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Effect of the microtubule inhibitor methyl benzimidazol-2-yl carbamate (MBC) on production and secretion of enzymes in Aspergillus nidulans
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Mycol. Res. 100 (11): 1375-1382 (1996) Printed ~n Great Britain
Effect of the microtubule inhibitor methyl benzimidazol-2-yl carbamate (MBC) on production and secretion of enzymes in Aspevgillus nidulans
A N D F. L A B O R D A Departamenfo de Microbiologia y Parasitologia, Universidad de Alcalk de Henares, Carrefera Madrid-Barcelona, km.33, E-28871 Alcala de Henares, Madrid, Spain
The effect of the antimicrotubular drug methyl benzimidazol-2-yl carbamate (MBC) on the production and secretion of acid phosphatase, a-galactosidase and P-galactosidase in Aspergillus nidulans was studied. A wild type and two benomyl resistant mutant (benAl0 and benC28) strains were used. All the strains secreted acid phosphatase and a-galactosidase into the culture medium, whereas P-galactosidase remained in the mycelium with a portion of its activity bound to the cell wall. When the wild type strain was incubated in the presence of a sublethal dose of MBC, a decrease of the activity of the enzymes studied was found. A reduction in the secretion of acid phosphatase and a-galactosidase into the culture medium was also observed. In addition, a decrease in the percentage of a and p-galactosidase activities bound to the cell wall was detected. The MBC dose used for the wild type strain did not modify either the total enzyme activities or the secretion of the enzymes studied in the benomyl resistant mutant benA and benC strains. However, when those strains were grown in the presence of a sublethal dose of MBC, a decrease in the total enzyme activities, as well as a reduction in acid phosphatase and a-galactosidase secretion was found. In addition, alterations in the percentage of enzyme activities bound to the cell wall were observed in both mutant strains. Results described in this work, clearly suggest that microtubules are involved in the polarized secretion of enzymes in A. nidulans.
Many filamentous fungi are characterized by their ability t o secrete enzymes into the external medium. This property is an essential feature of their lifestyle, as these enzymes have a role in pathogenesis (Laborda, Fielding & Byrde, 1973) or in saprotrophic growth on non-living material. Fungal exoenzymes are used in a variety of industries, especially in relation to food (Lowe, 1992). The ability of filamentous fungi to secrete enzymes into the culture medium, in contrast to yeasts, has also made them attractive candidates as hosts for heterologous protein production, since enzyme secretion facilitates purification. Moreover, filamentous fungi are eukaryotic hosts that are able to cany out protein glycosylation (Archer & Wood, 1994). Protein secretion has been extensively studied in yeast, due to the availability of temperature-sensitive secretory mutants (Schekman & Novick, 1982; Rexach et al., 1992). Despite its importance, the process of protein secretion in filamentous fungi is not yet well understood, although it has been proposed that secretory mechanisms in filamentous fungi share many features with yeast (MacKenzie et al., 1993; Peberdy, 1994). Vesicles containing proteins for export are translocated to the cell surface and release their contents following fusion with the cell membrane. Proteins may then either remain within the periplasmic space, be retained at the mature cell wall, or pass through the cell wall into the medium (Farkas, 1979; Gooday, 1994). In contrast to yeast, protein
secretion in filamentous fungi is a polarized process mainly restricted to the hyphal tips (Grove, 1978; Wosten ef al., 1991), where the cell wall is more porous than the mature wall, allowing the rapid diffusion of proteins (Chang & Trevithick, 1974). Little is known about the mechanism involved in polarized vesicle movement t o the hyphal tip, although several studies implicate cytoskeletal components in the process (Howard & Aist, 1980; Heath, 1994). A straight-forward approach t o determine the role of microtubules (MTs) is to disrupt them with specific inhibitors and to examine the effects of such treatments. Methyl benzirnidazol-2-yl carbamate (MBC), the active principle of the fungicide benomyl (Clemons & Sisler, 1969), is an inhibitor of M T assembly that acts by binding t o heterodimeric a- and P-tubdin molecules (Davidse & Flack, 1977). In Aspergillus nidulans, there are three known genes for benomyl resistance, b e d , benB and benC. The A. nidulans benA gene encodes P, and P, tubdins (Sheir-Neiss, Lai & Morris, 1978) and some benA mutants develop benomyl resistance by decreasing the afhnity of P-tubulin for the drug (Morris, 1986; Jung, Wilder & Oakley, 1992). However, it is not yet clear which proteins are encoded by the A. nidulans benB and benC genes (Morris ef al., 1984). Benomyl has been the most commonly used drug to study the role of microtubules in protein secretion by filamentous fungi. However, studies on this process have not yielded
1376
Enzyme secretion in Aspergillus nidulans conclusive results. Depending on the enzyme analysed, disruption of microtubules leads to an increase or a decrease in secretion (Monistrol, PCrez-Leblic & Laborda, 1988; Peterbauer et al., 1992; Pedregosa et al., 1995). A. nidulans is a good model for studying the role of fungal microtubules, especially because of the availability of benomyl resistant mutants. Since the effect of benomyl on secretion in A. nidulans has been only reported for invertase (Jochovi, Rupes & Peberdy, 1993), it seems interesting to extend this study to other enzymes in order to clarify the role of MTs in protein secretion. Because the cell wall plays an important role in controlling secretion in filamentous fungi (Peberdy, 1994), studies on this process should quantify not only the levels of enzyme released to the culture medium, but also the portion of cell-bound secreted protein. In this work, we studied and quantified the effect of MBC on the production and secretion of three hydrolytic enzymes from A. nidulans: a-galactosidase, which is secreted into the culture medium (Rios ef al., 1993), P-galactosidase, that does not reach the culture medium (Fantes & Roberts, 1973), and acid . phosphatase whose secretion into the culture medium . increases in the presence of MBC in Cladosporium cucurnerinum (Pedregosa et al., 1995). The results should help to determine whether MTs participate in enzyme secretion in A. nidulans. In order to confirm that the effects observed are mediated via MBC action on MTs, we compared the results in a wild type strain with the ones obtained in two benomyl resistant mutants (benA and a benC).
MATERIALS A N D METHODS Fungal strains and culture conditions Three strains of Aspergillus nidulans (Eidam) G. Winter were used in these experiments: A. nidulans 2.3 (wild type), and the benomyl resistant mutants 2.108 (benA10) and 2.110 (benC28). These strains were kindly provided by Professor J. F. Peberdy (Department of Life Science, University of Nottingham, U.K.) and were previously described by Van Tuyl (1977). Stock cultures were maintained on MYG agar (0.5 % malt extract, 0.25 % yeast extract, 1% glucose, 2 % agar). A. nidulans strains were grown in Aspergillus liquid complete medium (ACM) (Pontecorvo ef al., 1953) supplemented with lactose (1% w/v) as the carbon source, in the absence or presence of various concentrations of MBC. Suspensions of conidia were inoculated at lo6 conidia ml-' into 125 ml Erlenmeyer flasks containing 20 ml of liquid medium. The cultures were incubated at 28 OC on a rotary shaker at 200 rpm for 3 d. Whole cultures were harvested every 12 h. MBC (Sigma) was dissolved in dimethyl sulphoxide (DMSO). Concentrations of MBC from 0.1 to 2 yg ml-' were used to study the effect of this compound on growth of the strains used. To study the role of MTs in enzyme secretion, MBC was added to the cultures at the following sublethal doses: 0.25 yg ml-' for the wild-type strain 2.3; 0.25 and 1.1 ~g ml-' for strain 2.110; 0.25 and 1.8 yg ml-' for strain 2.108. Solvent concentration in the medium was never more than 0.1 % (vlv). Control experiments with DMSO did not show any detectable alteration as compared to the control without the solvent.
