryptophan, it is sortedto the vacuole to be degraded (31). erg6 cells, which are defective in a latestep of ergosterol biosynthesis, show decreased tryptophan importactivity and, consequently, a growth defect in synthetic medium con-taining even a standard concentration of tryptophan (20 �g/ml) (45).This growth defect was suppressed by the overexpression of TAT2,indicating that it was caused by the impaired function of Tat2p (31).These results suggested that the Cs� phenotype of gwt1-10 cells mightalso be caused by the impaired function of Tat2p. To examine thispossibility further, we determined whether the phenotype of gwt1-10cells is restored at a high concentration of tryptophan (200 �g/ml).Under such conditions, Tat2p is dispensable for cell growth, presum-

FIGURE 2. Decreased inositol acyltransferase activity in gwt1-10 cells. A, in vitro assayof the early steps in GPI anchor biosynthesis. Membrane fractions from wild-type (WT)and gwt1-10 cells were incubated with UDP-[14C]GlcNAc in the presence of ATP and CoAfor 1 h at the indicated temperatures. Radiolabeled lipids were extracted and analyzedby TLC in 10:10:3 (v/v) CHCl3/CH3OH/1 M NH4OH and analyzed by autoradiography. B, theincorporation of radiolabeled inositol into proteins is impaired in gwt1-10 cells. Afterincubating at 24 or 16 °C, wild-type and gwt1-10 cells were incubated in inositol-freemedium at each temperature for 30 min and then labeled with myo-[1,2-3H]inositol for2 h. The mannoprotein-enriched samples were isolated from whole lysates of labeledcells by concanavalin A-Sepharose chromatography. Purified glycoprotein extracts wereseparated by SDS-PAGE, and radiolabeled GPI-anchored proteins were visualized byautoradiography.

FIGURE 3. Multicopy suppressors of cold sensitivity in gwt1-10 cells and tryptophanuptake in gwt1-10 cells. A, effect of multicopy suppressor genes on the growth defectof gwt1-10 cells. gwt1-10 cells harboring a multicopy plasmid carrying the indicated geneor an empty vector were spotted onto SC�Leu medium and grown either for 3 days at 30or 37 °C or for 6 days at 16 °C. B, tryptophan uptake is impaired in gwt1-10 cells. Cells weregrown to the early logarithmic phase in YPAD medium at 28 °C and then for 8 h at 16 or28 °C. Uptake of [3H]tryptophan was measured at the indicated temperature and timepoints. The amount of imported tryptophan was normalized by the number of cells,which was determined from the A600 (OD). The results represent the means � S.D. fromthree independent experiments. WT, wild type.

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ably because another amino acid permease, possibly Tat1p, a low affin-ity tryptophan permease, can take up sufficient amounts of tryptophan.In support of this, the growth of erg6� cells is recovered at high concen-trations of tryptophan (31). As with erg6� cells, the growth of gwt1-10cells at 16 °C was restored at a high concentration of tryptophan (200�g/ml), whereas it did not recover at 37 °C (Fig. 4A), indicating againthat the cell death at 16 °C was due to the impaired uptake oftryptophan.It is reported that the severe tryptophan auxotrophy of erg6� cells is

suppressed by the disruption of BUL1, which encodes a component ofthe Rsp5p ubiquitin ligase complex (46) that is required for the polyubiquitination of Tat2p (31). In bul1� cells, the polyubiquitination ofTat2p is inhibited, and then free Tat2p is sorted to the plasma mem-brane (31). We, therefore, examined the effect of the BUL1 disruptionon the temperature sensitivity of gwt1-10 cells (Fig. 4B). The deletion ofBUL1 suppressed the Cs� phenotype but not the growth defect at 37 °C(Fig. 4B, bul1� gwt1-10). These results also suggest that the growthdefect of gwt1-10 cells at 16 °C is caused by the impaired function ofTat2p.

Export of Tat2p from the Endoplasmic Reticulum Is Impaired in CellsThat Are Defective in Several Steps of GPI Anchor Biosynthesis—At ahigh tryptophan concentration, Tat2p is transported from the trans-Golgi to the late endosomes through the early endosomes, which iswhere tryptophan-dependent sorting of Tat2p occurs (31). In erg6�cells, the impaired function of Tat2p is due to a defect in this sortingprocess that results in the missorting of Tat2p to the vacuole even at alow tryptophan concentrations (31), suggesting that ergosterol isrequired for the transport of Tat2p at the post-Golgi stage.To address whether the impaired function of Tat2p in gwt1-10 cells is

