Arch Microbiol (1992) 157: 104-106
Archives of
Hicrobiology ,9 Springer-Verlag 1992
Effects of light on protein secretion in Neurospora crassa Andreas Kallies, Saadat Mohsenzadeh, and Ludger Rensing Institut ffir Zellbiologie, Biochemie und Biotechnologie der Umversit/it, Postfach 330440, Bibliothekstral3e, W-2800 Bremen 33, Federal Republic of Germany Received May 13, 1991/Accepted September 9, 1991
Abstract. The relative c o n c e n t r a t i o n s of secreted proteins in liquid cultures of Neurospora crassa differ in c o n s t a n t darkness c o m p a r e d to c o n s t a n t light (2500 lx). Light reduces the c o n c e n t r a t i o n s o f some polypeptides m a r k e d ly and increases the c o n c e n t r a t i o n s o f protein species o f 67, 40, 18 and 13 k D a . The "blind" wc-2 m u t a n t o f Neurospora does n o t show light dependent differences in a m o u n t s o f secreted proteins. One o f the light-sensitive extracellular proteins is s h o w n to be a protease o f 17,5 k D a . Key words: Light effect Protease -
Extracellular proteins Neurospora crassa - F u n g i
-
Effects o f light on the synthesis rates o f intracellular proteins have been described in several organisms including Neurospora ( C h a m b e r s et al. 1985; R u s s o 1988; S o m m e r et al. 1989). In view o f the general influence o f light on metabolism, m e m b r a n e processes and m o r p h o genesis ( G r a a f m a n s 1977; M e t z e n b e r g 1979; Gressel 1980), we were interested in the question, whether light affects the secretion o f proteins, because extracellular polypeptides m i g h t play a role in intercellular signalling and hence the s y n c h r o n i z a t i o n o f circadian clocks within a mycelium. A n o t h e r reason for light dependent changes in protein secretion m a y be seen in the light-induction o f differentiation and the subsequent changes in the n u m b e r o f aerial hyphae. Aerial h y p h a e differ f r o m vegetative h y p h a e by there completely aerobic m e t a b o lism (Turian a n d Bianchi 1971). Thus, the different needs for nutrients m a y cause different proteins to be synthesized and secreted. This change m a y resemble the induction o f extracellular proteins by different nutrients in the m e d i u m ( C o h e n a n d D r u c k e r 1977; N a h a s et al. 1982; H a s u n u m a 1983; F u r u k a w a et al. 1987). In order to test the effect o f light, we collected proteins in the m e d i u m after several days o f light, light-dark or darkness and analyzed the protein pattern by one dimensional Offprint requests to: L. Rensing
gelelectrophoresis. The results showed clear evidence for an effect o f light - either on the induction and repression o f extracellular protein synthesis a n d / o r secretion by light.
Materials and methods In most of these experiments we used the "band" (bd) mutant of Neurospora crassa, which shows a clear circadian rhythmicity of conidiation on agar in constant darkness. The wild-type and the "white collar" (we-2) mutant, which is blind for light effects, was used in one experiment. Stock cultures were maintained on Horowitz complete medium (Horowitz 1947). 1,6 x 107 Conidia were inoculated into 25 ml autoclaved 2% Vogel-medium containing 3% sucrose and 2 mM Ca 2 +. The temperature was kept at 25 ~ cultures in constant light or a 12:12 h light-dark cycle were illuminated with 2500 lx white light (Radium NL 18W/25). The liquid medium of cultures was filtered through a membrane filter (SS 595 - Schleicher and Schfill, Dassel, FRG) without any mechanical stress in order to prevent damage to the hyphae. The medium containing the secreted proteins was quickly frozen in N 2 to - 80 ~ and lyophilized for 24 h, 500 gl distilled water was added to the lyophilized probe to further analyse the secretory proteins. In order to allow optimal separation of the proteins on SDS-gels, it was necessary to dialyse the probes for 12 h at 4 ~ against double distilled water (100-fold volume). Dialysis together with a protease inhibitor (30 gM/ml phenylmethylsulfonylfluoride) resulted in the same protein pattern. After dialysis the proteins were again lyophilized. Protein concentration was measured by the method of Lowry et al. (1951). The proteins were separated by SDS-PAGE according to Laemmli (1970) and the gels stained with Coomassie G250 (Neuhoff et al. 1985). The lyophilized protease probes were desalted on a NAP-25 column (Pharmacia) with Sephadex G-25 and eluted with 50 mM Tris-HC1 buffer (pH 7.2). The proteolytie activity were examined by non-denaturing polyacrylamlde gel electrophoresis (Heussen and Dowdle 1980). The hydrolysis of gelatine required 2 mM CaC12 and alkaline pH milieu between 7.4 and 8.5. The basic experiments were repeated more than five times with the same results.
