Journal of General Microbiology (1987), 133, 439-443.
Printed in Great Britain
439
Involvement of Cell-surface Proteins in Sexual Cell-Cell Interactions of
Tremella mesentericu, a Heterobasidiomycetous Fungus
By T O K I C H I M I Y A K A W A , * Y O S H I A K I A Z U M A , E I K O T S U C H I Y A A N D
SAKUZO FUKUI
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University,
Higashi- Hiroshima 724, Japan
(Received 6 May I986 ;revised 26 June 1986)
The role of cell-surface proteins in the sexual cell-cell interactions between haploid cells of two
mating types of Tremella mesenterica was studied. Gamete cells (mating-pheromone-treated
cells) of the two mating types immediately associated to form large sexual agglutination
complexes in a mating-type-specific manner. Vegetative cells, on the other hand, agglutinated
after a lag period that corresponded to the time required for mating tube formation. Sexual
agglutination was inhibited by the presence of various proteases. Polypeptides with binding
activity specific for gamete cells of the opposite mating type were released from gamete cells of
the two mating types by digestion with thermolysin.
INTRODUCTION
Change of the mode of growth from haploid yeast to dikaryotic mycelium in the life cycle of
Tremella mesenterica, a heterobasidiomycete, is achieved by the mating of two yeast cells having
compatible mating types, ab and A B (Bandoni, 1963, 1965). Before the mating, each cell is
induced to differentiate to a gamete cell by the action of a polyisoprenylated peptide mating
pheromone (Sakagami et al., 1979, 1981) secreted by the cell of the opposite mating type.
Pheromone-induced sexual differentiation is characterized by the arrest of vegetative growth in
the GI phase of the cell division cycle and the subsequent formation of mating tubes. (The
process is similar to that described by Abe et al. (1975) for the heterobasidiomycetous yeast
Khodosporidium toruloides.) Cell-cell interactions for mating appear to be facilitated by
biochemical and morphological changes induced by the pheromone. Using lactoperoxidasecatalysed iodination of proteins as a cell-surface probe, we have shown that the cells undergo a
major change in surface features when they are stimulated by the pheromone, and we suggested
that new surface protein species which appear during sexual differentiation may be important in
the specific cell-cell interactions (Miyakawa et al., 1982). In this study, we present data that
suggest direct involvement of surface proteins of the gamete cell in cell-cell recognition for
mating.
METHODS
Micro-organisms and growth conditions. Haploid strains of Tremella mesenterica UBC 6 106-1 (mating type A B )
and 6106-2 (mating type ab) were used. Cells were grown aerobically at 28 "C in liquid minimal medium (MM) as
described by Miyakawa et al. (1984).
Preparation and assay oj'mating pheromones. Culture filtrates of AB and ab cells, prepared as described by
Miyakawa et al. (1984), were used as a source of the mating pheromones. The culture filtrates usually contained
about 30 U mating pheromone ml-I. Biological activity of mating pheromones was assayed by the serial dilution
method (Miyakawa et al., 1984).
Abbreviations : MM, minimal medium ; T-GSP, thermolysin fragments of gamete cell-surface proteins; T-VSP,
thermolysin fragments of vegetative cell-surface proteins.
0001-3440 0 1987 SGM
Downloaded from www.microbiologyresearch.org by
IP: 78.47.19.138
On: Sun, 02 Oct 2016 20:48:25
440
T. M I Y A K A W A A N D O T H E R S
Assay ojsexual agglutination. Gamete cells were prepared by treating the vegetative cells ( A B and ab) (2 x
lo7 cells ml-I) with the opposite mating pheromone (10 U ml-I) for 14 h. Pheromone-containing medium used for
induction of sexual differentiation was prepared by 1 : 2 dilution of the culture filtrate with fresh MM. Cultures of
the gamete cells were centrifuged at 4000 g for 5 min and the cell pellets were suspended in fresh M M to the above
cell concentration. Equal volumes (0.25 ml) of the cell suspensions of the two mating types were mixed and
incubated at 28 "Cwith gentle shaking for 2-4 h unless otherwise noted. For pretreatment with various reagents or
proteases, the gamete cells of each mating type were incubated under the conditions indicated, washed with MM
by centrifugation and then suspended in the same volume of MM. Suspensions of the pretreated cells were mixed
and incubated as above. The extent of cell agglutination was determined by microscopy as follows. Cells which
existed singly or in a small clump of less than five cells were counted in a haemocytometer (unagglutinated cells).
