Biochimica et Biophysica Acta, 1038 (1990) 277-285

277

Elsevier BBAPRO 33624

The cell cycle modulated glycoprotein GP115 is one of the major yeast proteins containing glycosylphosphatidylinositol Marina Vai, Laura Popolo, Rita Grandori, Emanuela Lacanh and Lilia Alberghina Sezione di Biochimica Comparata, Dipartimento di Fisiologia e Biochimica Generali, Univerxit~ di Milano, Milano (Italy)

(Received 23 August 1989) (Revised manuscript received11 January 1990)

Key words: Glycoprotein;Acylation;Glycosylphosphatidylinositol;Membraneanchor; (S. cerevisiae)

The cell cycle modulated protein gpll5 (115 kDa, isoelectric point about 4.8-5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (pl00), dynamically related to the mature gpll5 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with ]35S]methionine, [3I-l]palmitic acid and myo-[alt]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gpll5 is one of the major palmitate- and inositoi-containing proteins in yeast. These results, and the susceptibility of gpll5 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.

Introduction Many biosynthetic pathways are similar in yeast and mammalian cells, in particular one of the most highly conserved is that leading to N-glycosylation of proteins [1], which in yeast is often coupled to secretion. The investigations on N-glycosylation and secretion pathways have taken advantage of the availability of conditional lethal mutants (sec mutants) in yeast, in which secretion is blocked at restrictive temperature [2-5]. Different sec mutants accumulate proteins either in the rough endoplasmic reticulum (ER), in the Golgi or in secretory vesicles. The completion of the entire secretory pathway requires at least 27 gene products [2,6], some of which have already been cloned [7-12].

Abbreviations: 2-D, two-dimensional; ER, endoplasmic reticulum; GPI, glycosylphosphatidylinositol;VSG, variant surface glycoprotein; PI-PLC, phosphatidylinositol-specificphospholipase C; EndoF, endo fl-N-acetylglucosaminidaseF; TM, tunicamycin;YEPD, mediumcontaining 1~ yeast extract, 2~ bacto-peptone and 2~ glucose; TBS, Tris-buffered saline. Correspondence: L. Alberghina, Dipartimento di Fisiologia e Biochimica Generali, Universit/ldi Milano-ViaCeloria26, 20133 Milano, Italy.

Other post-translational modifications occurring along the biosynthetic pathways of several proteins in higher eukaryotic cells have also been found to be conserved in yeast. A direct covalent attachment of fatty acids to the polypeptide chain has been found in a wide variety of eukaryotic and viral proteins [13]. This can occur in two ways: (i) post-translational attachment of fatty acid (primarily palmitate) in a thioester or ester bond to cysteine, serine or threonine residues or (ii) cotranslational myristoylation of proteins on amino terminal glycine via an amide linkage [13]. In 1984 the acylation of protein was discovered in yeast, using a secretory mutant [14]. Since then, the palmitoylation of a few proteins has been well-characterized, for example for the YPT1 protein [15], ras proteins [16] and a-factor [17,18]. Moreover, homologies between the process of acylation in yeast and mammalian cells have been suggested since the correct myristoylation of the p60 v-src expressed in yeast has also been reported [19]. Another type of fatty acid linkage to eukaryotic proteins has been described in the past few years [20,21]. It is the carboxy-terminal addition of a glycosylphosphatidylinositol (GPI) to proteins shortly after the completion of the polypeptide chain biosynthesis [13,21]. This type of modification has been reported for about 30 eukaryotic proteins, among them the variant

0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)

278 surface glycoprotein (VSG) of the protozoal parasite Trypanosoma brucei [21]. More recently, the attachment of fatty acid through GPI has also been identified in a 125 kDa glycoprotein of yeast [22]. GPI appears to function as a membrane-anchoring domain with the attractive role in signal transduction suggested by some pleiotropic effects of insulin in mammalian cells. These effects appear to be mediated by a low molecular mass compound, similar to GPI, that acts as a second messenger [20,21]. The present study is concerned with a more detailed analysis of gpll5 (115 kDa, isoelectric point 4.8-5), a yeast glycoprotein whose synthesis is modulated by the cell cycle [23]. An intermediate precursor form of 100 kDa has been previously identified by pulse-labeling experiments [23,24]. One additional form of 100 kDa has been found to be specific of the transition G1/S in cdc25 cells releasing from a cell cycle arrest at 'start' [23,25]. In the present paper we show by high-resolution two-dimensional (2-D) gel electrophroesis that gpll5 is one of the major palmitate- and inositol-containing proteins, gpll5 behaves as an integral membrane protein and its susceptibility to phosphatidylinositolspecific phospholipase C (PI-PLC) treatment strongly suggests that gpll5 contains a GPI structure which is added during its maturation through the secretory pathway.

