The Neurospora crassa Carotenoid Biosynthetic Gene (Albino 3 ...

THEJOURNAL of BIOLOGICAL CHEMISTRY

Vol. 266, No. 9, Issue of March 25, pp. 5854-5859, 1991 Printed in U.S.A.

(0 1991 by The American Society for Biochemistry and Molecular Biolopy, Inc

The Neurospora crassa Carotenoid Biosynthetic Gene (Albino3) Reveals Highly ConservedRegions among Prenyltransferases” (Received for publication, September 14, 1990)

Alessandra CarattoliS, Nicoletta Romano$, Paola Ballarioj, Giorgio MorelliT, and Giuseppe MacinoSII From the $Dipartimento diBiopatologia Umana, Sezione diBiologia Cellulare Policlinico Umberto 1, Uniuersita di RomaLa Sapienza 00161, YUnita’ di Nutrizione Sperimentale, Zstituto Nazionale della Nutrizione Via Ardeatinu 546001 78,and the SDipartimento di Genetica eBiologia Molecolare, Centro di Studio per gli Acidi Nucleici, Uniuersita di Roma La Sapienza, Piazzale Aldo Mora5 00185, Roma, Italy

Inthe filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light. Here we report the characterization of the albino-3 (al3) gene of N. crassa, which encodes the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthetase. This is the first geranylgeranyl-pyrophosphate synthetase gene isolated. Nucleotide sequence comparison of al-3 genomic and cDNAclones revealed that the al-3 gene is not interrupted by introns. Transcription of the al-3 gene has been examined in darkgrown and light-induced mycelia. The analysisrevealed that the al-3 gene is not expressed in the dark and that its transcription is induced by bluelight (Nelson, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G . (1989) Mol. Cell. Biol. 9, 1271-1276). The al-3 gene encodes a polypeptide of 428 amino acids. Comparison of the deduced amino acid sequence of al3 with the sequences of prenyltransferases of other species, from bacteria to humans, showed three highly conserved homologous regions. These homologous regions may beinvolved in the formation of the catalytic site of the prenyltransferases.

Carotenoids are synthetized by bacteria, plants, fungi, and algae (2). While their primary functions are in photoprotection and asaccessory pigments in photosynthesis, carotenoids also serve as precursors for vitamin A biosynthesis in animals and for abscissic acid biosynthesis in plants. In Neurospora crassa the biosynthesis of carotenoids is regulated by blue light in the mycelium but is constitutive inthe asexual spores (3-5). The photoinduction of carotenogenesis in the mycelia requires the de nouo synthesis of at least threeenzymes which have been shown to be the products of the albino (al) genes (5). Three a1 mutants have been characterized in N. crassa, each of which is defective in one step of carotenogenesis. * This work was supported by grants from the Minister0 Agricoltura e Foreste, Piano Nazionale Tecnologie Applicate alle Piante, and Instituto Pasteur-Fondazione Cenci Bolognetti. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence($ reported in thispaper has been submitted totheGenBankTM/EMBLDataBankwith accession number(s) x53979. 11 To whom correspondence should be addressed. Tel.: 6-445-2806; Fax: 6-446-2891.

Albino 3 (al-3) mutants are defective in GGPP’ synthetase (5), while albino 2 and albino 1 mutantsare defective in phytoene synthetase (6) and phytoene dehydrogenase (7), respectively. In previous work we isolated the gene encoding GGPP synthetase from N . crmsa by complementation of the al-3 mutant;expression studies showed that thetranscription of the al-3gene is controlled by light regulation (1). The pathway of carotenoid biosynthesis shares some steps with the biosynthetic pathways of other isoprenoid compounds. GGPP synthetase andseveral other enzymes of these complex pathways are members of the prenyltransferase family. Prenyltransferases catalyze the transfer of an isoprenoid diphosphate to another isoprenoid diphosphate or to a nonisoprenoid compound through a 1’-4 condensation reaction to produce various prenyl compounds that areprecursors of such diverse products as steroids, carotenoids, chlorophylls, heme a, prenylated proteinsand tRNAs, glycosyl carrier lipids, plant hormones, and the side chains of quinones (8-10). Prenyltransferases produce a wide range of products, from the simple dimer geranyl pyrophosphate to thecomplex structure of rubber which is thousands of monomers long. GGPP synthetase catalyzes the trans addition of three molecules of IPP onto DMAPP to form geranylgeranyl pyrophosphate. Here we present the sequence of the al-3 gene andits deduced amino acid sequence. The al-3 protein sequence is compared with those of other known prenyltransferases. MATERIALS AND METHODS AND RESULTS* DISCUSSION