Results The relative concentrations of secreted proteins in liquid cultures of Neuroapora crassa differ in c o n s t a n t darkness
105 a
C
iDa
kI)a
66
67
b
c
d
kDa
66
52 45
45
4O
36 31
29 24
36 29 24 20.1
20,1 18
14.2
17.5
14.2 Fig. 3. Secrctor~ protcna~ (100 lag) ol wdd-type and wc-2 culture
Fig. 1. SecretoryprotemsofNeuro~polacrassa (bcLmutant) analysed by SDS-PAGE. Lane a: Secretory proteins of mycelia grown at constant light. Lane b: Secretory proteins grown at constant dark. Lane c: Secretory proteins of mycelia kept in a 12:12 h light-dark regimen (last 6 h in light). All cultures were 120 h old and kept at a temperature of 25 ~ Each lane was loaded with 100 gg protein. The molecular weights of the marker proteins are given on the left side.
compared to constant light (Fig. 1). In constant darkness, relatively high concentrations of protein species with molecular masses of 72, 68, 62, 58, 39.5, 30.5 and 17.5 kDa and less prominent bands of 52.5 and 37 kDa are detected. In constant light the concentrations of some polypeptides are markedly reduced, in particular the concentration of the 17.5 kDa protein. Light induces new protein species of 67, 40, 31, 18 and 13 kDa. The secretory proteins of mycelia kept in 12:12 h light-dark regimes are similar to those detected in constant light. In a few cases a mixture of light- and
a
b
c
d
iDa
66 45
filtrates on SDS-PAGE. All mycelia were 120 h old and kept at a temperature of 25 ~ Lane a: Secretory proteins from wild-type mycelia at constant light. Lane b: Wild-type myceha at constant dark. Lane c: wc-2 mycelia at constant light. Lane d: wc-2 mycelia at constant dark. The marker proteins are shown on the left side dark-dependent proteins was observed: in the range of 72/62 kDa and 17/18 kDa. Analysis of the secretory proteins for their proteolytic activity indicates the presence of a protease of about 17.5 kDa, which shows light sensitive changes (Fig. 2): light conditions decrease the proteolytic activity. A similar activity, however less well expressed, is observed in a 22-kDa band of the gelatine gel. Other protease species of approximately 30, 53 and 68 kDa seem to have different activities depending on the age of the culture (40h versus 100h). In 100h-old cultures a 30-kDa protease is activated and a 68-kDa protease inhibited by light. In order to test whether the light effects were mediated by the blue light response system, we analysed the secretory proteins of the wc-2 mutant. In constant light w c - 2 shows the same pattern as in constant darkness, which is identical to the wild-type pattern in co.nstant dark (Fig. 3). Similar results were found in the second '"white collar" mutant wc-1 (not shown). A 40-kDa polypeptid is secreted only in those cultures which synthesize carotenoids (wild-type-LL, bd-LL). Discussion
36 29 24 20.1 14.2 Fig. 2. bccrctor} protcascb of ,\c,~o,V~ola ~/a,~a (hal-mutant)Ol] SDS-polyacrylamide gelatine gel. Extracellular proteases from cultures grown for 40 h in constant hght (lane a) and constant dark-(lane b). Secretory proteases of mycelia grown for 120 h in constant light (lane c) and under constant dark (lane d). Each lane
was loaded with 30 gg protein. The marker proteins are shown on the left side
Among the light-repressible and inducible secretory proteins is a protease of 17.5 and a protein of 18 kDa, respectively, which may be identical to secretory proteases of 18 kDa described by Lindberg et al. (1982). These two proteases (called M-l, M-2) were activated by a medium pH lower or higher than 5.5, respectively. Ruythers (1980) provided evidence that light affects the carbohydrate metabolism and since in our experiments the medium pH in constant light was slightly higher than the pH in constant darkness, the secretion of these two proteins may be mediated by pH changes within and outside the cytoplasm. The 30-kDa protease activated by