caused by the same factor as in erg6� cells, we examined the localizationof Tat2p under tryptophan-free conditions using a fusion protein ofTat2p linked at its C terminus to mRFP (47) and expressed under con-

trol of the TAT2 promoter. Expression of the Tat2p-mRFP fusion sup-pressed the growth defect of tat2� cells, indicating that it is functional(data not shown). Tat2p-mRFP fluorescence was detected at the plasmamembrane in wild-type cells grown in tryptophan-free medium at thepermissive temperature, whereas the fluorescence was detected in theintracellular organelles in gwt1-10 cells (Fig. 5, A and B). The coexpres-sion of Tat2p-mRFP with GFP-HDEL, an ER marker protein, indicatedclearly that Tat2p-mRFPwas located at the ER in gwt1-10 cells (Fig. 5B).The localization of Tat2p to the ER in gwt1-10 cells was also observed atthe restrictive temperature, 16 °C (data not shown). These results indi-cate that Tat2p is not transported to the plasma membrane in gwt1-10cells. However, unexpectedly, the localization of Tat2p in gwt1-10 cellswas different from that in erg6� cells; Tat2p is localized to the ER ingwt1-10 cells, whereas it is distributed to the vacuole in erg6� cells (Figs.5, B and C). The localization of Tat2p in gwt1-10 cells suggests thatGWT1 is involved in its localization in a different way from ERG6.Furthermore, Tat2p-mRFP is localized to the ER in erg6� gwt1-10 dou-ble-mutant cells (Fig. 5D), indicating thatGWT1 functions at an earlierpoint than ERG6 in the Tat2p transport pathway.Next, we investigated whether the accumulation of Tat2p in the ER is

unique to gwt1-10 cells or is common to other GPI mutants. Tat2p-mRFP fluorescence was observed in gwt1-20 cells, which showed astrong defect in the biosynthesis of GPI-anchored protein at the restric-tive temperature but only a weak defect at the permissive temperature(15). In gwt1-20 cells in tryptophan-freemedium at the permissive tem-perature (25 °C), Tat2p was localized at the plasma membrane, similar

FIGURE 4. The cold sensitivity of gwt1-10 cells is suppressed by a high concentrationof tryptophan and disruption of BUL1. A, the growth defect of gwt1-10 cells at 16 °Cwas suppressed by the addition of tryptophan to the medium. Wild-type (WT), andgwt1-10 cells were grown in SC medium containing the indicated concentration of tryp-tophan (200 �g/ml corresponds to a high concentration and 20 �g/ml to a standardconcentration). B, effect of BUL1 deletion on the Cs� phenotype of gwt1-10 cells. Wild-type, gwt1-10, bul1� (YMO20), and bul1� gwt1-10 (YMO21) cells were grown on SC platessupplemented with a standard concentration of tryptophan (20 �g/ml) at the indicatedtemperatures. FIGURE 5. The export of Tat2p from the endoplasmic reticulum is impaired in

gwt1-10 cells. Cells harboring YCpTAT2-mRFP and YCpGFP-HDEL (YMO24-2, YMO25-2,YMO31-1, and YMO32-1) were grown in tryptophan-free SC medium at 25 °C. Fluores-cence from mRFP and GFP was observed with a confocal laser scanning microscope. Afusion protein, GFP-HDEL, was used as a marker for ER-associated protein. All strains usedin this experiment have the tat2� genetic background. A, in wild-type cells (WT) Tat2p-mRFP was localized at the cell surface. B, in gwt1-10 cells Tat2p-mRFP was mainly local-ized in the ER. C, in erg6� cells Tat2p-mRFP was missorted to the vacuole. D, in erg6�gwt1-10 cells, Tat2p-mRFP was distributed mainly in the ER and partially in the vacuole.

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to the case in wild-type cells. In contrast, Tat2p was localized in the ERof these cells when they were shifted to 37 °C for 30min (Fig. 6A). Theseresults indicate that the decrease in inositol acyltransferase activity gen-erally causes an impairment in the export of Tat2p from the ER.To further examine whether other mutant cells with defects in GPI-

anchored biosynthesis have the same phenotype as gwt1-10 cells, weexamined the cellular localization of Tat2p in gaa1-1 cells. Gaa1p is anessential component of GPI transamidase, which mediates attachmentofGPI anchor to proteins, and gaa1-1 cells are reported to have a greatlyreduced incorporation of inositol into proteins even at a permissivetemperature (9). In gaa1-1 cells, Tat2p-mRFP was observed in the ERand the vacuole under tryptophan-free conditions at a permissive tem-perature (25 °C) (Fig. 6B). We also found the same mislocalization ofTat2p in gpi7� cells (data not shown), which have defects in the additionof ethanolamine phosphate to theGPImoiety during the biosynthesis ofthe GPI anchor (48) and in the localization of daughter cell-specificGPI-anchored proteins (49). Moreover, we found that gpi7� cells also

show cold sensitivity at 16 °C and that the Cs� phenotype of gpi7� cellsis suppressed by the addition of a high concentration of tryptophan,similar to gwt1-10 cells (data not shown). Taken together, these resultsimply that GPI-anchored proteins are required for the transport ofTat2p, independent of ergosterol.