Preparation of enzyme fractions To test enzyme production and mycelial distribution, three enzyme fractions were derived from the cultures: two from the mycelium (soluble and insoluble mycelial fractions) and a third one from the culture filtrate. The mycelium was separated from the culture liquid by filtration through nylon membranes, washed in 0.5 M-Tris/phosphate buffer (pH 6.7), frozen and freeze-dried. The freeze-dried mycelium was weighed to determine its biomass. Cell extracts were obtained by grinding the freeze-dried mycelia in liquid nitrogen to a h e powder with a pestle and mortar. The powdered mycelia were resuspended in lysis buffer [075M-TrislphosphatepH 6-7, 10% (v/v) glycerol, 0.1 m~-dithiothreitol and 2.5 mMphenylmethylsulphonyl fluoride]. The cell extracts were centrifuged in an Eppendorf microcentrifuge at 13000 g for 30 min at 4'. The supernatant obtained was used as the soluble mycelial fraction. The pellet was washed three times in lysis buffer, resuspended in this buffer and used as the insoluble mycelial fraction (wall fraction) (Vainstein & Peberdy, 1991). All fractions were kept on ice during assays.
Determination of enzyme activity Three enzyme activities were determined in the culture filtrates and cell lysates: acid phosphatase (EC 3.1.3.2), a galactosidase (EC 3.2.1.22) and p-galactosidase (EC 3.2.1.23). The enzyme activities were assayed by the hydrolysis of a p-nitrophenyl derivative of the substrate releasing p-nitrophenol, which is detected by its absorbance at 420 nm at basic pH (Brightwell & Tappel, 1968). The following substrates were used at a concentration of 2 mM: pnitrophenyl phosphate dissolved in 0-1M-acetate buffer (pH 4) for acid phosphatase determination; p-nitrophenyl-a-D-galactopyranoside, dissolved in 0.1 M-acetate buffer (pH 4) for a-Dgalactosidase and p-nitrophenyl-P-D-galactopyranoside,dissolved in 50 m~-phosphatebuffer (pH 7.6) plus 2 m~-MgCl,, for p-D-galactosidase. The reaction mixture contained 150 PI of appropriately diluted enzyme solution and 150 yl of substrate dissolved in buffer. After incubation at 3T0, the reaction was stopped by addition of 750 yl of Clark & Lubs (1917) buffer (pH 9.8). The material suspended in the insoluble mycelial fractions was removed by centrifugation before spectrophotometric determinations to avoid the interference with the assays. Enzyme units are defined as the amount of enzyme liberating 1 ymol p-nitrophenol min-l. The total enzyme activity was calculated in units per mg of freeze-dried mycelium. The level of enzymes secreted into the culture medium or determined in the soluble or insoluble mycelial fractions was expressed as a percentage of the total activity. The results presented show the average of three independent experiments. Two replicate samples were analysed at each determination.
RESULTS Effect of MBC on growth of several strains of A. nidulans The effect of increasing doses of MBC on growth of a wildtype, a benA and a benC strain of A. nidulans was studied. In
S. Torralba and others
1377 Table 1. Total acid phosphatase, a-galactosidase and p-galactosidase activities determined at 36 h of growth of A. nidulans strains in the absence or presence of MBC
Control Strain 2.3 (wild type) 2.5 Acid phosphatase 120.8 a-Galactosidase 7.3 P-Galactosidase
+ 0.2 + 11 + 0.9
MBC
MBC
(0.25 pg ml-l)
(1.1/1.8 pg rnl-')'
+0.1 + 1.4 0.4 + 0 1.5
12.0
Strain 2.110 (benC28) 2.3 t 0.1 2.3 Acid phosphatase 10.6 1.2 9 8 a-Galactosidase 12.1 2.5 14.5 P-Galactosidase
+ +
Strain 2.108 (benA10) Acid phosphatase 2.3 a-Galactosidase 23.0 P-Galactosidase 1.3
+ 0.3 + 1.9 + 0.1
+ 0.2 + 0.8 + 1.5
+0.3 + 1.9 1.0 +0.1
2.8
222
1.8
+ 0.1
+0.5 3.2 + 0.3 4.6
+0.1 + 0.6 0.1 + 0 1.6
5.8
Total activity is the sum of the activities determined in the three fractions derived from the cultures (insoluble mycelial fraction, soluble mycelial fraction and culture filtrate) and is expressed as mU mg-' freeze-dried mycelium. Data are shown as mean+ S.E. * 1.1for strain 2.110, 1.8 for strain 2.108.
all cases, increasing amounts of this fungicide progressively reduced growth, although higher doses of MBC were required to obtain a similar inhibition in the benomyl resistant mutants compared with the wild type strain (Fig. I). Sublethal doses of MBC, which produced a 35% growth inhibition at the maximal growth time (60 h of incubation), were chosen for further studies. These doses were: 0.25 pg ml-l, 1.8 pg ml-l and 1.1pg ml-l for the wild-type, benA and benC strains respectively. In addition, the sublethal dose of MBC chosen for the wild-type strain (0-25 pg ml-') was also used for studies on the mutant strains.
Eflect of MBC on total production of enzymes in A. nidulans
0
12
24 36 48 Incubation time (h)
60
72
.,
Fig. 1. Growth of A. nidulans strains in the absence o r presence of MBC. (a),2.3 (wild type); (b),2.110 (benC28) and (c), 2.108 (benA10). 0, Control; m, MBC 0.1 pg ml-'; *, MBC 0.2 pg ml-'; MBC MBC 0.3 pg ml-'; I, MBC 0.4 pg ml-'; 0, MBC 0.25 pg ml-'; 0, 0.8 pg ml-'; 1.8 pg ml-'; mean.
A,MBC 1.1pg ml-l; V, MBC 1.4 pg ml-'; V,MBC X , MBC 2 pg ml-'. S.E.were less than 10% of the
The effect of MBC on production and distribution of several hydrolytic enzymes in A. nid~lans was studied during exponential growth (until 60 h of incubation). A. nidulans produced acid phosphatase during the whole incubation, although its total activity was slightly higher at 12 h of growth. Total a-galactosidase and P-galactosidase activities increased significantly during the period tested. When the wild-type strain was incubated in the presence of MBC (0.25 pg ml-l), a reduction in the total activity of the enzymes studied during the period analysed was observed. This concentration of MBC did not inhibit the total enzyme activities in the benomyl resistant mutants. However, during growth in the presence of higher doses of this compound, a decrease in the level of enzyme activities was found in the mutant strains. Representative results of total acid phosphatase, a-galactosidase and p-galactosidase activities anal~sedduring incubation of the A. nidulans strains studied in the absence or presence of MBC are shown in Table I.
Enzyme secretion in Aspergillus nidulans
Incubation time (h) Fig. 2. Distribution of acid phosphatase (a, b, c), a-galactosidase (d, e, f ) and p-galactosidase (g, h, i) in different fractions of A. nidulans 2.3 cultures during incubation in the absence or presence of MBC. (a,d, g), culture filtrate; (b, e, h), mycelial soluble fraction; (c,f, i ) , mycelial insoluble fraction. 0, Control; m, MBC 0.25 yg ml-l. S.E.were less than 10% of the mean.
