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Effect of antimicrotubular drugs on the secretion process of extracellular proteins in Aspergillus nidulans
Mycol. Res 97 (8): 961-966 (1993)
Printed
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961
Great Bntarn
Effect of antimicrotubular drugs on the secretion process of extracellular proteins in Aspergillus nidulans
J. R. DE LUCAS, I. F. MONISTROL AND F. LABORDA'" Departamento de Microbiolog{a y Parasitolog{a, Universidad de Alcald de Henares, Campus Universitario, Carretera Madrid-Barcelona, KM. 33, Alcald de Henares, Madrid, Spain
E-28871
The role of microtubules (MTs) in the protein secretion process in Aspergillus nidulans has been studied using two antimicrotubular drugs, methyl benzimadazol-2-yl carbamate (MBC) and griseofulvin, and two strains of A. nidulans: a wild-type and a benomyl resistant-mutant (ben AID). Sublethal doses of MBC and griseofulvin reduced the total amount of proteins secreted by the wildtype strain and modified the electrophoretic pattern of the extracellular proteins, inhibiting the secretion of certain proteins and favouring the secretion of several others that were not secreted in the control cultures. Some of these modifications were the same with both antimicrotubular· compounds. MBC did not alter either the amount of proteins secreted or the electrophoretic patterns of proteins in the A. nidulans benomyl-resistant strain, demonstrating that its action is mediated via the microtubules. Our results suggest the involvement of MTs in the polarized secretion process of A. nidulans.
A characteristic of many filamentous fungi is their ability to secrete large amounts of proteins to the culture medium. Cytoplasmic vesicles containing proteins for secretion move from the Golgi apparatus and fuse with the plasmatic membrane liberating their contents to the periplasmic space (Farkas, 1979; Gooday, 1983). Proteins for secretion must cross the apical cell wall in order to be really extracellular proteins (Chang & Trevithick, 1974). The polarized transport towards the hyphal tip (apex) is not yet well understood. Several studies implicate cytoskeleton components, microtubules (MTs) and/or microfilaments (MFs), in this process (Howard & Aist, 1980; McKerracher & Heath, 1987). MTs take part in mitosis, intracellular transport and maintenance of the cell shape (Weatherbee & Morris, 1984), and cytoplasmic MTs of several ascomycete and basidiomycete species appear to play an important role as maintainers of the polarity in the apical cell (Raudaskoski, Rupes & Timonen, 1991). In filamentous fungi the movement of vesicles towards the cell apex could be associated with MTs tracks similar to the directional axonal transport (Howard, 1983; Hoch & Staples, 1985). Moreover, the presence of actin caps in normal growing tips suggest that other cytoskeleton components have a role in apical growth too (Heath, 1987). A simple approach to determine the cytoplasmic MT functions is to disrupt them with specific inhibitors and to examine the effect of these compounds. MBC (active principle of benomyl) is an MT assembly inhibitor that acts by binding to heterodimeric 0- and l3-tubulin molecules (Davidse & Flach,
• Corresponding author. 61
1977). The Aspergillus nidulans ben A gene encodes 131- and 132-tubulins and some ben A mutants develop benomyl resistance by diminishing the affinity of l3-tubulin for the drug (Morris, 1986). Griseofulvin is an MT-disorganizing agent that binds to tubulin (Wehland, Herzog & Weber, 1977) or more probably to microtubule associated proteins (MAPs) influencing the stability of MTs (RooboL Gull & Pogson, 1977). Although the effect of antimicrotubular drugs on differentiation and vegetative growth of filamentous fungi is now well established (Ton-That et al., 1988); only a few studies correlate the effect of these compounds with protein secretion and intracellular vesicle transport (MonistroL Perez-Leblic & Laborda, 1988; Rossier, Hoang-Van & Turian, 1989; Jochova, Rupes & Peberdy, 1993). We report here the effect of methyl benzimidazol-2-yl carbamate (MBC), the active principle of benomyl (Clemons & Sisler, 1969), and griseofulvin on protein synthesis and secretion in two strains of A. nidulans, a wild-type and a benomyl-resistant mutant (ben AlO). The results should help to determine whether MTs are necessary in these processes and whether the observed effects are mediated via drug action on MTs.
MATERIALS AND METHODS Fungal strains A. nidulans (Eidam) Winter G-I059 (wild-type strain) was donated by The Departamento de Microbiologia, Genetica, Medicina Preventiva y Salud Publica de la Universidad de Salamanca, Spain. The benomyl resistant mutant A. nidulans 2108 (ben AlO) was provided by J. F. Peberdy (Botany MYC 97
Protein secretion in A. nidulans 600
962 YEO solid medium (10 g 1-1 yeast extract 10 g 1-1 glucose, 20 g 1-1 agar-agar). Conidia were inoculated at 10 6 ml- 1 into 500 mI flasks with 100 ml of liqUid medium and incubated at 28°C on a rotary shaker at 200 rpm for 3 d. MBC (kindly provided by Dr Richmond, Long Ashton Research Station, Bristol, U.K.) and griseofulvin (Sigma Chemical Co.) were dissolved in dimethyl sulfoxide (DMSO) (1 mg ml- I and 70 mg ml- I stock solutions respectively), MBC and griseofulvin were added to culture media at final sublethal doses of 0'25 and 400 IJg ml- I respectively. 0'1 % (v Iv) DMSO was added to some flasks without antifungal compounds as a control.
(a)
400
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Growth and protein determination
2
Mycelia from 1, 2 and 3 d incubation were harvested by filtration through Whatman No.1 paper, washed with distilled water and dried to constant weight at 80 0. Culture filtrates were dialyzed and freeze-dried before the protein content was measured by the method of Lowry et al. (1951).