Tat2p Is Not Sorted to the DRMs in Mutant Cells Defective in theGPI-anchored Proteins—It is reported that the association of Tat2pwithDRMs is required for the delivery of Tat2p to the plasma membrane(31). In yeast, DRMs are rich in phosphoinositol-based sphingolipidsand ergosterol (25). In erg6� cells, Tat2p cannot associate with DRMs,probably because their properties are altered by the accumulation ofergosterol intermediates (31). Because GPI-anchored proteins are alsocontained in DRMs along with sphingolipids and ergosterol, we inves-tigated whether Tat2p associates with DRMs in gwt1-10 cells andgaa1-1 cells.Wild-type, gwt1-10, and gaa1-1 cells were mechanically disrupted

and then extracted with 20 mM CHAPS at 4 °C, and the extracts werefractionated by centrifugation on an Optiprep density gradient. Pma1p,a control DRM-associated protein, was located primarily in fractions 2and 3 in these strains. In wild-type cells, Tat2p is also found predomi-nantly in fractions 2 and 3, indicating its association with DRMs (Fig. 7,A and B). The quantity of Tat2p associated with DRMs was notablyreduced in gwt1-10 cells (Fig. 7A), and the association of Tat2p withDRMs was not maintained in gaa1-1 cells (Fig. 7B). These results indi-cate that GPI-anchored proteins may be required for the association ofTat2p with DRMs. However, we cannot exclude the possibility thatTat2p is unable to associate with DRMs in gwt1-10 and gaa1-1 cellsbecause it is not transported to the appropriate compartment.To address this possibility, we performed a pulse experiment with

35S-labeled amino acid in sec18-1 temperature-sensitive mutant cells(50) in which ER-to-Golgi trafficking is blocked at 37 °C (Fig. 8). Weincubated the sec18-1 cells at the permissive (24 °C) and restrictive(37 °C) temperatures and examined the detergent insolubility of Tat2p.As reported previously (25), Gas1p was present in the insoluble fraction

FIGURE 6. Phenotypes of the mutants with defects in GPI-anchor biosynthesis. A,wild-type (WT) and gwt1-20 cells harboring YCpTAT2-mRFP and YCpGFP-HDEL in thetat2� genetic background (YMO24-2 and YMO29-1) were grown in tryptophan-free SCmedium at 25 °C and then shifted to 37 °C for 30 min. In gwt1-20 cells Tat2p-mRFP waslocalized at the plasma membrane at 25 °C, but it was localized in the ER at 37 °C. B, thegaa1-1 cells harboring YCpTAT2-mRFP and YCpGFP-HDEL (YMO46-1) were grown intryptophan-free SC medium at 25 °C. Tat2p-mRFP was localized in the ER and thevacuole.

FIGURE 7. Analysis of DRMs in gwt1-10 and gaa1-1 cells. The cells were grown to theexponential phase at 25 °C in tryptophan-free SC medium. The cells were disrupted withglass beads, extracted with CHAPS, and subjected to Optiprep density gradient centrif-ugation. Nine fractions were collected and analyzed by Western blotting with antibodiesagainst HA, Pma1p, and Pho8p. Pma1p was used as a marker for DRM-associated pro-teins, and Pho8p was used as a marker for non-DRM-associated proteins. Fractions 2 and3 contained DRMs. A, wild-type (WT) and gwt1-10 cells harboring YCpTAT2–3HA andpRS314 in the tat2� genetic background (YMO24-1 and YMO25-1). B, wild-type (RH401-7D) and gaa1-1 (RH401-7C) cells harboring YCpTAT2-3HA (YMO45-2 and YMO46-2).