$288C cells (about 1 • 1 0 7 cells/ml) exponentially growing in YNB-glucose. After 2 h, extracts were prepared [23]. [9,10(n)-3H]Palmitic acid (20 ~tCi/ml, 52 Ci/mmol; Amersham) was added to cells exponentially growing or 15 min after the shift to 37 ° C. Labeling was performed in rich medium (YEPD) at a density of about 1-2.107 cells per ml. Cerulenin (3 /tg/ml of ethanol; Sigma) was added to improve incorporation of exogenous [3H]palmitate [28]. At the end of the labeling period (2 h) extracts were prepared for the analysis by 2-D gel electrophoresis [23,29] or for immunoprecipitation. In this case, labeled cells were lysed with glass beads in 300/~1 of 50 mM Tris-HC1 (pH 7.2), 150 mM NaC1, 1% SDS, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 1 0 / t g / m l pepstatin. The lysate was then diluted with RIPA buffer [30] containing 2 mM PMSF, 10 t~g/ml pepstatin and 10/~g/ml leupeptin, so that the final concentration of SDS was 0.1%. The clarified supernatant was immunoprecipitated first with preimmune rabbit serum (15 ~1) and then with anti-gpll5 antiserum (15 /~1). After elution from protein A-Sepharose (Pharmacia) with Laemmli buffer, proteins were analyzed by SDS-polyacrylamide gel electrophoresis [31]. The antiserum used was prepared by immunization of a rabbit with the 115 kDa spot excised from 2-D gels; this antiserum efficiently recognizes and immunoprecipitates gpll5 [32].

Materials and Methods

Strains and growth conditions Saccharomyces cerevisiae secretory mutant strain HMSF176 (MAT a, seclS-1, gal2) was kindly provided by Dr. Widmar Tanner (University of Regensburg, Regensburg, F.R.G.), while the wild-type strain $288C (MAT a, SUC2, mal, mel, gal2, CUP1) used throughout this study, was obtained from the Yeast Genetic Stock Center (Berkeley, CA). Cells were grown in YEPD medium containing 1% yeast extract, 2% bacto-peptone and 2% glucose, or in Difco yeast nitrogen base (YNB) medium (6.7 g/l) without amino acids and 2% glucose. All cultures were grown in batches in a water-shaking bath, at 30 °C for $288C cells and 25 °C for HMSF176 cells unless otherwise indicated. Tunicamycin was obtained from Sigma and dissolved in dimethyl sulfoxide (2 mg/ml). Extract preparation, labeling of cells and immunoprecipitation Total unlabeled protein extracts of yeast cells were prepared as previously described [26] and membrane fractions were according to Ref. 27. [35S]Methionine labeling (20 ~tCi/ml; Amersham) of $288C cells was performed as described in Ref. 24. myo-[3H]Inositol (20 /~Ci/ml, 90.5 Ci/mmol; Amersham) was added to

EndoF treatment Endo fl-N-acetylglucosaminidase F (EndoF; Boehringer) treatment was applied to cell lysates prepared as previously reported [14], except that lysis was carried out in 300/~1 of 100 mM sodium acetate (pH 6), 50 mM EDTA, 0.4% SDS and 2% 2-mercaptoethanol. Supernatants were mixed with equal volumes of 100 mM sodium acetate (pH 6), 50 mM EDTA, and about 1 /~g of EndoF (0.6 U//~g) was added per 100 ~tl sample volume. Both the treated and control samples were incubated at 37 °C for 18 h. Digestion was terminated by the addition of an equal volume of double-strength SDS sample buffer. Samples were denatured and analyzed by SDS electrophoresis. Electrophoretic procedures and immunoblotting Proteins were resolved by SDS electrophoresis [31] or by 2-D gel electrophoresis [29] on 8% polyacrylamide slab gels. Labeled gels, following electrophoresis, were treated with Enfightning (New England Nuclear), dried and exposed to Hyperfilms-MP (Amersham) at - 80 ° C. Electrophoretic transfer of proteins was carried out as reported by Towbin et al. [33] at 500 mA for 2 h. After transfer, blots were then processed as previously described [34]. Blots were incubated with anti-gpll5 antiserum diluted 1 : 1000 in TBS containing 5% bovine

279 serum albumin (BSA). Bound antibodies were revealed by 125I-labeled protein A (Amersham).