3
5VS-PAGE protein secretion analysis (b)
For electrophoretic analysis, culture filtrates were concentrated by ultrafiltration with YM-5 AMICON membranes (5000 d) (Amicon Corporation U.S.A.), freeze-dried and resuspended in 62'S mM Tris-HCl buffer (pH 6'8). Proteins (90 IJg sample-I) from culture filtrates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing 14 % polyacrylamide according to the procedure of Laemmli (1970). Pharmacia molecular weight markers were used. Gels were stained with silver nitrate as indicated by Morrisey (1981).
400
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[l55]methionine protein secretion analysis
2 Days
3
Fig. 1. Effect of MBC and griseofulvin on the growth of (a) A. nidulans G-1059 and (b) A. nidulans 2-108 (ben AW). Control; . - - . , MBC 0'25 I.lg ml- I ; A--A, griseofulvin 400 I.lg ml- I . Error bars represent S.E.M.
e--e,
Department, University of Nottingham, U.K.) and was preViously described by Van Tuyl (1977).
Culture conditions A. nidulans G-I059 was grown in CM-2 liquid medium (10 g 1-1 glucose, 0'2 g 1-1 adenine, 0'01 g -1 p-aminobenzoic acid) and A, nidulans 2-108 (ben AID) was grown in a complete liquid medium (5 g 1-1 glucose,S g 1-1 yeast extract,S g 1-1 casamino acids), Both media also contained salts (g 1-1) (5 Na N0 3, 0'5 MgS0 4, 0'5 KCI, 1'5 KH 2P0 4, 0'01 FeS0 4, 0'01 ZnS0 4 ), To produce conidia, the strains were grown on
For radioactive protein labelling, both strains were grown under the described conditions in 250 mI flasks with 60 ml culture media, After 44 h of incubation [35 S]methionine (38'1 MBq ml- I) (NEN) was added to the flasks. Aliquots (10 mI) were taken 1, 3, 5, 7 and 9 h after the pulse. Aliquots were used to determine mycelial dry-weight. Mycelia were separated from culture medium by centrifugation (17 000 g, 15 min, 4°) (rav 8'22 em). Extracellular proteins were precipitated by addition of 1 g I-I sodium deoxycholate (DOC) and 100 g 1-1 trichloroacetic acid (TCA), separated by centrifugation (27000 g, 30 min, 4°), and resuspended in 62'S mM Tris-HCI buffer (pH 6'8). The mycelium was washed twice with 50 roM Tris-HCI buffer (pH 6'9) and mycelial extracts were obtained after incubation with Novozym 234 (5 mg ml- I), 5 mM-EDTA and 2 mM-PMSF (60 min, 37°) in the same buffer in a rotary shaker. The cell debris was removed by centrifugation (27000 g, 30 min, 4°) and intracellular proteins were precipitated with 1 g I-I DOC and 100 g 1-1 TCA and separated from soluble intracellular material by centrifugation (27000 g, 30 min, 4°). Intracellular proteins were resuspended in 62'S mM Tris-HCI buffer (pH 6'8). Aliquots (20 IJI) of radiolabelled proteins were counted for radioactivity in a scintillation counter (Kontron Betamatic II)
J. R. De Lucas, I. F. Monistrol and F. Laborda
963
(a)
Days of fungal growth 3
2
94 ~l
67
43
-
-
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20
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TV
III
Days of fungal growth 3
2
94
.,n
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43
30
20 14
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1II
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I
If
III
IV
Fig. z. 50S-PAGE of extracellular proteins of (a) A. nidulans G-I059 and (b) A. nidulans 2-108 (ben AlO) after 1, 2 and 3 d of growth. (I) control; (II) MBC; (III) griseofulvin; (TV) molecular weight markers (kDa). For arrows see text. using 2'5 ml of Aquasol-2 scintillation fluid (NEN). Intra and extracellular radiolabelled proteins (20000 cpm sample-I) were analysed by SD5-PAGE followed by autoradiography. All experiments were done in triplicate. All chemicals were supplied by Merck, Sigma or Difco.
RESULTS Effeds of MBC and griseofulvin on growth in A. nidulans A sublethal dose of MBC (0'25 lAg ml- l ) partially inhibited 61-2
Protein secretion in A. nidulans
964
Effect of MBC and griseofulvin on the electrophoretic pattern of extracellular proteins in A. nidulans
(a)
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MBC and griseofulvin modified the electrophoretic pattern of secreted proteins by A. nidulans G-I059 (wild-type strain) compared with control. MBC reduced the amount of all extracellular proteins principally at day I of growth, and favoured the secretion, at days 2 and 3 of growth of 2 proteins with molecular weights of 16 and 70 kDa approx. (Fig. 2 a, see arrows I and m). The griseofulvin treatment did not alter the secretion of high molecular weight proteins. However, the effect of this compound coincided with MBC treatment inducing the secretion of a protein of 16 kDa approx. which was not secreted under control conditions (Fig. 2 a, see arrow m). Moreover both antifungals inhibited partially or totally the secretion of some proteins at days I, 2 and 3 of growth. When A. nidulans 2-108 (ben AIO) grew at control conditions in complete liquid medium it showed a different electrophoretic pattern of extracellular proteins from A. nidulans G-I059 (wild-type strain) growing in CM-2 medium. In A. nidulans 2-108 (ben AlO) griseofulvin stimulated the secretion of several proteins with molecular weight ranking from 43 kDa upwards at days I, 2 and 3 (Fig. 2 b, see arrow n), as well as, an increase in the secretion at days 2 and 3 of growth of two proteins, whose molecular weights were lower than 14 kDa (Fig. 2 b, see arrow p). Moreover this compound produced strong inhibition of the secretion of some proteins, particularly a protein of 38 kDa approx. which was released at control conditions (Fig. 2 b, see arrow 0). MBC did not modify the extracellular proteins pattern of the benomyl-resistant strain (ben AlO) (Fig. 2 b).