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(I in Fig. 8) at 37 °C as a 105-kDa ER form (precursor (p)). Tat2pwas alsopresent in the insoluble fraction even if the protein transport from theER to the Golgi was blocked at 37 °C, indicating that the association ofTat2p with DRMs occurs in the ER. Therefore, we concluded that adecrease of GPI-anchored proteins results in the failure of Tat2p toassociate with DRMs.

gwt1-10 Cells Are Defective in the Functions of Other DRM-associatedProteins—Wenext examinedwhether proteins other thanTat2p, which isassociatedwithDRMs, are affectedby thedefect inGPI-anchoredproteins.For these studies we focused on another transmembrane protein, Fur4p,which functions as a uracil permease. Fur4p is also known to be associatedwith DRMs (27), and in response to exogenous uracil, it is sorted directlyfrom the Golgi to the endosomal system via a process regulated by Rsp5p-dependent ubiquitination and without passage through the plasma mem-

brane (51). To examine the effects on Fur4p, we first investigated whethergwt1-10 and gpi7� cells exhibit resistance to 5-fluorouracil (5-FU), a toxicanalog of uracil. The fur4� cellswere resistant to 5-FUbecause they cannotimport it due to a lack of uracil permease. Interestingly, both gwt1-10 andgpi7� cells were also resistant to 5-FU (Fig. 9A), indicating that a defect inthe biosynthesis of the GPI anchor affects the function of Fur4p.To address whether the resistance to 5-FU in gwt1-10 and gpi7� cells

is caused by themislocalization of Fur4p, we examined the fluorescencein cells transformed with a high copy number plasmid for expressingGFP-tagged Fur4p under the control of the FUR4 promoter. We con-firmed that this plasmid is functional because the growth defect of fur4�cells in low uracil medium was suppressed by the introduction of theplasmid (data not shown). In exponentially growing wild-type cells,Fur4p-GFP fluorescence was present mainly at the plasma membranewith lesser amounts in the vacuole, whereas the fluorescence in gwt1-10cells was predominantly localized in the vacuole (Fig. 8B). The sameresults were obtained for gpi7� cells (data not shown).We also examined the cellular localization of Fur4p in gaa1-1 cells.

Unlike wild-type cells, Fur4p-GFP in gaa1-1 cells was mainly present inthe intracellular compartments (Fig. 9D). The gaa1-1 cells have beenreported to be defective in endocytosis and to accumulate an abnormalorganelles that seem to be endosomes (52). Therefore, we speculatedthat Fur4p-GFP is transported to abnormal endosomes and fragmentedvacuoles, which are probably formed as a result of defects in endocytosisin gaa1-1 cells. As shown in Figs. 9, B andD, the recruitment of Fur4p tothe plasma membrane was impaired in gwt1-10 and gaa1-1 cells. Thus,it is likely that the transport of Fur4p to the plasma membrane in thesemutant cells is defective due to the lack of GPI-anchored proteinsrequired for the transport of Fur4p.

FIGURE 8. Newly synthesized Tat2p associates with lipid rafts in the ER. The sec18-1cells harboring YCpTAT2-3HA (YMO41) were incubated at 24 or 37 °C for 10 min and thenradiolabeled for 40 min at 24 or 37 °C. The cells were disrupted with glass beads,extracted with CHAPS, and subjected to Optiprep density gradient centrifugation. Ninefractions were collected, and Tat2p-3HA and Gas1p were immunoprecipitated from frac-tions 2 and 3 (insoluble (I)) and from fractions 7 and 8 (soluble (S)) (see Fig. 7) and thenanalyzed by SDS-PAGE and phosphorimaging. ER-to-Golgi transport was monitored bychecking the processing of Gas1p from precursor (p) to mature (m) form, confirming thatits transport is blocked in sec18-1 cells shifted to 37 °C.

FIGURE 9. Influence of GPI-anchor mutations onthe localization of Fur4p. A, the indicated cells(YMO35-1, YMO34-1, gwt1-10, and MFY1-1) wereincubated for 3 days at 30 °C on uracil-free SCplates containing (5 �M) or lacking 5-FU. B, cellularlocalization of Fur4p in wild-type (WT) and gwt1-10cells. The cells harboring YEpFUR4-GFP (YMO35-2and YMO36-1) were grown in uracil-free SCmedium at 25 °C. In wild-type cells Fur4p-GFP wasmainly localized at the cell surface, whereas ingwt1-10 cells it was mainly localized in the vacuole.DIC, differential interference contrast. C, associa-tion of Tat2p with lipid rafts in wild-type andgwt1-10 cells. YMO35-2 and YMO36-1 cells weregrown under the same conditions as in panel B.The cells were analyzed as shown in Fig. 7 afterextraction with TX-100. Six fractions were col-lected from the gradient. Fraction 1 containedDRMs. D, cellular localization of Fur4p in wild-type(RH401-7D) and gaa1-1 (RH401-7C) cells. Cells har-boring YEpFUR4-GFP (YMO45-3 and YMO46-3)were grown under the same conditions as in panelB. In wild-type cells Fur4p-GFP was mainly local-ized at the cell surface, whereas in gaa1-1 cells itwas mainly localized in the vacuole. E, the associa-tion of Tat2p with lipid rafts in wild-type andgaa1-1 cells. DRM analysis was performed inYMO45-3 and YMO46-3 cells and as described inpanel C.