The number of unagglutinated cells was subtracted from the total cell number in the agglutination mixture and the
percentage of agglutinated cells was calculated. In some experiments, cell-wall chitin of one mating type was
stained with Calco-fluor White M2R (American Cyanide Co.) before cell mixing to distinguish between the two
mating types in the sexual agglutination complexes. All data shown here were obtained from experiments repeated
at least three times and representative results are shown.
Preparation of thermolysin fragments of gamete cell-surface proteins (T-GSP). Gamete cells were prepared by
incubation of haploid vegetative cells (mating type ab and A B ) (3 x lo7 cells ml-I) with appropriate mating
pheromone (10 U ml-I) for 10-12 h at 28 "C with shaking. The gamete cells were collected by centrifugation at
4000 g for 5 min and cell pellets were suspended (1.5 x lo8 cells ml-l) in 20 mM-sodium phosphate buffer, pH 7-2,
containing 25 pg thermolysin (Boehringer Mannheim) ml-' and 1 mM-CaC1,. Cell surface proteins were digested
by incubation of the cell suspension for 20 min at 28 "C and the protease was inactivated by the addition of
phosphoramidon (14 pg ml-I) (Protein Institute Inc.). The cell suspension was centrifuged in a microfuge
(Beckman) and clear supernatant was obtained (T-GSP). Thermolysin fragments of vegetative cell-surface
proteins (T-VSP) were prepared similarly from vegetative cells. Before agglutination assays, gamete cells were
treated with T-GSP as follows. Gamete cells prepared as described above were incubated in a mixture consisting
of 0.25 ml each of T-GSP preparation and MM (4 x lo7 cells ml-I) for 30 min at 28 "C. Sexual agglutination was
assayed by mixing 0.5 ml of each of the treated cell suspensions of both mating types as described in the preceding
section.
RESULTS A N D DISCUSSION
Sexual agglutination
To test whether gamete cells (pheromone-treated cells) of one mating type had the ability to
recognize gamete cells of the opposite mating type, pheromone-treated cells of mating types A B
and ab were mixed and the extent of cell agglutination was determined. The gamete cells readily
agglutinated, forming large clumps of 80-100cells (Figs 1 and 2). Although homotypic cell
agglutination of pheromone-treated A B cells occurred (Fig. l), the homotypic agglutination
complexes were much smaller (five cells or less) than those of heterotypic agglutination. In an
agglutination experiment in which the cell wall of one of the mating-types was stained with
Calcofluor White, the heterotypic cell agglutination complexes could be seen to consist of nearly
equal numbers of the two mating-type cells irrespective of the proportion of the two mating-type
cells mixed (mixing ratios from 1 :9 to 9 : 1 ; data not shown). The results suggested that matingtype-specific cell-cell recognition plays an important role in heterotypic cell agglutination.
When vegetative cells of the two mating types were mixed, the cells agglutinated only after a lag
period, corresponding approximately to the time for the appearance of the mating tube (Fig. 2),
indicating that cell agglutination due to constitutive cell surface properties is weak and that
sexual agglutination is mainly dependent on pheromone-induced changes of the cells.
Heterozygotes, as judged by the formation of clamp connections within the hyphal cells, were
produced from the cell aggregates after about 48 h cultivation (data not shown). Thus, it seems
likely that sexual agglutination of the gamete cells is a preparatory process for mating in the
liquid environment.