Effect of MBC and griseofulvin on secretion using radioactive protein labelling
2
2
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456
Hours
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9
Fig. 3. Effect of MBC and griseofulvin on extracellular newly synthesized radiolabelled proteins by (a) A. mdulans G-1059 and (b) A. nidulans 2-108 (ben AW). Control; . - - . , MBC 0'25 ~g ml- 1 ; .6.--.6., griseofulvin 400 ~g ml- 1 . Error bars represent S.E.M.
e--e,
growth of A. nidulans G-I059 (wild-type strain). This dose did not however, affect the resistant A. nidulans 2-108 (ben AlO). In the presence of a dose of 400 ~g ml- 1 of griseofulvin (usually used for this antifungal compound) growth stimulation was observed in both strains after 2 and 3 d incubation (Fig. I a, b).
MBC partially inhibited the secretion of labelled proteins between 44 and 53 h of growth in A. nidulans G-I059 (wildtype strain). The inhibition was greater than 50 % 9 h after the 35 [ S]methionine pulse. Griseofulvin slightly reduced the secretion of radiolabelled proteins into the culture medium (Fig. 3a). In the benomyl-resistant mutant strain A. nidulans 2-108 (ben AIO) griseofulvin decreased by 33 % the amount of proteins secreted 9 h after the pulse. However, MBC did not modify the levels of secreted proteins compared with control (Fig 3b). The proteins synthesized and secreted at several times after the radioactive label were resolved by SDS-PAGE and the different electrophoretic patterns obtained from the autoradiography were studied. MBC and griseofulvin produced a few alterations in the electrophoretic pattern of wild-type intracellular proteins, some of them being similar for both treatments (Fig. 4a, see arrows q, rand s). The autoradiography of extracellular proteins in the wildtype strain showed that only some of the newly synthesized proteins were secreted. After MBC treatment a protein band appeared (60 kDa approx.) which was not secreted at control conditions (Fig. 4a, see arrow u). Griseofulvin also favoured the secretion of a protein of 36 kDa approx. (Fig. 4a, see
J. R. De Lucas, 1. F. Monistrol and F. Laborda
965
Intracellular proteins (b)
(0)
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arrow v). In A. nidulans 2-108 (ben AlO) griseofulvin produced a few changes in the intracellular proteins pattern (Fig. 4 b), see arrow t) and stimulated the secretion of several extracellular proteins (Fig. 4 b, see arrows x, y and z). MBC did not significantly alter the electrophoretic pattern either intracellular or extracellular proteins in A. nidulans 2-108 (ben AlO) (Fig.4b).
DISCUSSION 43
30
,-r
20 14
I
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Extracellular proteins (b)
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,-U
,-X
.-v
'-y
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,-Z
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I
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Fig. 4. Autoradiography of SDS-PAGE of mtracellular and extracellular proteins labelled with [35S1methionine of A. mdulans G1059 (a) and A. nidulans 2-108 (ben AW) (b) (I) control; (II) MBC; (III)
griseofulvin; (IV) molecular weight markers. For arrows see text.
Cytoskeletal inhibitors have been widely used to probe the specific components involved in vegetative growth and differentiation processes or in organelle movement in filamentous fungi (Richmond & Pring, 1971; McKerracher & Heath, 1987). In these assays we have used sublethal doses of two antimicrotubular compounds, MBC and griseofulvin, with the purpose of studying the involvement of the microtubule cytoskeletal component in the protein secretion process in the filamentous fungus A. nidulans. The reduction observed in the vegetative growth of A. nidulans G-1059 (wild-type strain) in the presence of MBC has already been described in different species of filamentous fungi as a consequence of its inhibitory effect on fungal microtubules (Davidse, 1986). However, vegetative grown in A. nidulans 2108 (ben AlO) was resistant to the same dose of MBC. In both strains, griseofulvin increased the mycelial dry weight at 2 and 3 d although it had reduced early growth. This compound promotes hyphal branching on fungi that have chitin in their wall (Valla, 1979) as A. nidulans has. MBC and griseofulvin reduced the amount of proteins that were secreted by A. nidulans G-1059 (wild-type strain) as observed after radioactive protein labelling. In this same species it has been reported that invertase enzyme secretion is significantly reduced when hypha are treated with benomyl, whereas the protoplasts are not affected Oochova et aI., 1993). These observations suggest that the inhibition of the protein secretion could be a consequence of the impairment of polarized vesicle transport from the Golgi apparatus to the hyphal tip, caused by cytoplasmic microtubule depolymerization (Howard & Aist, 1980; Monistrol et al., 1988). Moreover, in the wild-type strain, both microtubular inhibitors caused an abnormal protein secretion in the culture filtrate, emphasizing the reduction or complete inhibition of secretion of some particular proteins. In addition, both compounds abnormally released other proteins into the culture supernatant. Although protein release could be due to stress caused by microtubule inhibitors (Aisemberg et aI., 1989), the results obtained in the present work suggest an irregular process of protein secretion when cytoplasmic microtubules are disorganized. In this way, the larger effect, from MBC rather than from griseofulvin, on protein secretion observed in A. nidulans G1059 (wild-type strain) could be due to a more specific action on MTs (De Carli & Larizza, 1988). The more intense protein bands observed in the autoradiography of wild-type intracellular proteins, which coincided in both antimicrotubular treatments, could be the result of the accumulation of several proteins in the cell due to the partial blocking of secretion by these compounds.
Protein secretion in A. nidulans Nevertheless, the changes in protein synthesis or secretion might be a stress response, e.g. by an effect on mitosis as a consequence of antimicrotubular compound treatments. Further work will be performed to determine whether these intracellular proteins, accumulated under drug influence, are a consequence of an inhibitor effect on the transport throughout the fungal hypha. Protein secretion was unaltered when A. nidulans 2-108 (benomyl-resistant strain) was grown in the presence of MBC. Since the ben A gene mutation prevents MBC action on microtubules (Davidse & Flach, 1977), the results obtained confirm that the effect of MBC on protein secretion in the wild-type strain is mediated via its action on microtubules. Griseofulvin altered the synthesis and secretion processes in the benomyl-resistant strain A. nidulans 2-108, reflecting the different target site of the drug (De Carli & Larizza, 1988). Although actin filaments play an important role in hyphal wall formation and related processes with the apical growth Gackson & Heath, 1989), the abnormal protein secretion observed during growth in the presence of both antimicrotubular drugs confirms the participation of cytoplasmic microtubules in the process of polarized secretion that occurs in the filamentous fungus A. nidulans, as has been described in other eukaryotic cells with polarized protein secretion (Schliwa, 1984; AchIer et at 1989), and recently in the same species Gochova et at 1993). We thank Professor Geoffrey Turner for helping to correct the manuscript. J. R. de Lucas was supported by a grant from the Diputaci6n de Guadalajara (Spain).