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Next, to investigate whether the mislocalization of Fur4p in gwt1-10and gaa1-1 is caused by a failure of Fur4p to associate with DRMs, weexamined the solubility of Fur4p in 1%TX-100 at 4 °C in cells expressingthe Fur4p-GFP fusion protein. As reported previously, in wild-typecells, we found that Fur4p is present in the detergent-resistant fraction(fraction 1 in Fig. 9C). This fraction also contained Pma1p, a knownraft-associated protein. In gwt1-10 cells, Fur4p was hardly detected inthe detergent-soluble fraction (fractions 5 and 6 in Fig. 9C) and was notdetected in the detergent-resistant fraction (fraction 1). Rather, wedetected low molecular weight proteins corresponding in size to freeGFP in the detergent-soluble fraction (fractions 4–6). As described pre-viously (27), it is conceivable that the free GFP arises from the degrada-tion of Fur4p-GFP in the vacuole because GFP tends to be resistant tothe vacuolar protease. Although free GFP was detected in both wild-type and gwt1-10 cells, the amount of free GFP was higher in gwt1-10cells than in wild-type cells (Fig. 9C), indicating that, in gwt1-10 cells,Fur4p-GFP cannot associate with DRMs and is degraded in the vacuole.We also obtained the same results in gaa1-1 cells. Although there was ahigh level of Fur4p-GFP in the detergent-resistant fraction (fraction 1 inFig. 9E) of wild-type cells, it was not detected in any of the fractions ingaa1-1 cells; instead, free GFP was detected in the detergent-solublefraction (fractions 4–6 in Fig. 9E), as observed in gwt1-10 cells (Fig. 9C).Therefore, we concluded that GPI-anchored proteins are required forthe transport of both Fur4p and Tat2p via DRMs.Finally, we ascertained whether the defect of GPI-anchored protein

biosynthesis is specific to the DRM-associated membrane proteins. Wefocused on the hexose transporter, Hxt1p, which is not associated withDRMs (33). We constructed cells carrying chromosomal Hxt1p fusedC-terminally to GFP and used them to examine the cellular localizationof Hxt1p. In both wild-type and gwt1-10 cells, Hxt1p-GFP was specifi-cally localized at the cell surface (Fig. 10), indicating that GPI-anchoredproteins are required for the transport ofDRM-associated proteins suchas Tat2p and Fur4p but not generally required for the transport of thenon-DRM-associated membrane protein, Hxt1p.

DISCUSSION

In the current studies we investigated the role of GPI-anchored pro-teins by analyzing gwt1-10 cells, which show temperature sensitivity atboth 37 and 16 °C. In gwt1-10 cells, inositol acyltransferase activity isdecreased in vitro, and the transfer of the GPI anchor onto proteins isdecreased even at a permissive temperature in vivo. Many kinds ofmutant cells known to be defective in the biosynthesis of GPI anchors

show a severe defect in the cell wall at their restrictive temperatures (6).Therefore, in such cells, it is difficult to find a newphysiological functionfor GPI-anchored proteins other than the maintenance of cell wallintegrity. In this report we showed that the Ts� phenotype of gwt1-10cells is caused by cell wall defects similar to other mutant cells defectivein the biosynthesis of GPI anchors. However, Cs� suppressor genescould not suppress theTs� phenotype, indicating that the causes of coldand high temperature sensitivity are different. Therefore, we consideredthat investigating the cause of Cs� phenotype in gwt1-10 cells shouldhelp determine what other physiological roles are played by GPI-an-chored proteins besides maintenance of cell wall integrity.