106 100 h of light m a y be identical to an alkaline protease of 30.5 k D a (Lindberg et al. 1981) or, possibly, an alkaline protease of 31 k D a ( H a n s o n and M a r z l u f 1973). O t h e r light-repressible secretory proteins can tentatively be related to k n o w n extracellular enzymes, such as a 7 0 - k D a alkaline p h o s p h a t a s e ( F u r u k a w a et al. 1987) and a 6 8 - k D a nuclease ( H a s u n u m a 1973). The same is true for light-induced proteins: the 6 6 - k D a laccase (Tamaru and I n o u e 1989) a n d a 11.5-kDa ribonuclease (Hashim o t o et al. 1971) m a y be close to the molecular welgtit of secretory proteins observed in o u r study. O n e w a y to interpret o u r findings is to assume that light directly controls the expression of genes coding for secretory proteins as s h o w n for a n u m b e r of intracellular enzymes (Richter 1984; C h a m b e r s et al. 1985; S o m m e r et al. 1989). An alternative interpretation is that light affects synthesis and release of these proteins indirectly. Indirect influences of light m a y stem from the effect of light on cellular metabolism ( G r a a f m a n s 1977; PiskorzBincycka 1978; R u y h t e r s 1980; Gressel and R a u 1983), on c o r r e s p o n d i n g changes of the intra- and extracellular p H (Lindberg et al. 1982) and on the effect of light on the differentiation of h y p h a e (Fiema 1979). Aerial h y p h a e differ in their m e t a b o l i s m f r o m their vegetative counterparts such that they utilize aerobic p a t h w a y s exclusively (Turian and Bianchi 1971). Differences in the metabolic state m a y create needs for other nutrients, which causes a r e p r o g r a m m i n g of the recruiting extracellular proteins. Direct or indirect effects of light seem to be mediated by the blue light response system. In some cases the light-inducible and repressible proteins differ only slightly in their molecular weight. This m a y be explained either by the existence of isoenzymes with slightly different molecular weight, or by assuming posttranslational m o d i fications of one enzyme. 9
I
Acknowledgement. We are grateful to V. E. A. Russo for valuable discussions and for providing mutant strains9
References Chambers JAA, Hinkelammert K, Russo VEA (1985) kightregulated protein and poly-(A+)-mRNA synthesis in Neurospora crassa. EMBO J 13:3649 3653 Cohen BL, Drucker H (1977) Regulation of exocellular protease in Neurospora crassa: induction and repression under conditions of nitrogen starvation. Arch Biochem Biophys 182:601-613 Faema J (1979) The hydroxyproline content in the mycelium of AspergiIlusglganteus rout. alba. Bull Acad Pol Sci 27 : 121 124 Furukawa K, Hasunuma K, Shinohara Y (1987) Characterization of Pcrepressible enzymes secreted in culture media by Neurospora crassa wild-type cells and null-type mutants9 J of Bacteriol 169:4790-4795 Graafmans WDJ (1977) Effect of blue light on metabolism in Penicillium isariiforme. J Gen Microbiol 101 : 157-161
Gressel J (1980) Blue light and transcription, In: Senger H (ed) The blue light syndrome. Springer, Berlin Heidelberg New York, pp 133 153 Gressel J, Rau W (1983) Photocontrol of fungal development, In: Shropshire W, Mohr H (eds) Photomorphogenesis. Encyclopedia of plant physiology 16b. Springer, Berlin Heidelberg New York, pp 603-639 Hanson MA, Marzluf GA (1973) Regulation of a sulfur-controlled protease in Neurospora crassa. J Bacteriol 116:785 789 Hashimoto J, Uchida T, E gami F (1971) Purification of ribonuclease U1 and some properties of ribonucleases U1 and NI9 J Biochem 78:903-911 Hasunuma K (1973) Repressible extracellular nucleases in Neurospora crassa. Biochim Biophys Acta 319:288-293 Hasunuma K (1983) Repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities in Neurospora crassa. J Bacteriol 156 : 291 - 300 Heussen C, Dowdle EB (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels contaimng dodecyl sulfate and copolymerized substrates. Anal Biochem 102: 196-202 Horowitz NH (1947) Methionine synthesis in Neurospora. J Biol Chem 171:255-264 Laemmli HK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature 227:680-685 Lindberg RA, Eirich LD, Price JS, Wolfinbarger L, Drucker H (1981) Alkaline protease from Neurospora crassa. J Biol Chem 256:811-814 Lindberg RA, Rhodes WG, Eirich LD, Drucker H (1982) Extracellular acid proteases from Neurospora crassa. J Bacteriol 150: 1103 1108 Lowry OH, Rosebrough NJ, Farr AL, Randall, RJ (1951) Protein measurement with the phenol reagent9J Biol Chem 139: 265-275 Metzenberg RL (1979) Implications of some