We have determined the nucleotide sequence of the al-3 gene, which encodes the carotenoid biosynthetic enzyme GGPP synthetase. It is known that blue light induces the biosynthesis of carotenoids in N. crmsa mycelia and that the activity of GGPP synthetase increases after light treatment (8). The al-3 gene encodes an mRNA of 1683 nucleotides, which is colinear with the al-3 gene, as revealed by cDNA sequence and S1 nuclease mapping analysis. We analyzed the expression of the al-3 gene and found that its mRNA is not present in dark-grown mycelia but is induced by blue light after a shortpulse of illumination. The abbreviations used are: GGPP, geranylgeranyl pyrophosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; FGSC, Fungal Genetic Stock Center; PIPES, 1,4 Piperazinediethanesulfonic acid; FPP, farnesyl pyrophosphate. Portions of this paper (including “Materials and Methods,” “Results,” Table 1, and Figs. 1-4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.

5854

Neurospora crassa Geranylgeranyl Pyrophosphate Synthetase Gene Dornaln I

mod 5

Domeln II

al-3

crtE FPPS

mod5

u#

I [ F O I ~ NL

-LGE

F Q l ODDVL

C

PEP FQRLDDRVDD

Domln I

Domaln II

Dornaln 111

al-3

crlE

,*1,,111,,,11,

FPPS

,,,,,,,,,,,,,,

mod5

,,,,,,,,,,,,,,

:.-:;.*. :.*:.-.*.

4 ...\..

..... ..\... \

FIG. 5. Alignment of the deduced al-3 protein sequence with the S.cerevisiae FPPS and Rhodobacter capsulatus crtE proteins. Domains 1, 11, and 111show amino acid alignment of the al-3, FPPS, and crtEproteins. Conserved amino acid residues present in a t least two of the three sequences are boxed. The distance between domains is indicated. Domains I and I11 of the 5’. cereuisiae mod5 protein and domain I1 of avian liver FPPS are also shown. The asterisk indicates the avian FPPS arginine residue discussed under “Results.” The three domains of the al-3, FPPS, crtE, and mod5 proteins and their relative positions within the genes are shown diagrammatically at thebottom (not drawn to scale).

The polypeptide encoded by the al-3 gene has a molecular mass of 47,876 daltons, is weakly basic and hydrophilic, and does not possess any hydrophobic membrane-spanning regions. This is in agreement with the finding that the GGPPS activity, isolated from various sources, is present inthe soluble fraction of cellular extracts (8, 36, 37). GGPP synthetase is a prenyltransferase that catalyzes the 1’-4 condensation of dimethylallyl pyrophosphate with three isopentenyl pyrophosphates. We therefore compared the al-3 polypeptide with other known prenyltransferases. Comparison with the FPP synthetase from human (29), rat (30), and Saccharomyces cereuisiae (28) showed significant homologies in three different regions of these proteins. The relative positions of the homologous regions were the same in all the proteins analyzed. Furthermore, the three domains were localized in the proteins at very similar distances. These facts suggest that the homologous regions may be involved in the formation of the catalytic site of the prenyltransferases. Comparison of domains I andI11 showed the presence of the motif DDXXD. These aspartateresidues could be responsible for the binding of the cations Mg2+ or Mn2+,shown to be important for the catalytic activities of the prenyltransferases (38, 39). The analysis of the conserved amino acids found in the three domains suggested that in all three domains not only the aspartate residues but also the positively charged amino acids may be important for enzyme activity. One of the major biological functions of arginine residues is to interact with phosphorylated metabolites. Lysine residues may also serve this function and indeed are known to be important in a