Distribution of enzymes in A. nidulans 2.3 cultures during fungal growth in the absence or presence of MBC
The distribution of the activity acid phosphatase, a galactosidase and P-galactosidase in the three fractions obtained from the fungal cultures during the incubation of strain 2.3 with or without 0-25 pg ml-' of MBC is shown in Figure 2. The activity determined in the culture filtrates, soluble mycelial and cell wall fractions is expressed as the percentage of the total activity. In control experiments, acid phosphatase activity was maximal at 12 and 60 h of incubation. In the presence of MBC, a great reduction in the percentage of the enzymatic activity secreted into the culture medium was found (Fig. La). However, an accumulation of this enzyme activity in the
soluble mycelial and cell wall fractions occurred in the presence of this fungicide (Fig. 2 b, c). Secretion of a-galactosidase into the culture medium increased with culture age. When the fungus grew in the presence of MBC, a great decrease in the secretion of this enzyme into the culture medium, as well as a decrease on the activity located in the cell wall fraction was found (Fig. 2d, f ). In contrast, in the presence of this antifungal compound, an accumulation of a-galactosidase activity in the soluble mycelial fraction was found (Fig. 2e). No activity of a cell-bound enzyme such as p-galactosidase was detected in the culture filtrates during the incubation time analysed in the absence or presence of MBC. In control cultures, approximately 20% of the total P-galactosidase activity was detected in the insoluble mycelial fraction.
S. Torralba and others
.,
Incubation time (h)
Fig. 3. Distribution of acid phosphatase (a, b, c), a-galactosidase (d, e, f ) and P-galactosidase (g, h, i ) in different fractions of A. nidulans 2.110 (benC28) cultures during incubation in the absence or presence of MBC. (a, d, g), culture filtrate; (b, e, h), mycelial soluble fraction; (c,J, i ) , mycelial insoluble fraction. 0, Control; MBC 0 2 5 vg ml-I; A,MBC 1-1 pg ml-I. S.E. were less than 1 0 % of the mean.
However, in the presence of MBC, only 5 % of the total activity appeared bound to the cell wall (Fig. 2i). However, accumulation of this activity in the soluble mycelial fraction was observed in MBC-treated cultures (Fig. 2 h). Disfribution of enzymes in A. nidulans benA and benC culfures during fungal growfh in fhe absence or presence of MBC
As described above, to study the effect of MBC on the distribution of enzymes in the benA and benC strains, two doses of this fungicide were used. A concentration of 0.25 pg ml-' of MBC did not greatly alter the distribution of the enzymes in the three fractions studied with respect to the control in both benomyl resistant strains. However, when either mutant was incubated in the presence of a higher dose
of MBC, alteration in the pattern of distribution of the enzyme activities was observed (Figs 3, 4). In the presence of sublethal doses of MBC (1.8 and 1.1yg ml-') for each mutant strain, an inhibition of the secretion of acid phosphatase into the culture medium was found (Figs 3a, 4a), along with an increase in activity bound to the cell wall (Figs 3c, 4c). No significant variations were observed in the levels of acid phosphatase activity in the soluble fraction of mycelium (Figs - 3 b, 4 b). When grown in the presence of a sublethal MBC dose, a decrease in the secretion of a-galactosidase into the culture medium as well as a reduction in activity in the mycelial insoluble fraction of each strain was found (Figs 3d, f, 4d, f ) . Accumulation of this enzyme activity in the soluble mycelial fraction, with respect to control cultures, was detected during the period studied (Figs 3e, 4e).
Enzyme secretion in Aspergillus nidulans
Incubation time (h) Fig. 4. Distribution of acid phosphatase (a, b, c), a-galactosidase (d, e, f ) and p-galactosidase (g, h, i ) in different fractions of A . niduhns 2.108 (benA10) cultures during incubation in the absence or presence of MBC. (a, d, g), culture filtrate; (b, e, h), mycelial soluble fraction; (c,f, i), mycelial insoluble fraction. 0, Control; H,MBC 0 2 5 pg ml-'; V,MBC 1.8 pg mI-'. S.E.were less than 10% of the mean.
No P-galactosidase activity was found in the culture medium during the exponential growth of strains 2.108 and 2.110 as reported for the wild-type strain. While in strain 2.108, a dose of 1.8 pg ml-' of MBC increased the levels of P-galactosidase activity bound to the cell wall (Fig. 4 i ) , in strain 2.110 the MBC dose of 1.1 pg ml-' reduced the amount of this enzyme activity in the ceII waII fraction (Fig. 3 i).
DISCUSSION The growth of an A. nidulans wild-type strain, and benAIO and benC28 mutant strains was reduced by MBC, the active principle of benomyl. However, doses that inhibited growth of the wild-type strain did not affect the growth of either mutant strain. The inhibition of fungal growth by benornyl is well known (Clemons & Sisler, 1971; De Lucas, Monistrol & Laborda, 1993; Pedregosa et al., 1995) and is a consequence of
its inhibitory effect on fungal microtubule polymerization (Howard & Aist, 1980; Davidse, 1986). In this work, we report the production and distribution of several hydrolytic enzymes produced during the trophophase of A. nidulans incubated in the absence or presence of MBC. In this way, we determined the levels of acid phosphatase, agalactosidase and p-galactosidase activities in several culture fractions. This approach allowed us to quantify the overall activity of these enzymes during the growth stage analysed. In addition, we could quantify the relative levels of secretion either into the culture medium or bound to the cell wall, as well as the portion of these enzymes in the soluble mycelial fraction (Vainstein & Peberdy, 1991). Since the term 'secreted enzyme' includes all enzyme activity external to the cell membrane (Archer & Wood, 1994; Gooday, 1994), we measured both the activity present in the culture filtrate and the activity bound to the cell wall to
S. Torralba and others quantify secretion. A disadvantage of this approach is the inability to measure the portion of the secreted enzyme activity localized in the periplasmic space. An alternative approach to measuring the total level of enzyme secretion requires protoplast formation and determination of the activity in the supernatant. However, since it has already been shown that MBC only affects processes dependent on apical growth (Jochovi ef al., 1993), which includes the secretion process, we decided to study the distribution of enzymes in different culture fractions instead of using protoplasts. During exponential growth of A. nidulans, acid phosphatase and a-galactosidase activities were detected in the culture medium and in the insoluble mycelial fraction. Secretion of acid phosphatase and a-galactosidase by A. nidulans has already been reported by Dorn & Rivera (1966) and Rios et al. (1993) respectively. The pH of the medium affects the expression of phosphatases in A. nidulans (Caddick Brownlee & Arst, 1986). No significant differences in the pH of culture medium during incubation of A. nidulans in the absence or presence of MBC were observed (data not shown). In agreement with previous work on A. nidulans (Fantes & Roberts, 1973; Fiedurek & Ilczuk, 1990) our results show that P-galactosidase activity was not released into the culture medium, although it was detected in the cell wall fraction. Recent studies from our group (Diaz, 1995; unpublished) have located this enzyme activity external to the cell membrane, demonstrating that it should be considered as a secreted enzyme (Archer & Wood, 1994; Gooday, 1994) although it is not released into the culture medium. When A . nidulans was incubated in the presence of MBC we observed a remarkable decrease in the total activity of the enzymes analysed (acid phosphatase, a-galactosidase and Pgalactosidase). This effect could be due to impairment of protein synthesis in the presence of this fungicide, which has already been observed in this species (De Lucas et al., 1993). When strain 2.3 (wild-type) was grown in the presence of