Fatty acid analysis The [3H]palmitate gp115, immunoprecipitated as described above, was eluted from protein A-Sepharose with 1% SDS and concentrated in a Centricon centrifugal microconcentrator (Amicon) equipped w i t h a YM30 membrane. The recovered 3H-labeled protein was hydrolyzed in 0.1 M KOH in methanol for 60 min at 25 ° C. After acidification with 1 M HC1, lipids were extracted with CHC13/CH3OH/H20 (8 : 4 : 3) according to Ref. 35. The released material (about 90% of the radioactivity present in the immunoprecipitated gpll5) was analyzed by thin-layer chromatography on precoated plates (silica gel 60, Merck) with hexane/ether (9 : 1) as developing solvent. Palmitic acid and methylpalmitate (Sigma) were used as unlabeled internal standards and detected with 1 mM 8-anilino-l-naphthalenesulfonic acid (ANS) [36]. Chromatograms were sprayed with Enlightning (New England Nuclear) and exposed to Hyperfilms-MP at -80°C. Phospholipase C treatment of detergent phases Yeast cells were lysed in 0.15 M NaC1, 0.01 M Tris (pH 7.4) containing 0.2 mM EDTA, 10/xg/ml pepstatin, 20/~g/ml leupeptin and an equal volume of glass beads. After centrifugation at 300 x g for 5 rain, Triton X-114 (Serva) was added to the supernatant to give a final concentration of 1%. After 30 min on ice, the insoluble material was removed at 10 000 rpm for 5 rain at 4 °C and the supernatant was warmed to 32 °C for 3 min. The detergent and the aqueous phases were separated by centrifugation at 10 000 rpm for 20 s at room temperature and then re-extracted by further phase separations according to Ref. 37. The treatment with phospholipase C from Bacillus cereus (Sigma, Type III) was performed on the detergent phase re-extracted three times at 50 U / m l for 1 h at 30 °C as described in Ref. 37.

shown in Fig. 1A, a diffused band of about 115 kDa corresponding to the gp115 is evident in cells grown at 25 °C (permissive temp.). In the restrictive condition (2 h at 37 o C), the immunoreactive band of about 100 kDa is present and indicates the accumulation of the intermediate precursor of the gp115 in sec18 cells. The gp115 made before the onset of the sec18 block is also detected, since the mature protein is rather stable (about 10% of degradation per h) (data not shown). Following treatment with EndoF, both gp115 and pl00 disappear, while a polypeptide of about 89 kDa becomes detectable (Fig. 1A). Total protein extracts of sec18 cells grown at 25°C or incubated for 2 h at 37 o C were also subjected to 2-D gel electrophoresis (Fig. 1B). The immunodecoration indicates that at the permissive temperature the gp115 (pI 4.8-5) is found and, following the sec18 gene block, a 100 kDa protein of about the same pI, is further

25*(3 ENDOF-

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-I-

gp115.-~ 89kd-,~

IEF

IEF

Results

Processing of the glycoprotein gp l 15 Post-translational modifications occurring on the gpll5 protein (115 kDa) have been investigated. By use of the secretory secl8 mutant, it is possible to identify a 100 kDa (pl00) intermediate precursor of gpll5 [32]. The presence of N-linked core-oligosaccharide chains in the pl00 protein, is further supported by treatment of this protein with EndoF. The reaction was performed on cellular lysates of secl8 cells. Proteins were then fractionated by SDS electrophoresis, transferred to nitrocellulose sheets and immunodecorated with the anti-gpll5 antiserum and t25I-labeled protein A. As

B Fig. 1. Immunoblot analysis of gp115 processing in secl8 mutant strain and EndoF treatment. (A) Cellular lysates of exponentially growing sec18 cells and after a 2 h incubation at the indicated temperature were treated with EndoF as described under Materials and Methods. Analysis was carried out with fraciionation of proteins by SDS electrophoresis followed by electrophoretic transfer and immunodecoration with anti-gp115 antiserum and 125I-labeled protein A. (B) Total protein extracts as in A were subjected to 2-D gel electrophoresis. Immunoblot attalysis as in A. The closed arrow indicates the gp115, while the pl00 precursor is indicated by the open arrow. The pH range of the acidic section is from about 5.6 (left) to 4 (right). (kd is kDa).