genetic control mechanisms in Neurospora. Microbiol Rev 43:361-383 Nahas E, Terenzi HF, Rossi A (1982) Effect of carbon source and pH on the production and secretion of acid protease (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Neurospora crassa. J Gen Mlcrobiol 128:2017-2021 Neuhoff V, Stamm R, Eibl HJ (1985) Intensive Proteinf/irbung ohne Hintergrund mit Coomassie Blau G-250 in Polyacrylamidgelen: Theorie und Praxis9 Electrophoresis 6 : 8 7 - 9 1 and 330-334 Piskorz-Bincycka B (1978) Studies on the organic acids content of the mycelia of Penicillium isariiforme. Acta Biol Cracov: 21-22 Richter G (1984) Blue light effects on the level of translation and transcription. In: Senger H (ed) The blue hght effects in biological systems. Springer, Berlin Heidelberg New York, pp 253 262 Russo VEA (1988) Blue light induces circadian rhythms in the bd mutant of Neurospora crassa: double mutants bd, wc-1 and bd, wc-2 are blind. J Photochem Photobiol 2:59 65 Ruythers G (1980) Effects of blue light on enzymes. In: Senger H (ed) The blue light syndrome. Springer, Berlin Heidelberg New York, pp 309-318 Sommer T, Chambers JAA, Eberle J, Lauter FR, Russo VEA (1989) Fast light-regulated genes of Neurospora crassa. Nucleic Acids Res 14:5713-5723 Tamaru H, Inoue H (1989) Isolation and characterization of a laccase-derepressed mutant of Neurospora crassa. J Bacteriol 171 : 6288-6293 Turian G, Bianchi DE (,1971) Conidiation in Neurospora crassa. Arch Mikrobiol 77:262-274
Archives of
Hicrobiology ,9 Springer-Verlag 1992
Effects of light on protein secretion in Neurospora crassa Andreas Kallies, Saadat Mohsenzadeh, and Ludger Rensing Institut ffir Zellbiologie, Biochemie und Biotechnologie der Umversit/it, Postfach 330440, Bibliothekstral3e, W-2800 Bremen 33, Federal Republic of Germany Received May 13, 1991/Accepted September 9, 1991
Abstract. The relative c o n c e n t r a t i o n s of secreted proteins in liquid cultures of Neurospora crassa differ in c o n s t a n t darkness c o m p a r e d to c o n s t a n t light (2500 lx). Light reduces the c o n c e n t r a t i o n s o f some polypeptides m a r k e d ly and increases the c o n c e n t r a t i o n s o f protein species o f 67, 40, 18 and 13 k D a . The "blind" wc-2 m u t a n t o f Neurospora does n o t show light dependent differences in a m o u n t s o f secreted proteins. One o f the light-sensitive extracellular proteins is s h o w n to be a protease o f 17,5 k D a . Key words: Light effect Protease -
Extracellular proteins Neurospora crassa - F u n g i
-
Effects o f light on the synthesis rates o f intracellular proteins have been described in several organisms including Neurospora ( C h a m b e r s et al. 1985; R u s s o 1988; S o m m e r et al. 1989). In view o f the general influence o f light on metabolism, m e m b r a n e processes and m o r p h o genesis ( G r a a f m a n s 1977; M e t z e n b e r g 1979; Gressel 1980), we were interested in the question, whether light affects the secretion o f proteins, because extracellular polypeptides m i g h t play a role in intercellular signalling and hence the s y n c h r o n i z a t i o n o f circadian clocks within a mycelium. A n o t h e r reason for light dependent changes in protein secretion m a y be seen in the light-induction o f differentiation and the subsequent changes in the n u m b e r o f aerial hyphae. Aerial h y p h a e differ f r o m vegetative h y p h a e by there completely aerobic m e t a b o lism (Turian a n d Bianchi 1971). Thus, the different needs for nutrients m a y cause different proteins to be synthesized and secreted. This change m a y resemble the induction o f extracellular proteins by different nutrients in the m e d i u m ( C o h e n a n d D r u c k e r 1977; N a h a s et al. 1982; H a s u n u m a 1983; F u r u k a w a et al. 1987). In order to test the effect o f light, we collected proteins in the m e d i u m after several days o f light, light-dark or darkness and analyzed the protein pattern by one dimensional Offprint requests to: L. Rensing
gelelectrophoresis. The results showed clear evidence for an effect o f light - either on the induction and repression o f extracellular protein synthesis a n d / o r secretion by light.