MBC, changes in enzyme distribution in the culture fractions were observed. A reduction in the secretion of acid phosphatase, and in a - and P-galactosidase activities was observed. The major reduction in secretion was observed in the levels of enzymes secreted into the culture medium. Although studies on the effect of antimicrotubular compounds in filamentous fungi other than A. nidulans have not demonstrated a clear role for microtubules in protein secretion (Rossier, Hoang-Van & Turian, 1989), our results together with previous work on invertase secretion (Jochovi et al., 1993) clearly suggest that disruption of microtubules in A. nidulans inhibits the process of enzyme secretion. This could be due to an effect on the polarized migration of vesicles carrying enzymes destined for secretion (Howard & Aist, 1980; De Lucas ef al., 1993) when cytoplasmic microtubules are depolymerized. In this way, the increase in enzyme activities in the soluble fraction of mycelium observed after MBC treatment could be regarded as a consequence of enzyme accumulation inside the cell due to blockage of the secretion process. Alternatively, the changes in enzyme synthesis or secretion might be a stress response, e.g. due to an effect on mitosis. Further work will be performed to determine whether this intracellular accumulation of enzymes
1381 is a consequence of an inhibitor effect on transport throughout the fungal hypha. In this work we also analysed the distribution of enzymes studied in benA and benC mutant strains. Results in both strains show that mutations at the benA or at the benC locus not only confer resistance to MBC for vegetative growth, but also allow a normal enzyme secretion process in A . nidulans. Moreover since benA and benC mutations prevent MBC action on microtubules (Davidse & Flack, 1977) our results c o n h that the effect of MBC observed in the wild-type strain is a specific action of MBC on microtubules. The benA gene has been shown to be essential for several processes such as nuclear division or nuclear migration during vegetative growth in A. nidulans (Oakley & Moms, 1980, 1981). The tubC gene which encodes P3 tubulin has been shown to be involved in conidiation (Weatherbee ef al., 1985). Although very little is known on the role of the benC gene in A. nidulans (Morris et al., 1984), Pinto ef al. (1994) showed that conidiation in the benC28 strain was not affected by MBC, suggesting that the benC and tubC could be the same genes. Since similar results in the presence of MBC were obtained in benA and benC strains, our experiments suggest that the mutations in both loci conferred resistance to MBC. May (1989) has shown that P-tubulins are functionally interchangeable in this fungus. The fact that sublethal MBC doses, which inhibited growth in the benA and benC strains, also reduced enzyme secretion, clearly suggests that depolymerization of microtubules leads to an altered secretion process in A . nidulans. The suggested role of actin filaments in the control of polarity in hyphal growth and localization of the site of exocytosis (Akashi, Kanbe & Tanaka, 1994; Gow, 1994; Heath, 1994) should also be taken into account. In addition, it has recently been demonstrated that a novel myosin I in A. nidulans is required for polarized growth and secretion in this fungal species (McGoldrick, Gruver & May, 1995). Therefore, further studies should be done to establish the definite role and possible interrelation of microtubules, actin and myosin I during protein secretion. S. Torralba was supported by a grant from the Universidad de Alcali de Henares.
REFERENCES Akashi, T., Kanbe, T. & Tanaka, K. (1994). The role of the cytoskeleton in the polarized growth of the germ tube in Candida albicans. Microbiology 140, 271-280. Archer, D. B. & Wood, D. A. (1994). Fungal exoenzyrnes. In The Graving Fungus (ed. N . A. R. Gow & G . M. Gadd), pp. 137-162. Chapman & Hall: London, U.K. Brightwell, R. & Tappel, A. (1968). Lysosomal acid pyrophosphatase and acid phosphatase. Archioes of Biochemishy and Biophysics 124, 333-334. Caddick, M.X., Brownlee, A. G. & Arst, H. N. (1986). Regulation of gene expression by pH of the growth medium in Aspergillus nidutans. Molecular and General Genefirs 188, 490-493. Chang, P.L. & Trevithick, I. R. (1974). How important is secretion of exoenzymes through apical cell walls of fungi? Arckiues of Microbiology 101, 281-293. Clark, W. L. & Lubs, H. A. (1917). The colorimetric determination of hydrogen iron concentration and its applications in Bacteriology. Journal of Bacteriology 2, 1.
Enzyme secretion in Aspergillus nidulans Clemons, G. P. & Sisler, H. D. (1969). Formation of a fungitoxic derivative from benlate. Phytopathology 59, 705-706. Clemons, G. P. & Sisler, H. D. (1971). Localization of the site of action of a fungitoxic benomyl derivate. Pesticide Biochemistry and Physiology 3, 317-320. Davidse, L. C. (1986). Benzimidazole fungicides: Mechanism of action and biological impact. Annual Review of Phytopathology 24, 43-65. Davidse, L. C. & lack, W. (1977). Differential binding of methylbenzimidazole-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of AspergiNw nidulans. Journaf of Cell Bioiogy 72, 174-193. De Lucas, J. R., Monistrol, I. F. & Laborda, F. (1993).Effect of antimicrotubular drugs on the secretion process of extracellular proteins in Aspergillw nidulans. Mycological Research 97,961-966. Diaz, M. S. (1995). Study of P-galactosidase as a model for protein secretion in Aspergillus nidulans. Ph.D. Thesis, University of Alcali, Spain. Dom, G. & Rivera, W. (1966). Kinetics of fungal growth and phosphatase formation in Aspergillw nidulans. Journal of Bacteriology 92, 1618-1622. Fantes, P. A. & Roberts, C. F. (1973). P-Galactosidase activity and lactose utilization in Aspergillus ilidulans. Journal of General Microbiology 77, 471-486. Farkas, V. (1979). Biosynthesis of cell walls of fungi. Microbiological Reviews 43, 117-144. Fiedurek, J. & Ilczuk, Z. (1990). Screening of microorganisms for improvement of P-galactosidase production. Acta Microbiologica polonica 39, 37-42. Gooday, G. W. (1994). Cell walls. In The Growing Fungus (ed. N. A. R. Gow & G. M. Gadd), pp. 4 3 4 2 . Chapman & Hall: London, U.K. Gow, N. A. R. (1994). Tip growth and polarity. In The Growing Fungus (ed. N. A. R. Gow & G. M. Gadd), pp. 278-299. Chapman & Hall: London, U.K. Grove, S. N. (1978). The cytology of hyphal tip growth. In The Filamentous Fungi, vol. I11 (ed. J. E. Smith & D. R. Beny), pp. 28-50. Amold: London, U.K. Heath, I. B. (1994). The cytoskeleton. In The Growing Fungus (ed. N . A. R. Gow & G. M. Gadd), pp. 10C-134. Chapman & Hall: London, U.K. Howard, D. R. J. & Aist, J. E. (1980). Cytoplasmic microtubules and fungal morphogenesis: ultrastructural effects of methyl benzimidazole-2.~1carbamate determinated by freeze-substitution of hyphal tip cells. Journal of Cell Biology 87,5 5 4 4 . Jochovd, J., Rupes, I. & Peberdy, J. F. (1993).Effect of the microtubule inhibitor benomyl on protein secretion in Aspergillw nidulans. Mycological Research 97, 22-27. Jung, M. K.. Wilder, I. B. & Oakley, B. R. (1992). Amino acid alterations in the benA (P-tubulin) gene of Aspergillus nidulans that confer benomyl resistance. Cell Motrlity and the Cytoskelefon 22, 17G174. Laborda, F., Fielding, A. H. & Byrde, R. J. W. (1973). Extra and intracellular aL-arafinofuranosidaseof Sclerotinia fructigena. Journal of General Microbiology 79,321-329. Lowe, D. A. (1992). Fungal enzymes. In Handbook of Applied Microbiology, vol. 4 (ed. D. K. Arora, R. P. Elander & K. G. Mukerji), pp. 681-706. Marcel Dekker: New York. MacKenzie, D. A,, Jeenes, D. J., Belshaw, N. J. & Archer, D. B. (1993). Regulation of secreted protein production by filamentous fungi: recent developments and perspectives. journal of General Microbiology 139, 2295-2307. May. G. S. (1989). The highly divergent beta-tubulins of Aspergillus nidulans are functionally interchangeable. Journal of Cell Biology 109, 2267-2274. McGoldrick, C. A., Gruver, C. & May, G. S. (1995). M y o A of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth. The journal of Cell Biology 128,577-587.