280

a

b

c

de

f

g gp115 p88 p86

! !fill !~i!!~ i

A

B

Fig. 2. Effect of TM on gp115 maturation. (A) Exponentiallygrowing cells (strain $288C) were treated with TM (10 ~tg/ml) and protein extracts were prepared at different times after antibiotic addition. Exponentiallygrowingcells (lane a); TM-treatedcells for 30 rain (lane b), 60 min (lane c), 90 min (lane d), 120 min (lane e), 150 min (lane f) and 180 min (lane g). (B) secl8 cellswere treated with TM (10 ttg/ml) for 30 min at 25 °C and then shifted to 37 o C. After 2 h protein extracts were prepared. After SDS electrophoresis,immunoblotanalysis was performed as in Fig. 1. The autoradiogram in (B) has been deliberatelyoverexposed. detected. This result is in agreement with previous experiments which have indicated a close relationship between g p l l 5 and two 100 kDa proteins [23,24]. On 2-D gels the two 100 kDa polypeptides differ only with respect to charge, and by proteolytic digestion they were shown to be multiple forms of a precursor processed by glycosylation to g p l l 5 [23,24]. The fact that one of the 100 kDa forms is now detected in the secl8 mutant is further evidence that this protein is the intraceUular core-glycosylated precursor of the mature gpll5. The result of EndoF treatment also agrees with the results of experiments with tunicamycin (TM), an inhibitor of N-glycosylation of proteins [38], which have preliminarily shown that the unglycosylated precursor of gpll5 is a polypeptide with a rather diffuse electrophoretic mobility in SDS electrophoresis (molecular mass about 86-88 kDa) [32]. Here, we have investigated in more detail the effect of T M on g p l l 5 precursor formation. Exponentially growing wild-type cells (strain $288C) were treated with T M (10 ttg/ml), and total extracts were made at different times after the addition of the inhibitor. An immunoblot developed with the anti-gpll5 antiserum is shown in Fig. 2A. Following TM addition, an immunoreactive form with the mobility of about 86 kDa (p86) is detected after 30 min (Fig. 2A, lane b), while for longer periods of treatment another form with a higher molecular mass (88 kDa, p88) is also accumulated, in increasing amounts (Fig. 2A, lanes c-g). As shown in Fig. 2A, lanes d-g, the rather diffuse mobility and the increasing accumulation of p88 mask p86, thus making difficult the detection of p86 as occurred in our previous analysis. Following immunoprecipitation the two forms of the precursor appear to be EndoF resistant (data not

shown), thus confirming that both these proteins represent two unglycosylated precursors of gpll5. Moreover, the difference in molecular mass between the 88 and the 89 kDa band produced by EndoF treatment can be tentatively attributed to the N-acetylglucosamine residues still linked to the asparagines after enzymatic digestion [39]. Since T M acts in the ER by preventing core-oligosaccharides building up, we also examined the effects of this antibiotic on the g p l l 5 core-glycosylated precursor. secl8 cells exponentially growing at 2 5 ° C were pretreated with T M (10 # g / m l ) for 30 min before shifting to 37°C; after 2 h of incubation at the restrictive temperature total protein extracts were made. As shown in Fig. 2B the pretreatment with T M at 25 ° C prevents the accumulation of the 100 kDa protein that normally occurs at 37 ° C (see Fig. 1A), and causes the accumulation of the two immunoreactive bands of 86 and 88 kDa already detected in exponentially growing cells treated with T M (Fig. 2A). These data confirm that the 100 kDa protein is the core-glycosylated intermediate of the fully glycosylated g p l l 5 and that 86 and 88 kDa polypeptides are its precursors. One possible hypothesis to explain the existence of these two forms is that the 86 kDa protein is subjected to a post-translational modification unaffected by TM, resulting in a slight increase in its molecular mass and in a rather diffused mobility in SDS electrophoresis. This hypothesis has been further examined in detail (see the following paragraph).