REFERENCES AchIer, c.. Filmer, D., Merte, C &< Drenckhahn, D. (1989). Role of microtubules in polarized delivery of apical membrane proteins to the brush border of the intestinal epitheliwn. journal of Cell BIOlogy 109, 179-189. Aisemberg, G. 0., Grotewold, E., Tacciolo, G. E. &< Judewicz, N. D. (1989). A major transcript in the response of Neurospora crassa to protein synthesis inhibition by cyclohexamide. Experimental Mycology 13, 121-128. Clemons, G. P. &< Sisler, H. D. (1969). Formation of a fungitoxIC denvative from benlate. Phytopathology 59, 705-706 Chang, P. L. &< Trevithick, J. R. (1974). How Important is secretion of exoenzymes through apical cell walls of fungi: Archives of MIcrobIOlogy 101, 281-293. Davidse, L. C (1986). Benzimidazole fungicides: Mechanism of action and bIOlogical impact. Annual Review of Phytopathology 24, 43--65. Davldse, L. C &< Flach, W. (1977). Differential bindmg of methyl benzim,dazol2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nrdulans. journal of Cell Biology 72, 174-193 De Carli, L. &< Lanzza, L. (1988). Gnseofulvin. Mutation Research 195, 91-126 Farkas, V. (1979). Biosynthesis of cell walls of fungi. MicrobiologIcal ReVIews 43, 117-144
(Accepted 14 December 1992)
966 Gooday, G. W (1983). The hyphal tip In Fungal DIfferentIatIOn; a Contemporary SyntheSIS (ed. J. E. Smith), pp. 315-356. Marcel Dekker Inc: New York. Heath, I. B. (1987). Preservation of a labile cortical array of actm filaments in growing hyphal hps of the fungus Saprolegnra ferax. European journal of Cell BIOlogy 44, 10--16. Hoch, H. C &< Staples, R. C (1985). The microtubule cytoskeleton m hyphae of Uromyces phaseolz germlings: its relahonshlp to the region of nucleahon and to the F-actin cytoskeleton. Protoplasma 124, 112-122. Howard, R. J &< Aist, J. R. (1980). CytoplasmiC rmcrotubules and fungal morphogenesis: ultrastructural effects of methyl-benzlmldazole-2-yl carbamate determined by freeze-substituhon of hyphal tip cells. journal of Cell BIOlogy 87, 55--64. Howard, R. J (1983). CytoplasmiC transport m hyphae of Gzlbertella. MycologIcal SOCIety of Amencan Newsletter 34, 24. Jackson, S. L. &< Heath, I. B. (1989). Effects of exogenous calcIUm ions on tip growth, intracellular Ca 2 + concentration, and actin arrays in hyphae of the fungus Saprolegnra ferax. Experimental Mycology 13, 1-12. Jochova, L Rupes,l. &< Peberdy, J. F. (1993). Effect of the microtubule inhibitor benomyl on protem secretion m Aspergillus nidulans MycologIcal Research. (in the press). Laemmli, U. K. (1970) Cleavage of structural proteins dUrIng the assembly of the head of bactenophage T4. Nature 227, 680--685. Lowry, 0 H., Rosenbrough, N. J, Farr, A. L. &< Randall, R. J. (1951) Protem determinahon with the fohn phenol method reagent. journal of BiologIcal ChemIStry 193, 265-275. McKerracher, L. J. &< Heath, I. B (1987). Cytoplasmic migration and intracellular organelle movements during tip growth of fungal hyphae. Experimental Mycology 11, 79-100. Monistrol, I. F, Perez-Leblic, M. I. &< Laborda, F. (1988). Effect of sublethal dose of benomyl on extracellular enzyme produchon by Cladosporium cucumennum. Transachons of the Bntish Mycological SOCIety 90, 193-197. Morris, N. R. (1986) The molecular genetics of microtubule protems m fungi. Experimental Mycology 10, 77-82. Morrisey, J. H. (1981). Silver stam for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitIvity. AnalytIcal BIOchemistry 117, 307-310. Richmond, D. V. &< Pring, R. J. (1971). The effect of benomyl on the fine structure of Botrytls fabae. journal of General MicrobIOlogy 66, 79-94. Roobol, A., Gull, K. &< Pogson, CI. (1977). Evidence that gnseofulvin binds to a microtubule assOCiated protem. FEBS Letters 75, 79-94. Raudaskoski, M., Rupes, I. &< T,monen, S. (1991). Immunofluorescence microscopy of the cytoskeleton in filamentous fungi after quick-freezing and low-temperature fixahon. Experimental Mycology IS, 167-173. ROSSler, C, Hoang-Van, K. &< Turian, G. (1989). Secrehon of an M, 60000 protem by benomyl-treated cells of Neurospora crassa. European journal of Cell BIOlogy 50, 333-339. Schliwa, M. (1984) Mechanism of intracellular organelle transport. In Cell and Muscle MotilIty: The Cytoskeleton (ed. j. W. Shay), pp. 1-82. Plenum Press: New York. Ton That, T c.. Rossier, C, Bal'Ja, F., Tunan, G. &< Roos, U. P. (1988). Induction of multiple germ tubes in Neurospora crassa by antitubulin agents European journal of Cell BIOlogy 46, 68-79. Valla, G. (1979). Effects of griseofulvin on cytology, growth, mitosis and branching of Polyporus arculanus. TransactIOns of the British MycologIcal SoCIety 73, 135-139. Van Tuyl, J. M. (1977). Genetics of fungal resistance to systemic fungicides. Meded Landbouwogesch Wagenmgen 2, 1-137. Weatherbee, J. A. &< Moms, N. R. (1984). AspergIllus contains mulhple tubulin genes. journal of BiologIcal Chemistry 259, 15452-15459. Wehland, J, Herzog, W. &< Weber, K (1977). Interaction of gnseofulvm With microtubules, microtubule protein and tubulin. journal of Molecular BIOlogy 111, 329-342.