(Accepted 4 March 1 9 9 6 )
1382 Monistrol, I. F., Perez-Leblic, M. I. & Laborda, F. (1988). Effect of sublethal dose of benomyl on extracellular enzyme production by Cladosporium cuc~merinum.Transactions of the British Mycological Society 90, 193-197. Moms, N.R. (1986). The molecular genetics of microtubule proteins in fungi. Experimental Mycology 10, 77-82. Morris, N. R., Weatherbee, J. A,, Gambino, J. & Bergen, L. G. (1984). Tubulins of Aspergillw nidulans: genetics, biochemistry and function. In Molecular Biology of the Cytoskeleton (ed. G. C. Borisy, D. W. Cleveland & D. B. Murphy), pp. 211-222. Cold Spring Harbor Laboratory Press: New York, U.S.A. Oakley, B. R. & Morris, N. R. (1980). Nuclear movement is beta-tubulindependent in Aspergillus nidulans. Cell 19, 255-262. Oakley, B. R. & Morris, N. R. (1981). A beta-tubulin mutation in Aspergillus nidulans that blocks microtubule function without blocking assembly. Cell 24, 837-845. Peberdy. J. F. (1994). Protein secretion in filamentous fungi-trying to understand a highly productive black box. Trends in Biotechnology 12, 5G.57. Pedregosa, A.M., Rios, S., Monistrol, I. F. & Laborda, F. (1995). Effect of the microtubule inhibitor methyl benzimidazol-2-yl carbamate (MBC) on protein secretion and microtubule distribution in Cladosporium cucumerinum. Mycological Research 99, 43-48. Peterbauer, C. K., Heidenreich, E., Baker, R. T. & Kubicek, C. P. (1992). Effect of benomyl and benomyl resistance on cellulase formation by Trichoderma reesei and Trichoderma harzianum. Canadian journal of Microbiology 38, 1292-1297. Pinto, F., Pedregosa, A. M., Monistrol, I. F. & Laborda, F. (1993). Effect of antimicrotubular fungicides on conidiation in Aspergillus nidulans. Revista Iberoamericana de Micologlh 10, 10G104. Pontecowo, G., Roper, J. A,, Hemmons, L. M., MacDonalk, K. D. & Buffon, A. W. J. (1953).The genetics of Aspergillus nidulans. Advances in Genetics 5, 141-238. Rexach, M., d'Enfert, C., Wuestehube, L. & Schekman, R. (1992). Genes and proteins required for vesicular transport from the endoplasmic reticulum. Antonie van Leewenhoek 61,87-92. Rios, S.,Pedregosa, A. M., Monistrol, I. F. & Laborda, F. (1993). Purification and molecular properties of an a-galactosidase synthesized and secreted by Aspergillus nidulans. FEMS Microbiology Letters 112,35-42. Rossier, C., Hoang-Van, K. & Turian, G. (1989). Secretion of an M , 60000 protein in benomyl-treated cells of Neurospora crassa. European Journal of Cell Biology 50, 333-339. Schekman, R. & Novick, P. (1982). The secretory process and yeast cell surface assembly. In The Molecular Biology of the Yeast Saccharomyces. Metabolism and Gene Expression (ed. J. N. Strathem, E. W. Jones & J. R. Broach), pp. 361-398. Cold Spring Harbor Laboratory Press: New York, U.S.A. Sheir-Neiss, G., Lai, M. H. & Morris, N. R. (1978). Identification of a gene for P-tubulin in Aspergillw. Cell 15,639449. Vainstein, M. H. & Peberdy, J. F. (1991). Regulation of invertase in Aspergillw nidulans: effect of different carbon sources. Joumal of General Microbiology 137,315-321. Van Tuyl, J. M. (1977). Genetics of fungal resistance to systemic fungicides. Ph.D. thesis, Agricultural University of Wageningen, The Netherlands. Weatherbee, J. A,, May, G. S., Gambino, J. & Morris, N. R. (1985).Involvement of a particular species of P-tubulin (P,) in conidial development in Aspergillw nidulans. ]ournu1 of Cell Biology 101,706-711. Wosten, H. A. B., Moukha, S. M., Sietsma, J. H. & Wessels, J. G. H. (1991). Localization of growth and secretion of proteins in Aspergillus niger. Journal of General Microbiology 137,2017-2023.
Mycol. Res. 100 (11): 1375-1382 (1996) Printed ~n Great Britain
Effect of the microtubule inhibitor methyl benzimidazol-2-yl carbamate (MBC) on production and secretion of enzymes in Aspevgillus nidulans
A N D F. L A B O R D A Departamenfo de Microbiologia y Parasitologia, Universidad de Alcalk de Henares, Carrefera Madrid-Barcelona, km.33, E-28871 Alcala de Henares, Madrid, Spain
The effect of the antimicrotubular drug methyl benzimidazol-2-yl carbamate (MBC) on the production and secretion of acid phosphatase, a-galactosidase and P-galactosidase in Aspergillus nidulans was studied. A wild type and two benomyl resistant mutant (benAl0 and benC28) strains were used. All the strains secreted acid phosphatase and a-galactosidase into the culture medium, whereas P-galactosidase remained in the mycelium with a portion of its activity bound to the cell wall. When the wild type strain was incubated in the presence of a sublethal dose of MBC, a decrease of the activity of the enzymes studied was found. A reduction in the secretion of acid phosphatase and a-galactosidase into the culture medium was also observed. In addition, a decrease in the percentage of a and p-galactosidase activities bound to the cell wall was detected. The MBC dose used for the wild type strain did not modify either the total enzyme activities or the secretion of the enzymes studied in the benomyl resistant mutant benA and benC strains. However, when those strains were grown in the presence of a sublethal dose of MBC, a decrease in the total enzyme activities, as well as a reduction in acid phosphatase and a-galactosidase secretion was found. In addition, alterations in the percentage of enzyme activities bound to the cell wall were observed in both mutant strains. Results described in this work, clearly suggest that microtubules are involved in the polarized secretion of enzymes in A. nidulans.