2-D gel analysis indicates that gpll5 is one of the major palmitoylated yeast proteins A large number of proteins in eukaryotic cells are known to contain covalently bound fatty acid [13]. The majority of these acylproteins are localized in cellular membranes and contain the 16 carbon saturated fatty acid palmitic acid. Since the g p l l 5 is a membrane glycoprotein [24,32] we tested the possibility that it could also be modified by palmitic acid attachment. An analysis of palmitoylated yeast proteins has been performed using a wild-type strain ($288C). Cells exponentially growing on YEPD medium at 30 ° C were labeled with [3H]palmitic acid (20 ~ C / m l ) (Fig. 3B) and as a control with [35S]methionine (20/~Ci/ml) (Fig. 3A), as described under Materials and Methods. After 2 h of labeling, protein extracts were prepared and analyzed by 2-D gel electrophoresis. By comparison of the patterns shown in Fig. 3A and B g p l l 5 can be recognized as one of the more heavily [3H]palmitate-labeled proteins. A group of four other major palmitoylated proteins include polypeptides n.1 (120 kDa, p I 4.3-4.4), n.2 (64 kDa, p I about 4.3) and n.5 (40 kDa, p l 4.2-4.3). Other minor palmitate proteins are polypeptides n.3 (55 kDa, p l 4.7-4.8), not indicated in Fig. 3B (see Fig. 4A), because it becomes evident after overexposure of the

281

7.5

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i,, I'~.q

o

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gel electrophoresis. Fluorograms obtained for sec18 labeled proteins are shown in Fig. 4A and B. At 37 °C the gpll5 in this mutant is no longer detected as labeled protein, since the [3H]palmitic acid, added 15 min after the shift to the restrictive temperature, is incorporated only in the proteins synthesized during the secl8 block among which one can recognize, although barely, the 100 kDa precursor (Fig. 4B). The same behaviour of gpll5 characterizes the polypeptides n.2 and n.5 suggesting that they are also N-glycosylated proteins. On the other hand, other proteins (n.1, see Discussion, n.3 and n.6) are greatly increased as compared to the permissive condition, while a group of proteins of about 46 kDa (n.4) are specifically accumulated in secl8 at 37 o C. Treatment of the unprocessed labeled gels with neutral hydroxylamine, which is known to destabilize ester and thioester bonds [40], removes 3H label from the gpll5 and from the other labeled polypeptides (data not shown).

a (/)

7.5

IEF

4.1 kDo -200

Fig. 3. 2-D analysis of [35S]methionine- and [3H]palmitate-labeled proteins. Exponentially growing $288C cells were labeled with [35S]methionine (20 #Ci/ml) (A) and [3H]palmitic acid (20/LCi/ml) (B) as described under Materials and Methods. After 2 h total protein extracts were analyzed by 2-D gel electrophoresis. Fluorograms are shown. The arrow indicates the gpll5 and the circle the pl00. The other 3H-labeled proteins are indicated by the numbers.

-100 -92.5 -69

~

-46 -30

gel, and n.6 (32 kDa, p I 4.1-4.2). Due to the high specific activity of the endogenous [3H]palmitate pool that can be reached by use of cerulenin, the long-term label with [3H]palmitic acid allows the detection of the intermediate p~ecursor pl00 of gpll5. The presence of palmitic acid in pl00 provides the first indication that the modification involving fatty acid addition is already present in the core-glycosylated precursor. Moreover, the specificity of fatty acid labeling of proteins is apparent by comparing the pattern of methionine-labeled proteins with that of palmitatelabeled ones (Fig. 3). The two patterns are clearly distinct, thus making a metabolic conversion of the fatty acid into amino acids unlikely. Further data supporting the specificity of the palmitic acid labeling of gpll5 will be reported below. Palmitate-labeled proteins were also analyzed in the secretory mutant secl8, secl8 cells were labeled both at 24 and 37°C with [3H]palmitic acid, and at the end of the labeling period (2 h) proteins were subjected to 2-D

- 200 -100 -92.5 -69 o)

-46

-30 Fig. 4. 2-D analysis of [3H]palmitate-labeled proteins in the secl8 mutant. (A) Exponentially growing secl8 cells were labeled with [3H]palmitic acid (20 #Ci/ml) at 25°C for 2 h. At the end of the labeling period, total extracts were subjected to 2-D gel electrophoresis. The arrow indicates the gpll5 and the circle the pl00. (B) secl8 cells grown at 25°C were shifted to 37°C for 15 rain and then labeled with [3H]pah~tie acid (20 ~tCi/ml) for 2 h. 2-D analysis as in A. The pl00 is indicated by the circle. The other 3H-labeled proteins are indicated by the numbers.