5855

number of enzymes acting on phosphorylated substrates (40, 41). Furthermore, it has been demonstrated that theargininespecific reagent hydroxyphenylglyoxal is a powerful inhibitor of prenyltransferases (38, 42). Domain 11 is homologous to the region proposed to be the active site of avian FPP synthetase. Brems et al. (32) identified this region by a site-directed photoaffinity label on the purified enzyme and proposed thatthe arginine residue (whose position is indicated in Fig. 5 with an asterisk), conserved also in human and rat FPP synthetase (not shown) andsubstituted by lysine in all the other known prenyl transferases, could be responsible for the binding of the pyrophosphate group. These authors also showed evidence of the involvement of 2 arginine residues in the function of prenyltransferases. In view of the model that we propose for the function of these conserved domains, it is interesting that the yeast prenyltransferase (involved in tRNA modification) has some homology, involving arginyl residues, with the first domain and a stronger homology with the third domain, in which all 3 aspartate residues are conserved. We have found that the crtEgene product of Rhodobacter capsulatm also has an impressive homology with all three conserved domains. All the prenyltransferases considered here perform a 1 ’ 4 condensation reaction between an allylic and an homoallylic substrate. CrtE has been indicated to be a phytoene synthetase (31) and therefore it does not belong to the prenyltransferase family. Due to these homologies, we propose that the crtEenzyme may have a 1‘-4condensation activity. These similarities and homologies among prenyltransferases suggest that thegenes may have a phylogenetic relation. It is conceivable that other prenyltransferasescould share the same regions of homology with those considered here. The prediction that the conserved amino acids, in the three homologous regions, play an essential role in the enzyme functions, will be tested by site-directed mutagenesis experiments on the al-3 gene, making use of the albine 3 mutant as the recipient strain for the transformation of mutated sequences. Acknowledgments-We thank EnzaIlardi for providing us with the N. crassa cDNA clone bank. We are grateful to Uwe Bertsch and Eugenio Pontieri for assistance in the screening of the cDNA bank. We thank Tullio Aversa for technical support. We thank TomSchmidhauser, Mary Anne Nelson, and Gloria Coruzzi for invaluable advice and suggestions. Addendum-During the preparation of the manuscript, we learned from Dr. P. Edwards3 that S. cereuisiae hexaprenyl pyrophosphate synthetase has amino acid sequence homology with domains I and 111. REFERENCES 1. Nelson, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G. (1989) Mol. Cell. Biol. 9, 1271-1276 2. Goodwin, T. W. (1980) in The Biochemistry of Carotenoids (Chapman, and Hall, eds) Vol 1, Plants, New York 3. De Fabo, E. C., Harding, R. W., and Shropshire, W. (1976) Plant Physiol. 57,440-445 4. Hardine, R. W., and Shroushire,. W.,. Jr. (1980)Annu. Reu. Plant Physyol. 31, 217-238 5. Harding, R. W., and Turner,R. V. (1981) Plant Physiol. 68,745~

149 .

”

6. Kushwaha, S. C., Kates, M., Renaud, R. L., and Subden, R. E. (1978) Lipids 13,352-355 7. Goldie, A. H., and Subden, R. E. (1973) Biochem. Genet. 10,275284

