a agglutinin of Saccharomyces cerevisiae - Europe PMC

The EMBO Journal vol.7 no.5 pp.1483- 1488, 1988

Purification and characterization of the inducible a agglutinin of Saccharomyces cerevisiae

Manfred Watzele, Frans Klis' and Widmar Tanner Institut fur Botanik der Universitat Regensburg, Universitatsstrasse 31, 8400 Regensburg, FRG and 'Department of Plant Physiology, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands Communicated by G.Gerisch

A cell surface glycoprotein induced by the mating pheromone a factor in Saccharomyces cerevisiae a cells has been purified to homogeneity. At 4 x 10-9 M it strongly inhibits mating-type-specific agglutination between a and a cells. The protein is solely O-glycosylated. It consists of 29% carbohydrate and its apparent molecular mass is 22 kd on SDS gels. After HF treatment it behaves like a protein of 13 kd; therefore its true molecular mass probably is close to 18 kd. Mild periodate treatment destroys the biological activity of the purified protein. The protein contains one cysteine, no arginine, and 27% of the amino acids are serine and threonine residues, two thirds of which are glycosylated. With a polyclonal antibody the glycoprotein can already be detected at the cell surface 15 min after pheromone addition. The inducible antigen is not expressed in a specific phase of the cell cycle; it first appears exclusively on the growing bud. Mother cells express the antigen on their surface only after the daughter cells have separated; it is then localized at the tip of the pear-shaped 'shmoo'. Using the secretory tsmutant sec 18 it is shown that a mannosylated precursor of a agglutinin accumulates at the endoplasmic reticulum. Key words: agglutinin/a factor/glycoprotein/O-glycosylationlSaccharomyces cerevisiae

pounds. It is an efficient monovalent inhibitor of the agglutination reaction. The glycoprotein has been purified to homogeneity. Using a polyclonal antibody the expression of the a agglutinin on daugther and mother cells, as well as the biosynthesis of this O-glycosylated protein in a secretory mutant have been studied.

A

t-_ C

.2

04Q.5

i5

m Rt (min)

B

Introduction In a number of yeast species a specific agglutination reaction takes place between mating partners and precedes conjugation. Whereas the agglutination depends on cell surface glycoproteins constitutively expressed in several species (Crandall and Brock, 1968; Burke et al., 1980), cell agglutination in Saccharomyces cerevisiae is strongly enhanced by the mating pheromones a and a factor. Haploid a and a cells produce these pheromone peptides, which act upon the respective mating partner (Betz et al., 1981). Thus at factor added to haploid a cells causes an arrest in GI of the cell cycle, the formation of pear-shaped cells ('shmoos') and in addition changes of the cell surface, thereby increasing agglutinability with a cells (Betz et al., 1978; Fehrenbacher et al., 1978; Doi et al., 1979). The corresponding cell surface components involved in pheromone-induced agglutination have so far not been characterized. We have recently described a 22-kd glycoprotein the synthesis of which is induced in a cells by a factor (Orlean et al., 1986). This protein is easily removed from intact cells by SH-com©IRL Press Limited, Oxford, England

Rt (min)

40

Fig. 1. Separation of a-agglutinin activity on HPLC gel filtration column (TSK 2000). (A) Total DTT extract of induced a cells. (B) The 22-kd band eluted from SDS gel and rechromatographed by HPLC. For details see Materials and methods.

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M.Watzele, F.Klis and W.Tanner Table II. Sugar and amino acid composition of S. cerevisiae a

Table I. Purification of a agglutinin

agglutinin

Total protein Biol. activity Sp. Act

(jg) 9600 DTT extract 1300 SDS-PAGE HP gel filtration 330

(units)

(units/Ag)

Purification factor

42 000 28 000 24 500

4.4 21.5 74.2

1 5 17

The purification shows a typical extraction starting with 110 g wet weight of ca-factor-induced a cells (bar 1-1).

HF

Standards +

1

Sugar residues (29%) Mannose Di-mannoside Tri-mannoside Tetra-mannoside Total

Amino acids (13 kd) 11 7 1 1 20

Asx Thr Ser Glx Pro

4 16 15 17 7 7 4 I 7 2 7 8 7 6 3 5 0 116

Gly Mannosyl residues Total - 32

Ala Cys Val Met Ile Leu

Tyr Phe His

Lys Arg Total

The sugar composition of the purified [2-3H]mannose-labelled GP 22 was determined by ,B-elimination with 0.2 M NaOH (25 h, room temperature) and separation of the liberated oligosaccharides by paper chromatography (Orlean et al., 1986). The distribution of the different mannosyl species was calculated for a carbohydrate part of 5.5 kd. The amino acid composition was determined as detailed in Materials and methods.

El Fig. 2. a Agglutinin deglycosylated with HF. Silver-stained SDS-gel. For details see Materials and methods.