282

a

b

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.~-p88

A

a

b

MePal.-,~

Pal._~ OR-'-~ R Fig. 5. Immunoprecipitation with the anti-gpll5 antiserum of [3H]palmitate-labeled proteins and thin-layer chromatographyof the [3H]palmitate lipids of the gpll5. (A) secl8 cells exponentiallygrowing at 25 °C (lane a) or blocked at 37 °C (lane b) were labeled with [3H]palmitic acid (20/~Ci/ml) as in Fig. 4 and then immunoprecipitated with the anti-gpll5 antiserum. Flurogram is shown. Lane c: exponentially growing cells (strain $288C) were treated with TM (10 /~g/ml) for 30 min and then labeled with [3H]palmitate(20/~Ci/ml) for 2 h. Immunoprecipitation was carried out with the anti-gp115 antiserum. (B) The [3H]palmitategp115 after immunoprecipitationwa subjected to methanolic KOH as described under Materials and Methods. The released lipids (lane b) were analyzed by thin-layer chromatography with hexane/ether (9 : 1) as developing solvent. Reference lipids are palmitic acid (Pal) and methylpalmitate (MePal) (lane a); about 3 /~g of each standard was included in the labeled sample, OR = origin.

Results obtained also using secl mutant, which is blocked at a post-Golgi level, indicate that the gp115 is accumulated as a [3H]palmitate-labeled fully glycosylated protein in the secretory vesicles (data not shown). To demonstrate further that g p l l 5 incorporates [3H]palmitic acid, [3H]palmitate-labeled extracts obtained as described above, from exponentially growing cells and from secl8 cells at 3 7 ° C were subjected to immunoprecipitation with the anti-gpll5 antiserum. Fig. 5A shows that both the [3H]palmitate g p l l 5 (lane a) and the [3H]palmitate 100 kDa protein (lane b) are immunoprecipitated by the antiserum.

As shown previously, the anti-gp115 antiserum detects in cells, which were treated with TM for a prolonged period, a protein of about 88 kDa (Fig. 2A). We tested the hypothesis that p88 could also be modified by fatty acids. Exponentially growing cells were treated with T M (10/~g/ml) for 30 min and then labeled with [3H]palmitic acid (20/~Ci/ml). After 2 h, extracts were prepared and immunoprecipitated with the anti-gpll5 antiserum. As shown in Fig. 5A, lane c, the antiserum immunoprecipitates a labeled protein. By comparison with an immunoprecipitate from [35S]methioninelabeled cells treated for 2.5 h with TM, the apparent molecular mass of this band has been found to correspond to the 88 kDa polypeptide. This strongly indicates the presence of palmitic acid in the 88 kDa precursor. The specificity of fatty acid labeling of g p l l 5 was further analyzed. Following immunoprecipitation the [3H]palmitate g p l l 5 (Fig. 5A, lane a) was hydrolyzed with methanolic KOH, the lipids were extracted by c h l o r o f o r m / m e t h a n o l and subjected to thin-layer chromatography. As shown in Fig. 5B, lane b, the fatty acid label, recovered as such from the gpll5, comi.'grates with methylpalmitate, indicating that the fatty acid is linked to the g p l l 5 protein through an ester-type bond.

2-D electrophoretic analysis of yeast inositol-containing proteins Covalent addition of fatty acids can occur via a thioester bond to a cysteine residue, or attachment as part of a GPI. In this case, the palmitic acid is in an ester linkage to the glycerol moiety of the phosphatidylinositol [21]. To differentiate between the two types of attachment, yeast wild-type cells (strain $288C) were labeled at 3 0 ° C with myo-[3H]inositol for 2 h and total protein extracts analyzed by 2-D gel electrophoresis. As appears in Fig. 6, the major polypeptides detected are n.1, gpll5, n.3 and n.5, and minor ones are pl00 and n.6. Thus, g p l l 5 appears to satisfy one of the criteria adopted to demonstrate that a protein is modified by GPI attachment, that is the incorporation of fatty acid and inositol in the protein. The other polypeptides appear to contain moieties that can be labeled both by [3H]inositol and [3H]palmitic acid, suggesting that these proteins are also very likely modified by addition of a GPI structure. The only exception is the polypeptide n.2 which is not labeled by inositol. gpl l 5 is an integral membrane protein containing GPI as membrane anchor domain The plasma membrane glycoproteins of mammals and protozoa that are attached to the lipid bilayer through an inositol-containing phospholipid behave as integral membrane proteins and are typically released from the cells by treatment with phosphatidylinositolspecific phospholipase C (PI-PLC) [20].