Materials and methods In most of these experiments we used the "band" (bd) mutant of Neurospora crassa, which shows a clear circadian rhythmicity of conidiation on agar in constant darkness. The wild-type and the "white collar" (we-2) mutant, which is blind for light effects, was used in one experiment. Stock cultures were maintained on Horowitz complete medium (Horowitz 1947). 1,6 x 107 Conidia were inoculated into 25 ml autoclaved 2% Vogel-medium containing 3% sucrose and 2 mM Ca 2 +. The temperature was kept at 25 ~ cultures in constant light or a 12:12 h light-dark cycle were illuminated with 2500 lx white light (Radium NL 18W/25). The liquid medium of cultures was filtered through a membrane filter (SS 595 - Schleicher and Schfill, Dassel, FRG) without any mechanical stress in order to prevent damage to the hyphae. The medium containing the secreted proteins was quickly frozen in N 2 to - 80 ~ and lyophilized for 24 h, 500 gl distilled water was added to the lyophilized probe to further analyse the secretory proteins. In order to allow optimal separation of the proteins on SDS-gels, it was necessary to dialyse the probes for 12 h at 4 ~ against double distilled water (100-fold volume). Dialysis together with a protease inhibitor (30 gM/ml phenylmethylsulfonylfluoride) resulted in the same protein pattern. After dialysis the proteins were again lyophilized. Protein concentration was measured by the method of Lowry et al. (1951). The proteins were separated by SDS-PAGE according to Laemmli (1970) and the gels stained with Coomassie G250 (Neuhoff et al. 1985). The lyophilized protease probes were desalted on a NAP-25 column (Pharmacia) with Sephadex G-25 and eluted with 50 mM Tris-HC1 buffer (pH 7.2). The proteolytie activity were examined by non-denaturing polyacrylamlde gel electrophoresis (Heussen and Dowdle 1980). The hydrolysis of gelatine required 2 mM CaC12 and alkaline pH milieu between 7.4 and 8.5. The basic experiments were repeated more than five times with the same results.
Results The relative concentrations of secreted proteins in liquid cultures of Neuroapora crassa differ in c o n s t a n t darkness
105 a
C
iDa
kI)a
66
67
b
c
d
kDa
66
52 45
45
4O
36 31
29 24
36 29 24 20.1
20,1 18
14.2
17.5
14.2 Fig. 3. Secrctor~ protcna~ (100 lag) ol wdd-type and wc-2 culture
Fig. 1. SecretoryprotemsofNeuro~polacrassa (bcLmutant) analysed by SDS-PAGE. Lane a: Secretory proteins of mycelia grown at constant light. Lane b: Secretory proteins grown at constant dark. Lane c: Secretory proteins of mycelia kept in a 12:12 h light-dark regimen (last 6 h in light). All cultures were 120 h old and kept at a temperature of 25 ~ Each lane was loaded with 100 gg protein. The molecular weights of the marker proteins are given on the left side.