Characteristics of the sexual agglutination process
The sexual agglutinability of gamete cells was destroyed by treatment with various proteases
(Table 1). N-Ethylmaleimide and dithiothreitol were also inhibitory, suggesting that thiols and
disulphide groups of proteins are important in heterotypic cell-cell recognition.
Downloaded from www.microbiologyresearch.org by
IP: 78.47.19.138
On: Sun, 02 Oct 2016 20:48:25
Cell-cell interactions in T . mesenterica
44 1
Fig. 1. Sexual agglutination of T. mesenterica. (a) Gamete cells of mating type A B ; (6) gamete cells of
mating type ab; (c) mixture of A B and ab gamete cells. Bar, 100 pm.
Time (h)
Fig. 2. Time course of sexual agglutination. Suspensions of vegetative or gamete cells of the two mating
types were mixed and the extent of cell agglutination was determined as described in Methods. V and G
indicate vegetative and gamete cells, respectively. 0,
G(AB) G(ab): , V ( A B ) V(ab);A, G ( A B ) ;
A,W b ) ; 0,W B ) ;D, V(ab).
+
+
Table 1. Eject of various pretreatments of gamete cells on sexual agglutination
Pretreatment
None (control)
Pronase, 25 pg ml-l
Trypsin, 100 pg ml-*
Chymotrypsin, 200 pg ml-*
Themolysin, 200 pg ml-'
Dithiothreitol, 5 mM
N-Ethylmaleimide, 2 mM
Agglutination (%)
62.4
0.0
15.2
0.0
13.3
21.8
0.0
Polypeptides released from the surface of gamete cells into the medium by protease treatment
were collected and tested for their effects on the sexual agglutination of intact gamete cells.
Among the protease digestion products tested, only thermolysin fragments of gamete cellsurface proteins (T-GSP), of both A B and ab cell types, inhibited sexual agglutination
effectively. Table 2 shows the effect of pretreatment of the gamete cells with T-GSP on sexual
agglutination. Pretreatment of the gamete cells of one mating type with T-GSP from the
opposite mating type was sufficient for inhibition. The inhibitory activity of the T-GSP was
Downloaded from www.microbiologyresearch.org by
IP: 78.47.19.138
On: Sun, 02 Oct 2016 20:48:25
442
T. MIYAKAWA AND OTHERS
Table 2. Eflect of pretreatment of gamete cells with T-GSP and T-VSP on sexual agglutination
Addition
Agglutination (%)
Expt I
None (control)
T-GSP(AB )
T-GSP(ab)
T-GSP(AB)
T-GSP(ab)
T-VSP(AB) T-VSP(ab)
Expt I1
None (control)
T-GSP(AB)
Heat-treated T-GSP(AB)*
T-GSP(ab)
Heat-treated T-GSP(ub)*
+
+
61.3
18.5
25.5
10.1
60.0
58.3
20.1
60.2
19.9
45.6
* T-GSP was heated in a boiling water bath for 3 min in 20 mM-sodium phosphate buffer, pH 7.2.
Table 3. Eflect of incubation of T-GSP with gamete cells ( G ) on remaining inhibitory activity
Treatment
Control (no addition)
T-GSP(AB)
Preincubation with
Preincubation with
T-GSP(ab)
Preincubation with
Preincubation with
Agglutination (%)
65-8
G(AB )
G(ab)
G(AB)
G(ab)
18.5
44.8
68.6
25.5
46.8
22.0
Table 4 . Eflect of mixing T-GSP of the two mating types at various ratios on sexual
agglutination
Addition
Agglutination (%)
None
T-GSP(AB) :T-GSP(ab)
(ml)
(ml)
1 :1
0.66 :0.33
0.5 :0.5
0.33 :0.66
0.2 :0.8
68.0
0:l
14.9
44.8
38.3
63.0
47.0
21.9
destroyed by heat treatment (100 "C, 3 min), indicating that protein component(s) in the T-GSP
preparation is (are) responsible for the inhibition of sexual agglutination. Since corresponding
activities were not detectable in the media of thermolysin-treated vegetative cells of either
mating type (Table 2), it is suggested that the active polypeptides are derived from cell-surface
proteins specific for gamete cells. The inhibitory activities in the T-GSP preparations of both
mating types were removed after incubation with gamete cells of the opposite mating type
(Table 3). The extent of inhibition of sexual agglutination by T-GSP was dependent on the
amount of protein used (data not shown). For both types, 50% inhibition of sexual agglutination
was achieved by T-GSP prepared from cells 3 to 4 times the number of those used for the
agglutination inhibition assay. When, instead of pretreating the gamete cells with T-GSP before
cell mixing, T-GSP of the two mating types were mixed together before addition to the gamete
cells, their inhibitory effects were reduced significantly, the extent depending on the ratio of the
two T-GSP mixed (Table 4). Under the conditions used, the inhibitory effect was almost
completely lost when T-GSP from the two mating types were mixed at a ratio of 1:2.