Many filamentous fungi are characterized by their ability t o secrete enzymes into the external medium. This property is an essential feature of their lifestyle, as these enzymes have a role in pathogenesis (Laborda, Fielding & Byrde, 1973) or in saprotrophic growth on non-living material. Fungal exoenzymes are used in a variety of industries, especially in relation to food (Lowe, 1992). The ability of filamentous fungi to secrete enzymes into the culture medium, in contrast to yeasts, has also made them attractive candidates as hosts for heterologous protein production, since enzyme secretion facilitates purification. Moreover, filamentous fungi are eukaryotic hosts that are able to cany out protein glycosylation (Archer & Wood, 1994). Protein secretion has been extensively studied in yeast, due to the availability of temperature-sensitive secretory mutants (Schekman & Novick, 1982; Rexach et al., 1992). Despite its importance, the process of protein secretion in filamentous fungi is not yet well understood, although it has been proposed that secretory mechanisms in filamentous fungi share many features with yeast (MacKenzie et al., 1993; Peberdy, 1994). Vesicles containing proteins for export are translocated to the cell surface and release their contents following fusion with the cell membrane. Proteins may then either remain within the periplasmic space, be retained at the mature cell wall, or pass through the cell wall into the medium (Farkas, 1979; Gooday, 1994). In contrast to yeast, protein
secretion in filamentous fungi is a polarized process mainly restricted to the hyphal tips (Grove, 1978; Wosten ef al., 1991), where the cell wall is more porous than the mature wall, allowing the rapid diffusion of proteins (Chang & Trevithick, 1974). Little is known about the mechanism involved in polarized vesicle movement t o the hyphal tip, although several studies implicate cytoskeletal components in the process (Howard & Aist, 1980; Heath, 1994). A straight-forward approach t o determine the role of microtubules (MTs) is to disrupt them with specific inhibitors and to examine the effects of such treatments. Methyl benzirnidazol-2-yl carbamate (MBC), the active principle of the fungicide benomyl (Clemons & Sisler, 1969), is an inhibitor of M T assembly that acts by binding t o heterodimeric a- and P-tubdin molecules (Davidse & Flack, 1977). In Aspergillus nidulans, there are three known genes for benomyl resistance, b e d , benB and benC. The A. nidulans benA gene encodes P, and P, tubdins (Sheir-Neiss, Lai & Morris, 1978) and some benA mutants develop benomyl resistance by decreasing the afhnity of P-tubulin for the drug (Morris, 1986; Jung, Wilder & Oakley, 1992). However, it is not yet clear which proteins are encoded by the A. nidulans benB and benC genes (Morris ef al., 1984). Benomyl has been the most commonly used drug to study the role of microtubules in protein secretion by filamentous fungi. However, studies on this process have not yielded
1376
Enzyme secretion in Aspergillus nidulans conclusive results. Depending on the enzyme analysed, disruption of microtubules leads to an increase or a decrease in secretion (Monistrol, PCrez-Leblic & Laborda, 1988; Peterbauer et al., 1992; Pedregosa et al., 1995). A. nidulans is a good model for studying the role of fungal microtubules, especially because of the availability of benomyl resistant mutants. Since the effect of benomyl on secretion in A. nidulans has been only reported for invertase (Jochovi, Rupes & Peberdy, 1993), it seems interesting to extend this study to other enzymes in order to clarify the role of MTs in protein secretion. Because the cell wall plays an important role in controlling secretion in filamentous fungi (Peberdy, 1994), studies on this process should quantify not only the levels of enzyme released to the culture medium, but also the portion of cell-bound secreted protein. In this work, we studied and quantified the effect of MBC on the production and secretion of three hydrolytic enzymes from A. nidulans: a-galactosidase, which is secreted into the culture medium (Rios ef al., 1993), P-galactosidase, that does not reach the culture medium (Fantes & Roberts, 1973), and acid . phosphatase whose secretion into the culture medium . increases in the presence of MBC in Cladosporium cucurnerinum (Pedregosa et al., 1995). The results should help to determine whether MTs participate in enzyme secretion in A. nidulans. In order to confirm that the effects observed are mediated via MBC action on MTs, we compared the results in a wild type strain with the ones obtained in two benomyl resistant mutants (benA and a benC).
MATERIALS A N D METHODS Fungal strains and culture conditions Three strains of Aspergillus nidulans (Eidam) G. Winter were used in these experiments: A. nidulans 2.3 (wild type), and the benomyl resistant mutants 2.108 (benA10) and 2.110 (benC28). These strains were kindly provided by Professor J. F. Peberdy (Department of Life Science, University of Nottingham, U.K.) and were previously described by Van Tuyl (1977). Stock cultures were maintained on MYG agar (0.5 % malt extract, 0.25 % yeast extract, 1% glucose, 2 % agar). A. nidulans strains were grown in Aspergillus liquid complete medium (ACM) (Pontecorvo ef al., 1953) supplemented with lactose (1% w/v) as the carbon source, in the absence or presence of various concentrations of MBC. Suspensions of conidia were inoculated at lo6 conidia ml-' into 125 ml Erlenmeyer flasks containing 20 ml of liquid medium. The cultures were incubated at 28 OC on a rotary shaker at 200 rpm for 3 d. Whole cultures were harvested every 12 h. MBC (Sigma) was dissolved in dimethyl sulphoxide (DMSO). Concentrations of MBC from 0.1 to 2 yg ml-' were used to study the effect of this compound on growth of the strains used. To study the role of MTs in enzyme secretion, MBC was added to the cultures at the following sublethal doses: 0.25 yg ml-' for the wild-type strain 2.3; 0.25 and 1.1 ~g ml-' for strain 2.110; 0.25 and 1.8 yg ml-' for strain 2.108. Solvent concentration in the medium was never more than 0.1 % (vlv). Control experiments with DMSO did not show any detectable alteration as compared to the control without the solvent.
Preparation of enzyme fractions To test enzyme production and mycelial distribution, three enzyme fractions were derived from the cultures: two from the mycelium (soluble and insoluble mycelial fractions) and a third one from the culture filtrate. The mycelium was separated from the culture liquid by filtration through nylon membranes, washed in 0.5 M-Tris/phosphate buffer (pH 6.7), frozen and freeze-dried. The freeze-dried mycelium was weighed to determine its biomass. Cell extracts were obtained by grinding the freeze-dried mycelia in liquid nitrogen to a h e powder with a pestle and mortar. The powdered mycelia were resuspended in lysis buffer [075M-TrislphosphatepH 6-7, 10% (v/v) glycerol, 0.1 m~-dithiothreitol and 2.5 mMphenylmethylsulphonyl fluoride]. The cell extracts were centrifuged in an Eppendorf microcentrifuge at 13000 g for 30 min at 4'. The supernatant obtained was used as the soluble mycelial fraction. The pellet was washed three times in lysis buffer, resuspended in this buffer and used as the insoluble mycelial fraction (wall fraction) (Vainstein & Peberdy, 1991). All fractions were kept on ice during assays.
Determination of enzyme activity Three enzyme activities were determined in the culture filtrates and cell lysates: acid phosphatase (EC 3.1.3.2), a galactosidase (EC 3.2.1.22) and p-galactosidase (EC 3.2.1.23). The enzyme activities were assayed by the hydrolysis of a p-nitrophenyl derivative of the substrate releasing p-nitrophenol, which is detected by its absorbance at 420 nm at basic pH (Brightwell & Tappel, 1968). The following substrates were used at a concentration of 2 mM: pnitrophenyl phosphate dissolved in 0-1M-acetate buffer (pH 4) for acid phosphatase determination; p-nitrophenyl-a-D-galactopyranoside, dissolved in 0.1 M-acetate buffer (pH 4) for a-Dgalactosidase and p-nitrophenyl-P-D-galactopyranoside,dissolved in 50 m~-phosphatebuffer (pH 7.6) plus 2 m~-MgCl,, for p-D-galactosidase. The reaction mixture contained 150 PI of appropriately diluted enzyme solution and 150 yl of substrate dissolved in buffer. After incubation at 3T0, the reaction was stopped by addition of 750 yl of Clark & Lubs (1917) buffer (pH 9.8). The material suspended in the insoluble mycelial fractions was removed by centrifugation before spectrophotometric determinations to avoid the interference with the assays. Enzyme units are defined as the amount of enzyme liberating 1 ymol p-nitrophenol min-l. The total enzyme activity was calculated in units per mg of freeze-dried mycelium. The level of enzymes secreted into the culture medium or determined in the soluble or insoluble mycelial fractions was expressed as a percentage of the total activity. The results presented show the average of three independent experiments. Two replicate samples were analysed at each determination.