283 7.5 I

IEF

4.1 I

Y

kDa

Fig2 6. 2-D analysis of myo-[3H]inositol labeled proteins. Exponentially growing cells (strain $288C) were labeled with myo-[3H]inositol (20 /~Ci/ml) at 30°C. After 2 h extracts were analyzed by 2-D gel electrophoresis. The arrow indicates the gpll5 and the circle the pl00. The other 3H-labeled proteins are indicated by the numbers.

Previously described fractionation experiments have indicated that the mature gpll5 is a plasma membrane protein [24,32]. Experiments have been carried out to establish whether this glycoprotein is an integral membrane polypeptide and if it is sensitive to PI-PLC treat-

ment. Total membranes (prepared according to Ref. 27) were treated with ice-cold 0.1 M Na2CO 3 (pH 11.5) on ice for 30 min. This carbonate procedure successfully removes soluble and peripheral membrane proteins, while the integral ones, being amphipatic, are solubilized only by conditions that disrupt the lipid bilayer [41]. As shown in Fig. 7A, the gpll5 is not released in soluble form by the carbonate treatment (lane 2) but remains tightly associated with the membranes (lane 3). Other conventional methods, such as low pH and high salt concentrations [42], failed in releasing gpll5 from the membranes (data not shown). Moreover, as an integral membrane protein gpll5 is solubilized by the detergent Triton X-114 [43] and is recovered in the detergent phase after phase separation (Fig. 7B, lane 2). The Triton X-114 detergent phase containing gpll5 was subsequently treated with PI-PLC from B. cereus for 1 h at 30 °C in the presence of EDTA to inhibit phosphatidylcholine-specific PLC [44], and proteinase inhibitors to avoid any protein degradation. After phase separation gpll5 is totally released from the membranes and partitioned into the aqueous phase (Fig. 7B lane 3). The release of the detergent binding moiety apparently does not change the electrophoretic mobility of gpll5 and this can be ascribed to the anomalous migration of this protein in SDS electrophoresis [23,32] that makes it difficult to detect slight differences in the molecular masses. Discussion

1

2

3

1

2 3 4

gp 115 . - ~

A

D

A

A

D

+

--

| PHASE ~ PLC

B

Fig. 7. Chemical, detergent and enzymatic solubilization of gpl15. (A) About 40/~g of a membrane preparation (see Materials and Methods) (lane 1) were treated with 0.1 M Na2CO 3 (pH 11.5) for 30 min on ice. After centrifugation at 15000xg, pellet (lane 3) and supernatant (lane 2) were analyzed by immunoblot. (B) Exponentially growing cells were subjected to a phase separation in Triton X-114 and the aqueous (lane 1) and the detergent (lane 2) phases were analyzed by immunoblot with gpll5 antiserum. The detergent phase (lane 2) was then treated with PI-PLC (50 U/ml); phases were re-extracted and the aqueous (lane 3) and the detergent (lane 4) phases analyzed.

This paper focuses on two main aspects of gpll5 biosynthesis: the analysis of its precursors in conditions that impose a block in its maturation, and the acquisition of evidence about the presence of a GPI-structure linked to the protein. Using an immunoblot technique we detected an 86 kDa band during short-term treatment with the N-glycosylation inhibitor tunicamycin which, in agreement with the result of EndoF treatment, is the putative precursor of gpll5. However, a protracted inhibition of N-glycosylation (> 60 min Fig. 2A, lanes c-g) causes the appearance of a slightly higher molecular mass band of about 88 kDa, that could arise from the ongoing process of a modification unaffected by TM (see below). These results agree with those obtained by immunoprecipitation from [3SS]methionine-labeled extracts (data not shown) confirming the validity of the immunoblot technique in the analysis of gpll5 precursor. In secl8 mutant cells, which accumulate intermediary precursors blocked at the passage from ER to Golgi apparatus, the core-glycosylated precursor of gp115 has been detected corresponding to a 100 kDa (pl00) protein. Its rather low level in the condition of secl8 block, although the protein synthesis is not influenced in this condition, could be ascribed to an increased instability of the precursor at this specific