Results Purification of the a agglutinin The material released from a factor treated a cells by 1 mM dithiothreitol (DTT) was 20-40 times more active in inhibiting cell agglutination than the corresponding extract from an equal amount of control cells. The production of a agglutinin in the bar mutant was identical to that in the wild-type. On an HPLC sizing column (TSK 2000) the biological activity coincided with a protein peak of Rt 26 min (Figure 1A), corresponding to an apparent molecular mass of 40 kd. The a factor induced glycoprotein originally observed on SDS gels possessed an apparent molecular mass of 22 kd (Orlean et al., 1986). When the corresponding material, however, is cut out and eluted from the SDS gel, it also runs with an Rt of 26 min on the TSK 2000 column (Figure IB). Since this material retained its biological activity (Orlean et al., 1986), the purification procedure adopted consisted of preparative SDS -PAGE followed by a single HP gel filtration run (Table I). Under the conditions of the assay 13-ng protein of the purified material (sp. act. 74 units/i4g protein) inhibit the agglutination of 7.5 x 106 a cells with the same number of a cells in 250 Id. Assuming a molecular mass of the protein moiety of 13 kd (see below) the purified material inhibits at a concentration of 4 x 10-9 M or 105 molecules per a cell. The purification required to obtain homogenous

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Table HI. Inactivation of a agglutinin with 10 mM NaIO4 at 4°C

Time (h)

% Inactivation

0.5 1 2 4

25 50 70 85

Purified a-agglutinin was treated with 10 mM NaIO4 at 40C. At the times indicated aliquots were withdrawn and assayed for biological activity. For more details see Materials and methods.

material was only 17-fold; thus the agglutinin amounted to 6% of the protein released by DTT from the cell surface. Characterization of the purified a agglutinin The purity of the a agglutinin obtained in this way can be judged from the gel of Figure 2. The protein could not be stained with Coomassie blue, but could be visualized with either silver or PAS stain. When the protein was treated with HF, the molecular mass of the silver-stained material changed to 13 kd, representing the protein portion of the molecule. Since a careful determination of the total carbohydrate content of the intact molecule resulted in a value of 29%, the shift on the gel is considerably larger than expected. It seems most likely that the apparent molecular mass of the intact glycoprotein on the gel is an overestimation and that the true molecular mass is close to 18 kd. As shown previously (Orlean et al., 1986) the carbohydrate moieties of a agglutinin are released by :-elimination and 43% of it is recovered as dimannoside, 36% as mannoside and 15% as tri- and tetramannoside. From these data and the total sugar content the composition given in Table H can be calculated. Approximately 20 mono- and oligosaccharides seem to be attached to hydroxy amino acids -

Inducible a agglutinin of S.cerevisiae

2

4:

3

+ Periodate

68430

CN0

26-

Control

186.7

20

Rt(min)

a

@

333

Fig. 3. Chromatographic behaviour (HPLC gel filtration) of a agglutinin after treatment with periodate for 2 h. The control material was incubated for the same time and under the same conditions (see Materials and methods) without periodate.

of the protein. The amino acid composition (Table II) indicated 16 threonine and 15 serine residues, two thirds of which must be glycosylated, therefore. The cysteine content determined was 0.6% (post-column derivatization) and 0.4% (pre-column derivatization); the presence of at least one cysteine per molecule was further substantiated by in vivo incorporation of ['4C]cysteine into the a agglutinin (data not shown). The protein does not contain any arginine. It contains an excess of acidic amino acids, consistent with an observed pl of 4.9 (data not shown).

Inactivation of biological activity by mild periodate treatment

As shown in Table III up to 70% of the biological activity of the a agglutinin was lost when the glycoprotein was treated with 10 mM periodate at 4°C for 2 h. Since N-ethylmaleimide (NEM) or iodoacetamide did not inactivate the agglutinin, a possible destruction of cysteine residues by periodate could not account for the inactivation. In addition, periodate-treated a agglutinin, which had lost 50% of its biological activity has identical behaviour on the HPLC gel filtration column as compared with the untreated control (Figure 3). These data indicate an involvement of the 0linked sugars in cell -cell interaction.

Localization of the antigen on the cell surface The specificity of an antiserum against a agglutinin was tested with total SDS extracts of a-factor-induced and non-induced a cells (Figure 4, lanes 1 and 2). The antigen is only present in SDS extracts from induced cells. A strong reaction is also seen with DTT-extracted cell-surface components of factor-treated cells (lane 3), whereas only a very weak reaction is seen with corresponding extracts from non-induced cells (lane 4). Since this weak band is not visible in corresponding extracts from a cells (data not shown), it obviously is mating-type specific and may represent constitutively a-

Fig. 4. Western blot with anti-a-agglutinin antibody: total yeast extract (lanes 1 and 2) and DTT solubilized cell surface components (lanes 3 and 4). Lanes 1 and 3: cells treated with a factor. For details see Materials and methods.

synthesized a agglutinin. The antibody diluted 1:1000 also inhibits the agglutination of a which cells, each induced with the corresponding mating factor. To see at what time and how the antigen is expressed at the cell surface, protein synthesis of cells was stopped at various times after ca factor addition (20 Ag cycloheximide/ml), and the cells were treated with anti a agglutinin antibody and with FITC-anti-rabbit IgG. Figure 5, 1 and 2 shows the response after 15 min of treatment with factor. At that time the antigen has been deposited almost exclusively on growing buds. Even before bud initials are visible, the antigen is strictly localized at what may become a bud initial. After longer times non-budding GI cells on the other hand start to show polar exposition of antigen too (Figure 5, 3 and 4). After 90 min (when the number of budding cells was