RESULTS Effect of MBC on growth of several strains of A. nidulans The effect of increasing doses of MBC on growth of a wildtype, a benA and a benC strain of A. nidulans was studied. In
S. Torralba and others
1377 Table 1. Total acid phosphatase, a-galactosidase and p-galactosidase activities determined at 36 h of growth of A. nidulans strains in the absence or presence of MBC
Control Strain 2.3 (wild type) 2.5 Acid phosphatase 120.8 a-Galactosidase 7.3 P-Galactosidase
+ 0.2 + 11 + 0.9
MBC
MBC
(0.25 pg ml-l)
(1.1/1.8 pg rnl-')'
+0.1 + 1.4 0.4 + 0 1.5
12.0
Strain 2.110 (benC28) 2.3 t 0.1 2.3 Acid phosphatase 10.6 1.2 9 8 a-Galactosidase 12.1 2.5 14.5 P-Galactosidase
+ +
Strain 2.108 (benA10) Acid phosphatase 2.3 a-Galactosidase 23.0 P-Galactosidase 1.3
+ 0.3 + 1.9 + 0.1
+ 0.2 + 0.8 + 1.5
+0.3 + 1.9 1.0 +0.1
2.8
222
1.8
+ 0.1
+0.5 3.2 + 0.3 4.6
+0.1 + 0.6 0.1 + 0 1.6
5.8
Total activity is the sum of the activities determined in the three fractions derived from the cultures (insoluble mycelial fraction, soluble mycelial fraction and culture filtrate) and is expressed as mU mg-' freeze-dried mycelium. Data are shown as mean+ S.E. * 1.1for strain 2.110, 1.8 for strain 2.108.
all cases, increasing amounts of this fungicide progressively reduced growth, although higher doses of MBC were required to obtain a similar inhibition in the benomyl resistant mutants compared with the wild type strain (Fig. I). Sublethal doses of MBC, which produced a 35% growth inhibition at the maximal growth time (60 h of incubation), were chosen for further studies. These doses were: 0.25 pg ml-l, 1.8 pg ml-l and 1.1pg ml-l for the wild-type, benA and benC strains respectively. In addition, the sublethal dose of MBC chosen for the wild-type strain (0-25 pg ml-') was also used for studies on the mutant strains.
Eflect of MBC on total production of enzymes in A. nidulans
0
12
24 36 48 Incubation time (h)
60
72
.,
Fig. 1. Growth of A. nidulans strains in the absence o r presence of MBC. (a),2.3 (wild type); (b),2.110 (benC28) and (c), 2.108 (benA10). 0, Control; m, MBC 0.1 pg ml-'; *, MBC 0.2 pg ml-'; MBC MBC 0.3 pg ml-'; I, MBC 0.4 pg ml-'; 0, MBC 0.25 pg ml-'; 0, 0.8 pg ml-'; 1.8 pg ml-'; mean.
A,MBC 1.1pg ml-l; V, MBC 1.4 pg ml-'; V,MBC X , MBC 2 pg ml-'. S.E.were less than 10% of the
The effect of MBC on production and distribution of several hydrolytic enzymes in A. nid~lans was studied during exponential growth (until 60 h of incubation). A. nidulans produced acid phosphatase during the whole incubation, although its total activity was slightly higher at 12 h of growth. Total a-galactosidase and P-galactosidase activities increased significantly during the period tested. When the wild-type strain was incubated in the presence of MBC (0.25 pg ml-l), a reduction in the total activity of the enzymes studied during the period analysed was observed. This concentration of MBC did not inhibit the total enzyme activities in the benomyl resistant mutants. However, during growth in the presence of higher doses of this compound, a decrease in the level of enzyme activities was found in the mutant strains. Representative results of total acid phosphatase, a-galactosidase and p-galactosidase activities anal~sedduring incubation of the A. nidulans strains studied in the absence or presence of MBC are shown in Table I.
Enzyme secretion in Aspergillus nidulans
Incubation time (h) Fig. 2. Distribution of acid phosphatase (a, b, c), a-galactosidase (d, e, f ) and p-galactosidase (g, h, i) in different fractions of A. nidulans 2.3 cultures during incubation in the absence or presence of MBC. (a,d, g), culture filtrate; (b, e, h), mycelial soluble fraction; (c,f, i ) , mycelial insoluble fraction. 0, Control; m, MBC 0.25 yg ml-l. S.E.were less than 10% of the mean.
Distribution of enzymes in A. nidulans 2.3 cultures during fungal growth in the absence or presence of MBC
The distribution of the activity acid phosphatase, a galactosidase and P-galactosidase in the three fractions obtained from the fungal cultures during the incubation of strain 2.3 with or without 0-25 pg ml-' of MBC is shown in Figure 2. The activity determined in the culture filtrates, soluble mycelial and cell wall fractions is expressed as the percentage of the total activity. In control experiments, acid phosphatase activity was maximal at 12 and 60 h of incubation. In the presence of MBC, a great reduction in the percentage of the enzymatic activity secreted into the culture medium was found (Fig. La). However, an accumulation of this enzyme activity in the
soluble mycelial and cell wall fractions occurred in the presence of this fungicide (Fig. 2 b, c). Secretion of a-galactosidase into the culture medium increased with culture age. When the fungus grew in the presence of MBC, a great decrease in the secretion of this enzyme into the culture medium, as well as a decrease on the activity located in the cell wall fraction was found (Fig. 2d, f ). In contrast, in the presence of this antifungal compound, an accumulation of a-galactosidase activity in the soluble mycelial fraction was found (Fig. 2e). No activity of a cell-bound enzyme such as p-galactosidase was detected in the culture filtrates during the incubation time analysed in the absence or presence of MBC. In control cultures, approximately 20% of the total P-galactosidase activity was detected in the insoluble mycelial fraction.
S. Torralba and others
.,
Incubation time (h)
Fig. 3. Distribution of acid phosphatase (a, b, c), a-galactosidase (d, e, f ) and P-galactosidase (g, h, i ) in different fractions of A. nidulans 2.110 (benC28) cultures during incubation in the absence or presence of MBC. (a, d, g), culture filtrate; (b, e, h), mycelial soluble fraction; (c,J, i ) , mycelial insoluble fraction. 0, Control; MBC 0 2 5 vg ml-I; A,MBC 1-1 pg ml-I. S.E. were less than 1 0 % of the mean.
However, in the presence of MBC, only 5 % of the total activity appeared bound to the cell wall (Fig. 2i). However, accumulation of this activity in the soluble mycelial fraction was observed in MBC-treated cultures (Fig. 2 h). Disfribution of enzymes in A. nidulans benA and benC culfures during fungal growfh in fhe absence or presence of MBC
As described above, to study the effect of MBC on the distribution of enzymes in the benA and benC strains, two doses of this fungicide were used. A concentration of 0.25 pg ml-' of MBC did not greatly alter the distribution of the enzymes in the three fractions studied with respect to the control in both benomyl resistant strains. However, when either mutant was incubated in the presence of a higher dose
of MBC, alteration in the pattern of distribution of the enzyme activities was observed (Figs 3, 4). In the presence of sublethal doses of MBC (1.8 and 1.1yg ml-') for each mutant strain, an inhibition of the secretion of acid phosphatase into the culture medium was found (Figs 3a, 4a), along with an increase in activity bound to the cell wall (Figs 3c, 4c). No significant variations were observed in the levels of acid phosphatase activity in the soluble fraction of mycelium (Figs - 3 b, 4 b). When grown in the presence of a sublethal MBC dose, a decrease in the secretion of a-galactosidase into the culture medium as well as a reduction in activity in the mycelial insoluble fraction of each strain was found (Figs 3d, f, 4d, f ) . Accumulation of this enzyme activity in the soluble mycelial fraction, with respect to control cultures, was detected during the period studied (Figs 3e, 4e).