284 step of maturation or to the intrinsic reduced synthesis of gpll5 following the temperature shift-up (data not shown). Further elaboration of the N-linked oligosaccharide chains takes place in the Golgi. In fact, secl mutant which is blocked at a post-Golgi level (i.e., in the transport of the vesicles that mediate export of secretory and plasma membrane proteins [44,46]) accumulates a gp115 form indistinguishable from that of the mature gp115 (data not shown). The contribution of the carbohydrate moiety of the protein gp115 to its molecular mass can be approximately calculated to be around 27 kDa, the molecular mass of the outer chains being about 15 kDa, corresponding to the addition of about 80 mannose residues [47]. A 2-D gel analysis of the yeast proteins labeled with [3H]palmitic acid has been performed. The labeling pattern reflects the fatty acid acylation of proteins, since it is distinct from that observed when proteins are labeled with [35S]methionine, thus excluding a conversion of the 3H label to amino acids. In particular, the 3H label remains tightly bound to gp115 despite denaturating condition (shown also by immunoprecipitation experiments of Fig. 5A), but it can be released by hydroxylamine and alkaline methanolysis indicating that the [3H]palmitate is linked through an ester bond to gp115. The 2-D pattern shows that only a limited number of polypeptides are labeled, confirming previous SDS electrophoresis analysis [14,15], and allows the comparison with proteins whose position has already been established on a 2-D map. The acylated proteins migrate in a restricted region in the pH range of 5.2 to about 4.1 (Figs. 3B and 4). During exponential growth the gpll5 appears to be one of the most prominent palmitoylated proteins, while in the pattern obtained for secl8 blocked cells, as mentioned in the Results, gpll5 is no longer detectable. In the same restrictive condition, pl00 is present, although at a low level for the previously mentioned reasons. The polypeptide n.1 (120 kDa) appears to be the stress-inducible glycoprotein p118, recently identified in S. cerevisiae, whose expression is modulated by cAMP. More specifically a continuous synthesis of this protein is correlated with low levels of adenylate cyclase activity and with the arrest of cell proliferation [48,49]. This behaviour is opposite to that of gpll5 whose synthesis appears to be positively growth-related [23,49]. In our analysis, pl18 is detectable as [35S]methionine-labeled protein only after overexposure of 2-D gels (data not shown), in agreement with the reported low expression of pl18 in exponentially growing cells [48,49], while it is detectable as a palmitate-labeled protein probably due to the high specific activity of the precursor pools (see Results). The fact that pl18 is more heavily 3H-labeled in exponentially growing cells at 30 ° C than at 25 ° C may be in agreement with the characteristics of heat-shock pro-

tein displayed by p118 [48]. For the same reason, the polypeptide 1 (pl18) or its closing migrating intermediate precursor [48] is detected at a particularly high level in secl8 cells at 37°C (Fig. 4B). More ambiguous is the identification of polypeptide 1 as the GPI-containing glycoprotein (125 kDa) found in yeast, since for this protein only SDS electrophoresis analysis and no growth modulation experiments are reported [22]. The labeling with myo-[3H]inositol has made it possible to determine whether palmitic acid is linked directly to these proteins or through a GPI structure [21]. Polypeptides gpll5, 1, 3, 5 and 6 appear to incorporate myo-[3H]inositol in the $288C strain at 30°C (Fig. 6). pl00 has also been detected. This finding strongly supports the presence of a lipid moiety containing palmitic acid and inositol in these proteins, and it seems very likely from the chemical composition of GPI [21] that this type of modification is present in these proteins. Our data agree with previous suggestions that this modification occurs in the ER since pl00 is labeled by both radioactive inositol and palmitate. Since p88 is immunoprecipitated as labeled palmitate protein we tentatively suggest that this modification is already present in p88 and can be uncoupled from glycosylation, as occurs in other eukaryotic cells [50]. In conclusion, the overall picture of the 2-D analysis performed defines at least five major palmitate- and inositol-containing proteins. Among them gpll5, an integral plasma membrane glycoprotein, has been identified. This protein is also sensitive to PI-PLC treatments, since it becomes water soluble following the enzymatic treatment on the detergent solubilized protein. Taken together, these data indicate that gpll5 fulfils the criteria to be considered a GPI-anchor protein, although no detailed chemical characterization of its GPI moiety is presently available. The molecular cloning of gpll5 gene, now under way, besides contributing to the understanding of its biochemical function and to the significance of its cell cycle regulated synthesis, will provide information on the sequence requirement for addition of GPI in yeast.

Acknowledgments We wish to thank Mr. A. Grippo for preparing 'the figures. This work was partially supported by a grant from the Ministero Pubblica Istruzione 40% 1988 to L. Alberghina. E.L. is a recipient of a fellowship from Fondazione Anna Villa Rusconi.

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