Printed
rn
961
Great Bntarn
Effect of antimicrotubular drugs on the secretion process of extracellular proteins in Aspergillus nidulans
J. R. DE LUCAS, I. F. MONISTROL AND F. LABORDA'" Departamento de Microbiolog{a y Parasitolog{a, Universidad de Alcald de Henares, Campus Universitario, Carretera Madrid-Barcelona, KM. 33, Alcald de Henares, Madrid, Spain
E-28871
The role of microtubules (MTs) in the protein secretion process in Aspergillus nidulans has been studied using two antimicrotubular drugs, methyl benzimadazol-2-yl carbamate (MBC) and griseofulvin, and two strains of A. nidulans: a wild-type and a benomyl resistant-mutant (ben AID). Sublethal doses of MBC and griseofulvin reduced the total amount of proteins secreted by the wildtype strain and modified the electrophoretic pattern of the extracellular proteins, inhibiting the secretion of certain proteins and favouring the secretion of several others that were not secreted in the control cultures. Some of these modifications were the same with both antimicrotubular· compounds. MBC did not alter either the amount of proteins secreted or the electrophoretic patterns of proteins in the A. nidulans benomyl-resistant strain, demonstrating that its action is mediated via the microtubules. Our results suggest the involvement of MTs in the polarized secretion process of A. nidulans.
A characteristic of many filamentous fungi is their ability to secrete large amounts of proteins to the culture medium. Cytoplasmic vesicles containing proteins for secretion move from the Golgi apparatus and fuse with the plasmatic membrane liberating their contents to the periplasmic space (Farkas, 1979; Gooday, 1983). Proteins for secretion must cross the apical cell wall in order to be really extracellular proteins (Chang & Trevithick, 1974). The polarized transport towards the hyphal tip (apex) is not yet well understood. Several studies implicate cytoskeleton components, microtubules (MTs) and/or microfilaments (MFs), in this process (Howard & Aist, 1980; McKerracher & Heath, 1987). MTs take part in mitosis, intracellular transport and maintenance of the cell shape (Weatherbee & Morris, 1984), and cytoplasmic MTs of several ascomycete and basidiomycete species appear to play an important role as maintainers of the polarity in the apical cell (Raudaskoski, Rupes & Timonen, 1991). In filamentous fungi the movement of vesicles towards the cell apex could be associated with MTs tracks similar to the directional axonal transport (Howard, 1983; Hoch & Staples, 1985). Moreover, the presence of actin caps in normal growing tips suggest that other cytoskeleton components have a role in apical growth too (Heath, 1987). A simple approach to determine the cytoplasmic MT functions is to disrupt them with specific inhibitors and to examine the effect of these compounds. MBC (active principle of benomyl) is an MT assembly inhibitor that acts by binding to heterodimeric 0- and l3-tubulin molecules (Davidse & Flach,
• Corresponding author. 61
1977). The Aspergillus nidulans ben A gene encodes 131- and 132-tubulins and some ben A mutants develop benomyl resistance by diminishing the affinity of l3-tubulin for the drug (Morris, 1986). Griseofulvin is an MT-disorganizing agent that binds to tubulin (Wehland, Herzog & Weber, 1977) or more probably to microtubule associated proteins (MAPs) influencing the stability of MTs (RooboL Gull & Pogson, 1977). Although the effect of antimicrotubular drugs on differentiation and vegetative growth of filamentous fungi is now well established (Ton-That et al., 1988); only a few studies correlate the effect of these compounds with protein secretion and intracellular vesicle transport (MonistroL Perez-Leblic & Laborda, 1988; Rossier, Hoang-Van & Turian, 1989; Jochova, Rupes & Peberdy, 1993). We report here the effect of methyl benzimidazol-2-yl carbamate (MBC), the active principle of benomyl (Clemons & Sisler, 1969), and griseofulvin on protein synthesis and secretion in two strains of A. nidulans, a wild-type and a benomyl-resistant mutant (ben AlO). The results should help to determine whether MTs are necessary in these processes and whether the observed effects are mediated via drug action on MTs.
MATERIALS AND METHODS Fungal strains A. nidulans (Eidam) Winter G-I059 (wild-type strain) was donated by The Departamento de Microbiologia, Genetica, Medicina Preventiva y Salud Publica de la Universidad de Salamanca, Spain. The benomyl resistant mutant A. nidulans 2108 (ben AlO) was provided by J. F. Peberdy (Botany MYC 97
Protein secretion in A. nidulans 600
962 YEO solid medium (10 g 1-1 yeast extract 10 g 1-1 glucose, 20 g 1-1 agar-agar). Conidia were inoculated at 10 6 ml- 1 into 500 mI flasks with 100 ml of liqUid medium and incubated at 28°C on a rotary shaker at 200 rpm for 3 d. MBC (kindly provided by Dr Richmond, Long Ashton Research Station, Bristol, U.K.) and griseofulvin (Sigma Chemical Co.) were dissolved in dimethyl sulfoxide (DMSO) (1 mg ml- I and 70 mg ml- I stock solutions respectively), MBC and griseofulvin were added to culture media at final sublethal doses of 0'25 and 400 IJg ml- I respectively. 0'1 % (v Iv) DMSO was added to some flasks without antifungal compounds as a control.