compared to constant light (Fig. 1). In constant darkness, relatively high concentrations of protein species with molecular masses of 72, 68, 62, 58, 39.5, 30.5 and 17.5 kDa and less prominent bands of 52.5 and 37 kDa are detected. In constant light the concentrations of some polypeptides are markedly reduced, in particular the concentration of the 17.5 kDa protein. Light induces new protein species of 67, 40, 31, 18 and 13 kDa. The secretory proteins of mycelia kept in 12:12 h light-dark regimes are similar to those detected in constant light. In a few cases a mixture of light- and
a
b
c
d
iDa
66 45
filtrates on SDS-PAGE. All mycelia were 120 h old and kept at a temperature of 25 ~ Lane a: Secretory proteins from wild-type mycelia at constant light. Lane b: Wild-type myceha at constant dark. Lane c: wc-2 mycelia at constant light. Lane d: wc-2 mycelia at constant dark. The marker proteins are shown on the left side dark-dependent proteins was observed: in the range of 72/62 kDa and 17/18 kDa. Analysis of the secretory proteins for their proteolytic activity indicates the presence of a protease of about 17.5 kDa, which shows light sensitive changes (Fig. 2): light conditions decrease the proteolytic activity. A similar activity, however less well expressed, is observed in a 22-kDa band of the gelatine gel. Other protease species of approximately 30, 53 and 68 kDa seem to have different activities depending on the age of the culture (40h versus 100h). In 100h-old cultures a 30-kDa protease is activated and a 68-kDa protease inhibited by light. In order to test whether the light effects were mediated by the blue light response system, we analysed the secretory proteins of the wc-2 mutant. In constant light w c - 2 shows the same pattern as in constant darkness, which is identical to the wild-type pattern in co.nstant dark (Fig. 3). Similar results were found in the second '"white collar" mutant wc-1 (not shown). A 40-kDa polypeptid is secreted only in those cultures which synthesize carotenoids (wild-type-LL, bd-LL). Discussion
36 29 24 20.1 14.2 Fig. 2. bccrctor} protcascb of ,\c,~o,V~ola ~/a,~a (hal-mutant)Ol] SDS-polyacrylamide gelatine gel. Extracellular proteases from cultures grown for 40 h in constant hght (lane a) and constant dark-(lane b). Secretory proteases of mycelia grown for 120 h in constant light (lane c) and under constant dark (lane d). Each lane
was loaded with 30 gg protein. The marker proteins are shown on the left side
Among the light-repressible and inducible secretory proteins is a protease of 17.5 and a protein of 18 kDa, respectively, which may be identical to secretory proteases of 18 kDa described by Lindberg et al. (1982). These two proteases (called M-l, M-2) were activated by a medium pH lower or higher than 5.5, respectively. Ruythers (1980) provided evidence that light affects the carbohydrate metabolism and since in our experiments the medium pH in constant light was slightly higher than the pH in constant darkness, the secretion of these two proteins may be mediated by pH changes within and outside the cytoplasm. The 30-kDa protease activated by
106 100 h of light m a y be identical to an alkaline protease of 30.5 k D a (Lindberg et al. 1981) or, possibly, an alkaline protease of 31 k D a ( H a n s o n and M a r z l u f 1973). O t h e r light-repressible secretory proteins can tentatively be related to k n o w n extracellular enzymes, such as a 7 0 - k D a alkaline p h o s p h a t a s e ( F u r u k a w a et al. 1987) and a 6 8 - k D a nuclease ( H a s u n u m a 1973). The same is true for light-induced proteins: the 6 6 - k D a laccase (Tamaru and I n o u e 1989) a n d a 11.5-kDa ribonuclease (Hashim o t o et al. 1971) m a y be close to the molecular welgtit of secretory proteins observed in o u r study. O n e w a y to interpret o u r findings is to assume that light directly controls the expression of genes coding for secretory proteins as s h o w n for a n u m b e r of intracellular enzymes (Richter 1984; C h a m b e r s et al. 1985; S o m m e r et al. 1989). An alternative interpretation is that light affects synthesis and release of these proteins indirectly. Indirect influences of light m a y stem from the effect of light on cellular metabolism ( G r a a f m a n s 1977; PiskorzBincycka 1978; R u y h t e r s 1980; Gressel and R a u 1983), on c o r r e s p o n d i n g changes of the intra- and extracellular p H (Lindberg et al. 1982) and on the effect of light on the differentiation of h y p h a e (Fiema 1979). Aerial h y p h a e differ in their m e t a b o l i s m f r o m their vegetative counterparts such that they utilize aerobic p a t h w a y s exclusively (Turian and Bianchi 1971). Differences in the metabolic state m a y create needs for other nutrients, which causes a r e p r o g r a m m i n g of the recruiting extracellular proteins. Direct or indirect effects of light seem to be mediated by the blue light response system. In some cases the light-inducible and repressible proteins differ only slightly in their molecular weight. This m a y be explained either by the existence of isoenzymes with slightly different molecular weight, or by assuming posttranslational m o d i fications of one enzyme. 9