TheReason for theCold Sensitivity of gwt1-10Cells—Tounderstandwhygwt1-10 cells are sensitive to the cold, we focused on TAT2, which sup-presses the Cs� (16 °C) phenotype most effectively among several isolatedCs� suppressor genes. Tat2p is a high affinity tryptophan permease and isknown to be delivered from the trans-Golgi to the early endosomes, wheretryptophan-dependent sorting occurs. When there is a low concentrationof tryptophan in the medium, Tat2p is sorted to the plasma membrane,whereas at a high tryptophan concentration, Tat2p is polyubiquitinatedand sorted to the vacuole through the late endosomes (31). The polyubiq-uitination of Tat2p is performed by the Rsp5p-Bul1p ubiquitin ligase com-plex, and the deletion ofBUL1 is reported to cause a redirection ofTat2p tothe plasma membrane even at high tryptophan concentrations (31). Wefound that the uptake of tryptophan is decreased in gwt1-10 cells and thatthe Cs� phenotype is suppressed by the addition of tryptophan to themedium, suggesting that theCs�phenotype is causedby an impaired func-tion of Tat2p.Moreover, we found that, at low tryptophan concentrations,Tat2p is mainly localized to the ER rather than the plasma membrane ingwt1-10 cells. This indicates that a loss of Tat2p function in gwt1-10 cells isdue to itsmislocalization. BecauseTat2p is required for cell growth at a lowtemperature (15 °C) (53, 54), it is quite conceivable that theCs� phenotypeof gwt1-10 cells is caused by the reduced uptake of tryptophan due to themislocalization of Tat2p. If Tat2p is accumulated entirely in the ER, it isunexpected that the Cs� phenotype of gwt1-10 cells is suppressed by theBUL1 deletion. We suspect that Tat2p is not entirely blocked in the ER ingwt1-10 cells and that a small amount of it is sorted to the vacuole. In bul1�cells, this Tat2p would be sorted to the plasmamembrane.

GPI-anchored Proteins Are Required for the Recruitment to thePlasmaMembrane and DRMAssociation of Tat2p—The association ofTat2p with DRMs is required for its transport to the plasma membrane(31). In yeast, themajor components of DRMs are ergosterol and sphin-golipids. In erg6� cells and erg13� cells, which have defects in ergosterolsynthesis, Tat2p cannot associate with DRMs, and at a low concentra-tion of tryptophan, it is transported to the vacuole instead of the plasmamembrane (31). Ergosterol is thought to function in the post-Golgisorting of Tat2p. In this study, we found that the phenotype of thegwt1-10 cells was similar to that of erg6� cells, including reduced tryp-tophan uptake and suppression of the Cs� phenotype by a high concen-tration of tryptophan or the deletion ofBUL1.We also found that Tat2pcannot associate with DRMs in gwt1-10 or gaa1-1 cells. Gaa1p isrequired for the attachment of a completed GPI anchor to proteins. It ispossible that ergosterol or sphingolipids could be abnormal in gwt1-10and gaa1-1 cells, resulting in the failure of Tat2p to associate withDRMs. However, we think it unlikely based on the following two points.First, the localization of Tat2p is different between erg6� cells andgwt1-10 or gaa1-1 cells; Tat2pwasmainly localized to the ER in gwt1-10and gaa1-1 cells, whereas it is found in the vacuole in erg6� cells. Theseresults suggested that GPI-anchored proteins are required for the trans-port of Tat2p from the ER to the Golgi (Fig. 11, 1a) and that theyfunction independently of ergosterol in the transport of Tat2p (Fig. 11,

FIGURE 10. The transport of Hxt1p is normal even in gwt1-10 cells. WT (YMO37) andgwt1-10 (YMO38) cells expressing HXT1-GFP were grown in SC medium at 25 °C. Hxt1p-GFP was localized at the plasma membrane in both cells. DIC, differential interferencecontrast.

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1b). The finding that Tat2p is localized to the ER in erg6� gwt1-10 cellsclearly supports this possibility. Second, lipid analysis did not identifyany abnormalities of sphingolipids in gaa1-1 cells (9, 48) and gwt1-10cells.4 From these results we conclude that the dissociation of Tat2pfrom DRMs found in gwt1-10 and gaa1-1 cells is caused by defects inGPI-anchored proteins and not by defects in ergosterol or sphingolip-ids. Although someGPI-anchored proteins are associatedwithDRMs, itwas not known that GPI-anchored proteins are involved in the associ-ation of membrane proteins with DRMs as well as ergosterol and sphin-golipids. To our knowledge this is the first report suggesting that GPI-anchored proteins participate in the sorting of membrane proteins.Why does the dissociation of Tat2p from DRMs cause a delay in the

export of Tat2p from the ER in cells that are defective in GPI synthesis?GPI-anchored proteins are expected to be exported from theERby specificvesicles that are distinct from those for other secretory proteins, such as�-factor precursor and the general amino acid permease, Gap1p (55). Onthe basis of the following reports, it is predicted that ceramide-richmicrodomains are involved in the export of GPI-anchored proteins from