Downloaded from www.microbiologyresearch.org by
IP: 78.47.19.138
On: Sun, 02 Oct 2016 20:48:25
Cell-cell interactions in T. mesenterica
443
The results of the experiments with T-GSP are interpreted as follows. Proteins involved in
sexual cell-cell recognition on the surface of the cells of the two mating-types were released by
thermolysin into the medium in a form that still retained their specific binding activities toward
cells of the opposite mating type. Recognition molecules on the gamete cell surface were masked
by the binding of thermolysin-released ligands derived from the opposite mating type, resulting
in failure of sexual cell recognition. Since the inhibitory activities in T-GSP of the two mating
types were lost by mixing them (Table 4), it seems likely that the proteins released from the two
mating types interact in a complementary fashion. As T-GSPs themselves did not agglutinate
cell of the opposite mating-type (data not shown), the released molecules may have only one or
very few binding sites.
We thank M. Yasuda for performing some of the early experiments involved in this study. This work was
supported in part by a grant-in-aid for scientific research (no. 605601 17) from the Ministry of Education, Science
and Culture of Japan.
REFERENCES
ABE, K . , KUSAKA,I. & FUKUI,S. (1975). MorphoMIYAKAWA, T., KADOTA,
T., OKUBO, Y . ,HATANO,
T.,
logical change in the early stages of the mating of
TSUCHIYA,
E. & FUKUI,S. (1984). Mating pheromone-induced alteration of cell surface proteins in
Rhodosporidium toruloides. Journal of Bacteriology
122, 710-718.
the heterobasidiomycetous yeast, Tremella mesenterBANDONI,R. J. (1963). Conjugation in Tremella
ica. Journal of Bacteriology 158, 814-819.
mesenterica. Canadian Journal of Botany 41,467-474.
SAKAGAMI,
Y., ISOGAI,A., SUZUKI,A. & FUJINO,
M.
BANDONI,
R. J. (1965). Secondary control of conjuga(1979). Structure of tremerogen A-10, a peptidal
tion in Trernella mesenterica. Canadian Journal of
hormone inducing conjugation tube formation in
Botany 43, 621-630.
Tremella rnesenterica. Agricultural and Biological
MIYAKAWA,
T., OKUBO,Y. T., TSUCHIYA,
E., YAMAChemistry 43, 2643-2645.
SHITA, I. & FUKUI,S. (1982). Appearance of new
SAKAGAMI,
Y . , YOSHIDA,
M., ISOGAI,I. & SUZUKI,A .
protein species on the cell surface during sexual
(1981).Structure of tremerogen a-13, a peptidal sex
differentiation in the heterobasidiomycetous yeast,
hormone of Tremella mesenterica. Agricultural and
Tremella mesenterica. Agricultural and Biological
Biological Chemistry 45, 1045-1 047.
Chemistry 43, 2403-2405.
Downloaded from www.microbiologyresearch.org by
IP: 78.47.19.138
On: Sun, 02 Oct 2016 20:48:25