(a)
400
200
Growth and protein determination
2
Mycelia from 1, 2 and 3 d incubation were harvested by filtration through Whatman No.1 paper, washed with distilled water and dried to constant weight at 80 0. Culture filtrates were dialyzed and freeze-dried before the protein content was measured by the method of Lowry et al. (1951).
3
5VS-PAGE protein secretion analysis (b)
For electrophoretic analysis, culture filtrates were concentrated by ultrafiltration with YM-5 AMICON membranes (5000 d) (Amicon Corporation U.S.A.), freeze-dried and resuspended in 62'S mM Tris-HCl buffer (pH 6'8). Proteins (90 IJg sample-I) from culture filtrates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing 14 % polyacrylamide according to the procedure of Laemmli (1970). Pharmacia molecular weight markers were used. Gels were stained with silver nitrate as indicated by Morrisey (1981).
400
200
[l55]methionine protein secretion analysis
2 Days
3
Fig. 1. Effect of MBC and griseofulvin on the growth of (a) A. nidulans G-1059 and (b) A. nidulans 2-108 (ben AW). Control; . - - . , MBC 0'25 I.lg ml- I ; A--A, griseofulvin 400 I.lg ml- I . Error bars represent S.E.M.
e--e,
Department, University of Nottingham, U.K.) and was preViously described by Van Tuyl (1977).
Culture conditions A. nidulans G-I059 was grown in CM-2 liquid medium (10 g 1-1 glucose, 0'2 g 1-1 adenine, 0'01 g -1 p-aminobenzoic acid) and A, nidulans 2-108 (ben AID) was grown in a complete liquid medium (5 g 1-1 glucose,S g 1-1 yeast extract,S g 1-1 casamino acids), Both media also contained salts (g 1-1) (5 Na N0 3, 0'5 MgS0 4, 0'5 KCI, 1'5 KH 2P0 4, 0'01 FeS0 4, 0'01 ZnS0 4 ), To produce conidia, the strains were grown on
For radioactive protein labelling, both strains were grown under the described conditions in 250 mI flasks with 60 ml culture media, After 44 h of incubation [35 S]methionine (38'1 MBq ml- I) (NEN) was added to the flasks. Aliquots (10 mI) were taken 1, 3, 5, 7 and 9 h after the pulse. Aliquots were used to determine mycelial dry-weight. Mycelia were separated from culture medium by centrifugation (17 000 g, 15 min, 4°) (rav 8'22 em). Extracellular proteins were precipitated by addition of 1 g I-I sodium deoxycholate (DOC) and 100 g 1-1 trichloroacetic acid (TCA), separated by centrifugation (27000 g, 30 min, 4°), and resuspended in 62'S mM Tris-HCI buffer (pH 6'8). The mycelium was washed twice with 50 roM Tris-HCI buffer (pH 6'9) and mycelial extracts were obtained after incubation with Novozym 234 (5 mg ml- I), 5 mM-EDTA and 2 mM-PMSF (60 min, 37°) in the same buffer in a rotary shaker. The cell debris was removed by centrifugation (27000 g, 30 min, 4°) and intracellular proteins were precipitated with 1 g I-I DOC and 100 g 1-1 TCA and separated from soluble intracellular material by centrifugation (27000 g, 30 min, 4°). Intracellular proteins were resuspended in 62'S mM Tris-HCI buffer (pH 6'8). Aliquots (20 IJI) of radiolabelled proteins were counted for radioactivity in a scintillation counter (Kontron Betamatic II)
J. R. De Lucas, I. F. Monistrol and F. Laborda
963
(a)
Days of fungal growth 3
2
94 ~l
67
43
-
-
~-
30
.,m
20
14
II
II
III
(b)
1Il
II
TV
III
Days of fungal growth 3
2
94
.,n
67
43
30
20 14
II
1II
II
III
I
If
III
IV
Fig. z. 50S-PAGE of extracellular proteins of (a) A. nidulans G-I059 and (b) A. nidulans 2-108 (ben AlO) after 1, 2 and 3 d of growth. (I) control; (II) MBC; (III) griseofulvin; (TV) molecular weight markers (kDa). For arrows see text. using 2'5 ml of Aquasol-2 scintillation fluid (NEN). Intra and extracellular radiolabelled proteins (20000 cpm sample-I) were analysed by SD5-PAGE followed by autoradiography. All experiments were done in triplicate. All chemicals were supplied by Merck, Sigma or Difco.
RESULTS Effeds of MBC and griseofulvin on growth in A. nidulans A sublethal dose of MBC (0'25 lAg ml- l ) partially inhibited 61-2
Protein secretion in A. nidulans
964
Effect of MBC and griseofulvin on the electrophoretic pattern of extracellular proteins in A. nidulans
(a)
8
6
4
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2
;:
00
'0) ~
2
C
3
4
5
6
7
8
9
"0
'OSIl
S 0.. ..,u
(b)
0
-; 6
4
MBC and griseofulvin modified the electrophoretic pattern of secreted proteins by A. nidulans G-I059 (wild-type strain) compared with control. MBC reduced the amount of all extracellular proteins principally at day I of growth, and favoured the secretion, at days 2 and 3 of growth of 2 proteins with molecular weights of 16 and 70 kDa approx. (Fig. 2 a, see arrows I and m). The griseofulvin treatment did not alter the secretion of high molecular weight proteins. However, the effect of this compound coincided with MBC treatment inducing the secretion of a protein of 16 kDa approx. which was not secreted under control conditions (Fig. 2 a, see arrow m). Moreover both antifungals inhibited partially or totally the secretion of some proteins at days I, 2 and 3 of growth. When A. nidulans 2-108 (ben AIO) grew at control conditions in complete liquid medium it showed a different electrophoretic pattern of extracellular proteins from A. nidulans G-I059 (wild-type strain) growing in CM-2 medium. In A. nidulans 2-108 (ben AlO) griseofulvin stimulated the secretion of several proteins with molecular weight ranking from 43 kDa upwards at days I, 2 and 3 (Fig. 2 b, see arrow n), as well as, an increase in the secretion at days 2 and 3 of growth of two proteins, whose molecular weights were lower than 14 kDa (Fig. 2 b, see arrow p). Moreover this compound produced strong inhibition of the secretion of some proteins, particularly a protein of 38 kDa approx. which was released at control conditions (Fig. 2 b, see arrow 0). MBC did not modify the extracellular proteins pattern of the benomyl-resistant strain (ben AlO) (Fig. 2 b).