the ER (56); (i) in yeast, newly synthesized GPI-anchored protein Gas1p isrecruited to lipid rafts in the ER (25), (ii) Gas1p cannot associate with lipidrafts in lcb1-100 cells, which have a defect in ceramide biosynthesis (25),and (iii) ceramide is required for the transport of GPI-anchored proteinsfrom the ER to the Golgi (29, 30). Also, all precursors of sphingolipids aresynthesized in the ER. Based on these findings, we postulate that someGPI-anchored proteins are selectively recruited to ceramide-richmicrodo-mains (see Fig. 11) because of the physical properties of the GPI anchorsand that theyare thensorted toGPI-anchoredprotein-specific vesicles (Fig.11, 2). In this report we showed that Tat2p in the ER is already associatedwith DRMs (Fig. 8). If Tat2p normally associates with ceramide-richmicrodomains composed of GPI-anchored proteins in the ER, in mutantcells defective in GPI synthesis its export from the ER is likely delayedbecause it is not sorted to GPI-anchored protein-specific vesicles due to alack of association with ceramide-rich microdomains. In this case Tat2pmay not be associated with ceramide-richmicrodomains because they aredestabilized by the lack of GPI-anchored protein or because a particularGPI-anchored protein involved in the recruitment of Tat2p to microdo-mains is lacking (Fig. 11, 3). Thus, we suspect that certain GPI-anchoredproteins stabilize Tat2p-associated DRMs through their lipid portion orthat they recruit Tat2p to DRMs. In the latter case, if only one specificGPI-anchored protein is directly involved in the export of Tat2p from theER, it should have been possible to isolate it as the gene that suppresses thecold sensitivity of gwt1-10 cells. However, we did not isolate any GPI-an-chored proteins in our screens. Thus, another factor might act along withGPI-anchored proteins to recruit Tat2p to the DRMs.In addition to Tat2p, we found that Fur4p, a uracil permease that is

also recruited to DRMs in the ER (27), cannot associate with DRMs ingwt1-10 or gaa1-1 cells (Fig. 7, A and B). However, unlike Tat2p, Fur4pis mainly localized in the vacuole in gwt1-10 cells. This difference in thelocalization may be caused by a difference either in the mechanism oftheir transport or in the specific GPI-anchored proteins involved intheir transport. Alternatively, because we used a multicopy overexpres-sion system for Fur4p-GFP due to the inability to detect nativelyexpressed Fur4p-GFP from single copy plasmid, it is possible that weobserved an artificial localization of Fur4p in gwt1-10 cells as a result ofits overexpression. Also, Fur4p does not associate with DRMs and ismislocalized to the ER in lcb1-100 cells, indicating that ceramide isrequired for its transport to the membrane (27). Whatever the reason,these reports combined with our findings strongly suggest that Fur4p,like Tat2p, is transported through ceramide-rich microdomains con-taining GPI-anchored proteins.There is no direct evidence linking the formation of ceramide-rich

microdomains in the ER with the selective transport of GPI-anchoredproteins or indicating that Tat2p or Fur4p is cotransported with GPI-anchored proteins. To clarify the function of GPI-anchored protein inthe ER, it will be necessary to identifyGPI-anchored proteins involved inthe association of Tat2p or Fur4p with DRMs. In yeast, after transfer toprotein, the GPI-lipid moieties are remodeled from phosphatidylinosi-tol to ceramide or diacylglycerol carrying C26 fatty acid (57). Thisremodeling occursmainly in the ER and partially in theGolgi (58). It willbe interesting to determine whether ceramide remodeling of the GPIanchor is involved in the association of Tat2p and Fur4p with DRMs.

GPI-anchored ProteinsMay Be Required for the Transport ofMembraneProteins That Change Their Cellular Localization According to Environ-mentalConditions—It isnoteworthy thatGPI-anchoredprotein is involvedin the association of certain proteins with DRMs, such as Tat2p and Fur4pbut not Pma1p. This could be due to the structural features of the differentmembrane proteins. However, the homology between Tat2p and Fur4p isvery low, and the two proteins have different numbers of transmembrane4 M. Okamoto, T. Yoko-o, M. Umemura, K. Nakayama, and Y. Jigami, unpublished results.