I
Acknowledgement. We are grateful to V. E. A. Russo for valuable discussions and for providing mutant strains9
References Chambers JAA, Hinkelammert K, Russo VEA (1985) kightregulated protein and poly-(A+)-mRNA synthesis in Neurospora crassa. EMBO J 13:3649 3653 Cohen BL, Drucker H (1977) Regulation of exocellular protease in Neurospora crassa: induction and repression under conditions of nitrogen starvation. Arch Biochem Biophys 182:601-613 Faema J (1979) The hydroxyproline content in the mycelium of AspergiIlusglganteus rout. alba. Bull Acad Pol Sci 27 : 121 124 Furukawa K, Hasunuma K, Shinohara Y (1987) Characterization of Pcrepressible enzymes secreted in culture media by Neurospora crassa wild-type cells and null-type mutants9 J of Bacteriol 169:4790-4795 Graafmans WDJ (1977) Effect of blue light on metabolism in Penicillium isariiforme. J Gen Microbiol 101 : 157-161
Gressel J (1980) Blue light and transcription, In: Senger H (ed) The blue light syndrome. Springer, Berlin Heidelberg New York, pp 133 153 Gressel J, Rau W (1983) Photocontrol of fungal development, In: Shropshire W, Mohr H (eds) Photomorphogenesis. Encyclopedia of plant physiology 16b. Springer, Berlin Heidelberg New York, pp 603-639 Hanson MA, Marzluf GA (1973) Regulation of a sulfur-controlled protease in Neurospora crassa. J Bacteriol 116:785 789 Hashimoto J, Uchida T, E gami F (1971) Purification of ribonuclease U1 and some properties of ribonucleases U1 and NI9 J Biochem 78:903-911 Hasunuma K (1973) Repressible extracellular nucleases in Neurospora crassa. Biochim Biophys Acta 319:288-293 Hasunuma K (1983) Repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities in Neurospora crassa. J Bacteriol 156 : 291 - 300 Heussen C, Dowdle EB (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels contaimng dodecyl sulfate and copolymerized substrates. Anal Biochem 102: 196-202 Horowitz NH (1947) Methionine synthesis in Neurospora. J Biol Chem 171:255-264 Laemmli HK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature 227:680-685 Lindberg RA, Eirich LD, Price JS, Wolfinbarger L, Drucker H (1981) Alkaline protease from Neurospora crassa. J Biol Chem 256:811-814 Lindberg RA, Rhodes WG, Eirich LD, Drucker H (1982) Extracellular acid proteases from Neurospora crassa. J Bacteriol 150: 1103 1108 Lowry OH, Rosebrough NJ, Farr AL, Randall, RJ (1951) Protein measurement with the phenol reagent9J Biol Chem 139: 265-275 Metzenberg RL (1979) Implications of some genetic control mechanisms in Neurospora. Microbiol Rev 43:361-383 Nahas E, Terenzi HF, Rossi A (1982) Effect of carbon source and pH on the production and secretion of acid protease (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Neurospora crassa. J Gen Mlcrobiol 128:2017-2021 Neuhoff V, Stamm R, Eibl HJ (1985) Intensive Proteinf/irbung ohne Hintergrund mit Coomassie Blau G-250 in Polyacrylamidgelen: Theorie und Praxis9 Electrophoresis 6 : 8 7 - 9 1 and 330-334 Piskorz-Bincycka B (1978) Studies on the organic acids content of the mycelia of Penicillium isariiforme. Acta Biol Cracov: 21-22 Richter G (1984) Blue light effects on the level of translation and transcription. In: Senger H (ed) The blue hght effects in biological systems. Springer, Berlin Heidelberg New York, pp 253 262 Russo VEA (1988) Blue light induces circadian rhythms in the bd mutant of Neurospora crassa: double mutants bd, wc-1 and bd, wc-2 are blind. J Photochem Photobiol 2:59 65 Ruythers G (1980) Effects of blue light on enzymes. In: Senger H (ed) The blue light syndrome. Springer, Berlin Heidelberg New York, pp 309-318 Sommer T, Chambers JAA, Eberle J, Lauter FR, Russo VEA (1989) Fast light-regulated genes of Neurospora crassa. Nucleic Acids Res 14:5713-5723 Tamaru H, Inoue H (1989) Isolation and characterization of a laccase-derepressed mutant of Neurospora crassa. J Bacteriol 171 : 6288-6293 Turian G, Bianchi DE (,1971) Conidiation in Neurospora crassa. Arch Mikrobiol 77:262-274