Enzyme secretion in Aspergillus nidulans
Incubation time (h) Fig. 4. Distribution of acid phosphatase (a, b, c), a-galactosidase (d, e, f ) and p-galactosidase (g, h, i ) in different fractions of A . niduhns 2.108 (benA10) cultures during incubation in the absence or presence of MBC. (a, d, g), culture filtrate; (b, e, h), mycelial soluble fraction; (c,f, i), mycelial insoluble fraction. 0, Control; H,MBC 0 2 5 pg ml-'; V,MBC 1.8 pg mI-'. S.E.were less than 10% of the mean.
No P-galactosidase activity was found in the culture medium during the exponential growth of strains 2.108 and 2.110 as reported for the wild-type strain. While in strain 2.108, a dose of 1.8 pg ml-' of MBC increased the levels of P-galactosidase activity bound to the cell wall (Fig. 4 i ) , in strain 2.110 the MBC dose of 1.1 pg ml-' reduced the amount of this enzyme activity in the ceII waII fraction (Fig. 3 i).
DISCUSSION The growth of an A. nidulans wild-type strain, and benAIO and benC28 mutant strains was reduced by MBC, the active principle of benomyl. However, doses that inhibited growth of the wild-type strain did not affect the growth of either mutant strain. The inhibition of fungal growth by benornyl is well known (Clemons & Sisler, 1971; De Lucas, Monistrol & Laborda, 1993; Pedregosa et al., 1995) and is a consequence of
its inhibitory effect on fungal microtubule polymerization (Howard & Aist, 1980; Davidse, 1986). In this work, we report the production and distribution of several hydrolytic enzymes produced during the trophophase of A. nidulans incubated in the absence or presence of MBC. In this way, we determined the levels of acid phosphatase, agalactosidase and p-galactosidase activities in several culture fractions. This approach allowed us to quantify the overall activity of these enzymes during the growth stage analysed. In addition, we could quantify the relative levels of secretion either into the culture medium or bound to the cell wall, as well as the portion of these enzymes in the soluble mycelial fraction (Vainstein & Peberdy, 1991). Since the term 'secreted enzyme' includes all enzyme activity external to the cell membrane (Archer & Wood, 1994; Gooday, 1994), we measured both the activity present in the culture filtrate and the activity bound to the cell wall to
S. Torralba and others quantify secretion. A disadvantage of this approach is the inability to measure the portion of the secreted enzyme activity localized in the periplasmic space. An alternative approach to measuring the total level of enzyme secretion requires protoplast formation and determination of the activity in the supernatant. However, since it has already been shown that MBC only affects processes dependent on apical growth (Jochovi ef al., 1993), which includes the secretion process, we decided to study the distribution of enzymes in different culture fractions instead of using protoplasts. During exponential growth of A. nidulans, acid phosphatase and a-galactosidase activities were detected in the culture medium and in the insoluble mycelial fraction. Secretion of acid phosphatase and a-galactosidase by A. nidulans has already been reported by Dorn & Rivera (1966) and Rios et al. (1993) respectively. The pH of the medium affects the expression of phosphatases in A. nidulans (Caddick Brownlee & Arst, 1986). No significant differences in the pH of culture medium during incubation of A. nidulans in the absence or presence of MBC were observed (data not shown). In agreement with previous work on A. nidulans (Fantes & Roberts, 1973; Fiedurek & Ilczuk, 1990) our results show that P-galactosidase activity was not released into the culture medium, although it was detected in the cell wall fraction. Recent studies from our group (Diaz, 1995; unpublished) have located this enzyme activity external to the cell membrane, demonstrating that it should be considered as a secreted enzyme (Archer & Wood, 1994; Gooday, 1994) although it is not released into the culture medium. When A . nidulans was incubated in the presence of MBC we observed a remarkable decrease in the total activity of the enzymes analysed (acid phosphatase, a-galactosidase and Pgalactosidase). This effect could be due to impairment of protein synthesis in the presence of this fungicide, which has already been observed in this species (De Lucas et al., 1993). When strain 2.3 (wild-type) was grown in the presence of MBC, changes in enzyme distribution in the culture fractions were observed. A reduction in the secretion of acid phosphatase, and in a - and P-galactosidase activities was observed. The major reduction in secretion was observed in the levels of enzymes secreted into the culture medium. Although studies on the effect of antimicrotubular compounds in filamentous fungi other than A. nidulans have not demonstrated a clear role for microtubules in protein secretion (Rossier, Hoang-Van & Turian, 1989), our results together with previous work on invertase secretion (Jochovi et al., 1993) clearly suggest that disruption of microtubules in A. nidulans inhibits the process of enzyme secretion. This could be due to an effect on the polarized migration of vesicles carrying enzymes destined for secretion (Howard & Aist, 1980; De Lucas ef al., 1993) when cytoplasmic microtubules are depolymerized. In this way, the increase in enzyme activities in the soluble fraction of mycelium observed after MBC treatment could be regarded as a consequence of enzyme accumulation inside the cell due to blockage of the secretion process. Alternatively, the changes in enzyme synthesis or secretion might be a stress response, e.g. due to an effect on mitosis. Further work will be performed to determine whether this intracellular accumulation of enzymes
1381 is a consequence of an inhibitor effect on transport throughout the fungal hypha. In this work we also analysed the distribution of enzymes studied in benA and benC mutant strains. Results in both strains show that mutations at the benA or at the benC locus not only confer resistance to MBC for vegetative growth, but also allow a normal enzyme secretion process in A . nidulans. Moreover since benA and benC mutations prevent MBC action on microtubules (Davidse & Flack, 1977) our results c o n h that the effect of MBC observed in the wild-type strain is a specific action of MBC on microtubules. The benA gene has been shown to be essential for several processes such as nuclear division or nuclear migration during vegetative growth in A. nidulans (Oakley & Moms, 1980, 1981). The tubC gene which encodes P3 tubulin has been shown to be involved in conidiation (Weatherbee ef al., 1985). Although very little is known on the role of the benC gene in A. nidulans (Morris et al., 1984), Pinto ef al. (1994) showed that conidiation in the benC28 strain was not affected by MBC, suggesting that the benC and tubC could be the same genes. Since similar results in the presence of MBC were obtained in benA and benC strains, our experiments suggest that the mutations in both loci conferred resistance to MBC. May (1989) has shown that P-tubulins are functionally interchangeable in this fungus. The fact that sublethal MBC doses, which inhibited growth in the benA and benC strains, also reduced enzyme secretion, clearly suggests that depolymerization of microtubules leads to an altered secretion process in A . nidulans. The suggested role of actin filaments in the control of polarity in hyphal growth and localization of the site of exocytosis (Akashi, Kanbe & Tanaka, 1994; Gow, 1994; Heath, 1994) should also be taken into account. In addition, it has recently been demonstrated that a novel myosin I in A. nidulans is required for polarized growth and secretion in this fungal species (McGoldrick, Gruver & May, 1995). Therefore, further studies should be done to establish the definite role and possible interrelation of microtubules, actin and myosin I during protein secretion. S. Torralba was supported by a grant from the Universidad de Alcali de Henares.
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(Accepted 4 March 1 9 9 6 )
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