FIGURE 11. A model for the regulation of membrane proteins by GPI-anchored pro-teins. 1, GPI-anchored protein is required for the transport of Tat2p from the ER to theGolgi (1a), whereas ergosterol is required at the late Golgi (1b) (2). GPI-anchored proteinsmay be transported selectively via ceramide-rich microdomains. Membrane proteinTat2p may also be transported from the ER to the Golgi via ceramide-rich microdomainscontaining GPI-anchored protein (3). GPI-anchored proteins may stabilize Tat2p-associ-ated ceramide-rich microdomains or recruit Tat2p to the microdomains. In wild-type(WT) cells Tat2p is associated with ceramide-rich microdomains and then effectivelyexported from the ER. In cells defective in GPI synthesis (gpi mutant) Tat2p does notassociate with ceramide-rich microdomains because the microdomains are destabilizedby the lack of GPI-anchored proteins that compose the microdomains or because Tat2pis not recruited to the microdomains due to the lack of a GPI-anchored protein that isinvolved in its transport (4). Tat2p is transported to the plasma membrane at a lowtryptophan concentration. At a high tryptophan concentration, Tat2p is sorted to thevacuole via the late endosomes and polyubiquitinated by Rsp5p-Bul1p ligase. Thepolyubiquitination of Tat2p acts as a signal for sorting to the vacuole. Rsp5p-dependentubiquitination is required for the endocytosis of Tat2p. GPI-anchored protein may beinvolved in these systems regulated by Rsp5p-dependent ubiquitination.

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domains (Tat2p is predicted to contain the same 12 transmembranedomains as Hxt1p, a non-DRM associated protein, whereas Fur4p andPma1p contain 10 transmembrane domains). In yeast there are at least twodifferent raft-based membrane compartments on the plasma membrane;theyare thePma1p-richdomainandtheCan1p-richdomain (33).Recently,Fur4p was shown to be localized in the Can1p-rich domain but not thePma1p-rich domain (59). However, homologous regions have not beenfoundwithin Can1p and Fur4p. Therefore, it appears that the difference inbehavior among Tat2p, Fur4p, and Pma1p in cells with defective GPI-an-chored protein synthesis cannot be explained by their structural featuresalone. GPI-anchored proteins might function in the Can1p-rich domain,but whether Tat2p is localized in the Can1p-rich domain remainsunknown.Tat2p and Fur4p change their cellular localization according to the

concentration of nutrients, but the localization of Pma1p is extremelystable. At low tryptophan concentrations, Tat2p is transported to theplasma membrane, whereas at high tryptophan concentrations it ispolyubiquitinated by the Rsp5p ubiquitin ligase complex in early endo-somes and transported to the vacuole (Fig. 11, 4). Like Tat2p, Fur4p ispolyubiquitinated by Rsp5p ubiquitin ligase in the late Golgi and trans-ported to the vacuole rather than the plasma membrane in response touracil (51). The endocytosis of Tat2p and Fur4p is also regulated byRsp5p-dependent ubiquitination in response to nutrient levels (Fig. 11,4) (60, 61). Interestingly, Nedd4, the mammalian homolog of Rsp5p hasbeen reported to associate with lipid rafts (62). Therefore, Tat2p andFur4p may be polyubiquitinated by Rsp5p via DRMs in response to thepresence of certain nutrients. Together, the previous reports and ourcurrent data suggest that GPI-anchored proteins participate in the reg-ulatory system that determines the destination of Tat2p and Fur4pthrough Rsp5p-dependent ubiquitination in DRMs and, furthermore,that Pma1p acts independently of this system.

Acknowledgments—We thank Drs. Kyohei Umebayashi (National Institute ofGenetics), AkihikoNakano (RIKEN), Howard Riezman (University of Geneva),Roger Y. Tsien (University of California), Toru Sumita (National Institute ofAdvanced Industrial Science and Technology), Kappei Tsukahara (Eisai Co.,Ltd.), and Katsura Hata (Eisai Co., Ltd.) for providing plasmids, strains, andantibodies.We thankDr. RyogoHirata (RIKEN) for advice during the isolationof DRMs. We are indebted to Drs. Taro Kinoshita (University of Osaka) andMorihisa Fujita (University of Tsukuba) and the members of our laboratoryfor stimulating discussions.

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GPI-dependent Transport of DRM-associated Membrane Proteins

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Yoshifumi JigamiMichiyo Okamoto, Takehiko Yoko-o, Mariko Umemura, Ken-ichi Nakayama and

Fur4pDetergent-resistant Microdomain-associated Membrane Proteins Tat2p and Glycosylphosphatidylinositol-anchored Proteins Are Required for the Transport of

doi: 10.1074/jbc.M504684200 originally published online December 15, 20052006, 281:4013-4023.J. Biol. Chem. 

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