Effect of MBC and griseofulvin on secretion using radioactive protein labelling
2
2
3
456
Hours
7
8
9
Fig. 3. Effect of MBC and griseofulvin on extracellular newly synthesized radiolabelled proteins by (a) A. mdulans G-1059 and (b) A. nidulans 2-108 (ben AW). Control; . - - . , MBC 0'25 ~g ml- 1 ; .6.--.6., griseofulvin 400 ~g ml- 1 . Error bars represent S.E.M.
e--e,
growth of A. nidulans G-I059 (wild-type strain). This dose did not however, affect the resistant A. nidulans 2-108 (ben AlO). In the presence of a dose of 400 ~g ml- 1 of griseofulvin (usually used for this antifungal compound) growth stimulation was observed in both strains after 2 and 3 d incubation (Fig. I a, b).
MBC partially inhibited the secretion of labelled proteins between 44 and 53 h of growth in A. nidulans G-I059 (wildtype strain). The inhibition was greater than 50 % 9 h after the 35 [ S]methionine pulse. Griseofulvin slightly reduced the secretion of radiolabelled proteins into the culture medium (Fig. 3a). In the benomyl-resistant mutant strain A. nidulans 2-108 (ben AIO) griseofulvin decreased by 33 % the amount of proteins secreted 9 h after the pulse. However, MBC did not modify the levels of secreted proteins compared with control (Fig 3b). The proteins synthesized and secreted at several times after the radioactive label were resolved by SDS-PAGE and the different electrophoretic patterns obtained from the autoradiography were studied. MBC and griseofulvin produced a few alterations in the electrophoretic pattern of wild-type intracellular proteins, some of them being similar for both treatments (Fig. 4a, see arrows q, rand s). The autoradiography of extracellular proteins in the wildtype strain showed that only some of the newly synthesized proteins were secreted. After MBC treatment a protein band appeared (60 kDa approx.) which was not secreted at control conditions (Fig. 4a, see arrow u). Griseofulvin also favoured the secretion of a protein of 36 kDa approx. (Fig. 4a, see
J. R. De Lucas, 1. F. Monistrol and F. Laborda
965
Intracellular proteins (b)
(0)
94
67
arrow v). In A. nidulans 2-108 (ben AlO) griseofulvin produced a few changes in the intracellular proteins pattern (Fig. 4 b), see arrow t) and stimulated the secretion of several extracellular proteins (Fig. 4 b, see arrows x, y and z). MBC did not significantly alter the electrophoretic pattern either intracellular or extracellular proteins in A. nidulans 2-108 (ben AlO) (Fig.4b).
DISCUSSION 43
30
,-r
20 14
I
II
II
III
III
Extracellular proteins (b)
(0)
94
67
,-U
,-X
.-v
'-y
43
,-Z
30
20
14
II
1II
I
II
III
Fig. 4. Autoradiography of SDS-PAGE of mtracellular and extracellular proteins labelled with [35S1methionine of A. mdulans G1059 (a) and A. nidulans 2-108 (ben AW) (b) (I) control; (II) MBC; (III)
griseofulvin; (IV) molecular weight markers. For arrows see text.
Cytoskeletal inhibitors have been widely used to probe the specific components involved in vegetative growth and differentiation processes or in organelle movement in filamentous fungi (Richmond & Pring, 1971; McKerracher & Heath, 1987). In these assays we have used sublethal doses of two antimicrotubular compounds, MBC and griseofulvin, with the purpose of studying the involvement of the microtubule cytoskeletal component in the protein secretion process in the filamentous fungus A. nidulans. The reduction observed in the vegetative growth of A. nidulans G-1059 (wild-type strain) in the presence of MBC has already been described in different species of filamentous fungi as a consequence of its inhibitory effect on fungal microtubules (Davidse, 1986). However, vegetative grown in A. nidulans 2108 (ben AlO) was resistant to the same dose of MBC. In both strains, griseofulvin increased the mycelial dry weight at 2 and 3 d although it had reduced early growth. This compound promotes hyphal branching on fungi that have chitin in their wall (Valla, 1979) as A. nidulans has. MBC and griseofulvin reduced the amount of proteins that were secreted by A. nidulans G-1059 (wild-type strain) as observed after radioactive protein labelling. In this same species it has been reported that invertase enzyme secretion is significantly reduced when hypha are treated with benomyl, whereas the protoplasts are not affected Oochova et aI., 1993). These observations suggest that the inhibition of the protein secretion could be a consequence of the impairment of polarized vesicle transport from the Golgi apparatus to the hyphal tip, caused by cytoplasmic microtubule depolymerization (Howard & Aist, 1980; Monistrol et al., 1988). Moreover, in the wild-type strain, both microtubular inhibitors caused an abnormal protein secretion in the culture filtrate, emphasizing the reduction or complete inhibition of secretion of some particular proteins. In addition, both compounds abnormally released other proteins into the culture supernatant. Although protein release could be due to stress caused by microtubule inhibitors (Aisemberg et aI., 1989), the results obtained in the present work suggest an irregular process of protein secretion when cytoplasmic microtubules are disorganized. In this way, the larger effect, from MBC rather than from griseofulvin, on protein secretion observed in A. nidulans G1059 (wild-type strain) could be due to a more specific action on MTs (De Carli & Larizza, 1988). The more intense protein bands observed in the autoradiography of wild-type intracellular proteins, which coincided in both antimicrotubular treatments, could be the result of the accumulation of several proteins in the cell due to the partial blocking of secretion by these compounds.
Protein secretion in A. nidulans Nevertheless, the changes in protein synthesis or secretion might be a stress response, e.g. by an effect on mitosis as a consequence of antimicrotubular compound treatments. Further work will be performed to determine whether these intracellular proteins, accumulated under drug influence, are a consequence of an inhibitor effect on the transport throughout the fungal hypha. Protein secretion was unaltered when A. nidulans 2-108 (benomyl-resistant strain) was grown in the presence of MBC. Since the ben A gene mutation prevents MBC action on microtubules (Davidse & Flach, 1977), the results obtained confirm that the effect of MBC on protein secretion in the wild-type strain is mediated via its action on microtubules. Griseofulvin altered the synthesis and secretion processes in the benomyl-resistant strain A. nidulans 2-108, reflecting the different target site of the drug (De Carli & Larizza, 1988). Although actin filaments play an important role in hyphal wall formation and related processes with the apical growth Gackson & Heath, 1989), the abnormal protein secretion observed during growth in the presence of both antimicrotubular drugs confirms the participation of cytoplasmic microtubules in the process of polarized secretion that occurs in the filamentous fungus A. nidulans, as has been described in other eukaryotic cells with polarized protein secretion (Schliwa, 1984; AchIer et at 1989), and recently in the same species Gochova et at 1993). We thank Professor Geoffrey Turner for helping to correct the manuscript. J. R. de Lucas was supported by a grant from the Diputaci6n de Guadalajara (Spain).
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(Accepted 14 December 1992)
966 Gooday, G. W (1983). The hyphal tip In Fungal DIfferentIatIOn; a Contemporary SyntheSIS (ed. J. E. Smith), pp. 315-356. Marcel Dekker Inc: New York. Heath, I. B. (1987). Preservation of a labile cortical array of actm filaments in growing hyphal hps of the fungus Saprolegnra ferax. European journal of Cell BIOlogy 44, 10--16. Hoch, H. C &< Staples, R. C (1985). The microtubule cytoskeleton m hyphae of Uromyces phaseolz germlings: its relahonshlp to the region of nucleahon and to the F-actin cytoskeleton. Protoplasma 124, 112-122. Howard, R. J &< Aist, J. R. (1980). CytoplasmiC rmcrotubules and fungal morphogenesis: ultrastructural effects of methyl-benzlmldazole-2-yl carbamate determined by freeze-substituhon of hyphal tip cells. journal of Cell BIOlogy 87, 55--64. Howard, R. J (1983). CytoplasmiC transport m hyphae of Gzlbertella. MycologIcal SOCIety of Amencan Newsletter 34, 24. Jackson, S. L. &< Heath, I. B. (1989). Effects of exogenous calcIUm ions on tip growth, intracellular Ca 2 + concentration, and actin arrays in hyphae of the fungus Saprolegnra ferax. Experimental Mycology 13, 1-12. Jochova, L Rupes,l. &< Peberdy, J. F. (1993). Effect of the microtubule inhibitor benomyl on protem secretion m Aspergillus nidulans MycologIcal Research. (in the press). Laemmli, U. K. (1970) Cleavage of structural proteins dUrIng the assembly of the head of bactenophage T4. Nature 227, 680--685. Lowry, 0 H., Rosenbrough, N. J, Farr, A. L. &< Randall, R. J. (1951) Protem determinahon with the fohn phenol method reagent. journal of BiologIcal ChemIStry 193, 265-275. McKerracher, L. J. &< Heath, I. B (1987). Cytoplasmic migration and intracellular organelle movements during tip growth of fungal hyphae. Experimental Mycology 11, 79-100. Monistrol, I. F, Perez-Leblic, M. I. &< Laborda, F. (1988). Effect of sublethal dose of benomyl on extracellular enzyme produchon by Cladosporium cucumennum. Transachons of the Bntish Mycological SOCIety 90, 193-197. Morris, N. R. (1986) The molecular genetics of microtubule protems m fungi. Experimental Mycology 10, 77-82. Morrisey, J. H. (1981). Silver stam for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitIvity. AnalytIcal BIOchemistry 117, 307-310. Richmond, D. V. &< Pring, R. J. (1971). The effect of benomyl on the fine structure of Botrytls fabae. journal of General MicrobIOlogy 66, 79-94. Roobol, A., Gull, K. &< Pogson, CI. (1977). Evidence that gnseofulvin binds to a microtubule assOCiated protem. FEBS Letters 75, 79-94. Raudaskoski, M., Rupes, I. &< T,monen, S. (1991). Immunofluorescence microscopy of the cytoskeleton in filamentous fungi after quick-freezing and low-temperature fixahon. Experimental Mycology IS, 167-173. ROSSler, C, Hoang-Van, K. &< Turian, G. (1989). Secrehon of an M, 60000 protem by benomyl-treated cells of Neurospora crassa. European journal of Cell BIOlogy 50, 333-339. Schliwa, M. (1984) Mechanism of intracellular organelle transport. In Cell and Muscle MotilIty: The Cytoskeleton (ed. j. W. Shay), pp. 1-82. Plenum Press: New York. Ton That, T c.. Rossier, C, Bal'Ja, F., Tunan, G. &< Roos, U. P. (1988). Induction of multiple germ tubes in Neurospora crassa by antitubulin agents European journal of Cell BIOlogy 46, 68-79. Valla, G. (1979). Effects of griseofulvin on cytology, growth, mitosis and branching of Polyporus arculanus. TransactIOns of the British MycologIcal SoCIety 73, 135-139. Van Tuyl, J. M. (1977). Genetics of fungal resistance to systemic fungicides. Meded Landbouwogesch Wagenmgen 2, 1-137. Weatherbee, J. A. &< Moms, N. R. (1984). AspergIllus contains mulhple tubulin genes. journal of BiologIcal Chemistry 259, 15452-15459. Wehland, J, Herzog, W. &< Weber, K (1977). Interaction of gnseofulvm With microtubules, microtubule protein and tubulin. journal of Molecular BIOlogy 111, 329-342.