P. Edwards, Division of Cardiology, University of California at Los Angeles, personal communication.

5856

Neurospora crassa Geranylgeranyl Pyrophosphate

8. Brown, M. S., and Golstein, J. L. (1980) J. Lipid Res. 21, 505517 9. Anderegg, R. J., Betz, R., Carr, S. A,, Crabb, J. W., and Duntze, W. (1988) J. Biol. Chem. 2 6 3 , 18236-18240 10. Glomset, J. A., Gelb, M. H., and Farnsworth, C. C. (1990) Trends. Biochem. Sci. 15, 139-142 11. Davis, R. H., and De Serres, F. J. (1970) Biochemistry 1 8 , 52945299 12. Schweizer, M., Case, M. E., Dykstra, C.C., Giles, N. H., and Kushner, S. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,50865090 13. Kuiper, M. T. R., Akins, R. A., Holtrop, M., de Vries, H., and Lambowitz, A. M. (1988) J. Biol. Chem. 2 6 3 , 2840-2847 14. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular C1oning:A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 15. Vogel, H.J. (1964) Am. Nat. 98,435-446 16. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 1 8 , 5294-5299 17. Gliiin, V., Crkvenjakov, R., and Byus, C. (1974) Biochemistry 13,2633-2637 18. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A . 69, 1408-1412 19. Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977) Biochemistry 16,4743-4751 20. Cobianchi, F., and Wilson, S. H. (1987) in Methods Enzymol. 152,94-97 21. Sanger, F., Coulson, A.R., Barrel, B. G., Smith, A. J. H., and Roe, B. A. (1980) J. Mol. Biol. 143, 161-178 22. Devereux, J., Haeberli, P., and Smithies, 0. (1984) Nucleic Acids Res. 1 2 , 387-395 23. Ballance, D. J. (1986) Yeast 2 , 229-236 24. Orbach, M. J., Porro, E. B., and Yanofsky, C. (1986) Mol. Cell. Biol. 6 , 2452-2461 ~~

Synthetase Gene

25. Legerton, T. L., Yanofsky, C. (1985) Gene (Amst.) 39,129-140 26. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 27. Roberts, A. N., and Yanofsky, C. (1989) Nucleic Acids Res. 17, 197-214 28. Anderson, M. S., Yarger, J. G., Burck, C. L., and Poulter, C. D. (1989) J. Biol. Chem. 2 6 4 , 19176-19184 29. Sheares, B. T., White, S. S., Molowa, D. T., Chan, K., Ding, V. D.-H., Kroon, P. A,, Bostedor, R. G., and Karkas, J. D. (1989) Biochemistry 28,8129-8135 30. Clarke, C. F., Tanaka, R. D., Svenson, K., Wamsley, M., Fogelman, A. M., and Edwards, P. A. (1987) Mol. Cell. Biol. 7,31383146 31. Armstrong, G. A., Alberti, M., Leach, F., and Hearst, J. E. (1989) Mol. Gen. Genet. 216,254-268 32. Brems, D. N., Bruenger, E., and Rilling, H. C. (1981)Biochemistry 20,3711-3718 33. Najarian, D., Dihanich, M. E., Martin, N. C., and Hopper, A. K. (1987) Mol. Cell. Biol. 7, 185-191 34. Anderson, M. S., Muehlbacher, M., Street, I. P., Proffitt, J., and Poulter, C. D. (1989) J. Biol. Chem. 264,19169-19175 35. Powell. G.K.. and Morris. R. 0.(1986) Nucleic Acids Res. 14, 255512565 36. Brinkhaus. F. L.. and Rilline. H. C. (1988) . , Arch. Biochem. Biophys. 266, 607-612 37. Dogbo, O., and Camara, B. (1987) Biochim. Biophys. Acta 920, 140-148 38. Dogbo, O., Laferriere, A., d'Harlingue, A., and Camara, B. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7054-7058 39. King, H. L., Jr., and Rilling, H. C. (1977) Biochemistry 16,38153819 40. Riordan, J. F., McElvany, K. D., and Borders, C. L., Jr. (1977) Science 195,884-886 41. Xia, Z., and Storm, D. R. (1990) J. Biol. Chem. 266,6517-6520 42. Barnard, G.F., and Popjak, G. (1980) Biochim.Biophys.Acta 617, 169-182 '

I

I

Neurospora crassa Geranylgeranyl Pyrophosphate Synthetase Gene

5857

Neurospora crassa GeranylgeranylPyrophosphate Synthetase Gene

AC.5

01A T U GX GN3 GX 01A T K 2066

-

Fiq.2: DNA sepuence of the al-3 gene and its predicted qene product. Nucleotide sequence of the 2.0 kbp HindIII-AccI frsment of Fig. 2. Containing the codinq region Of Che al-3 gene. The putative TATA Sequence and polyadenylation recopnition signal are underlined. The arrows indicate the 5 ' and 3 . ends of the W A a8 determined by SI. nuclease mapping. The Open arrow shows the m N A 3' end determined by =DNA sequence. The sequence of the oligonucleotide used for labeling the probe for 5' end studies 1s also shorn.

I" a previous mper (1) we demnstrated that the al-3 m A level increases about 15 fold in mycelia, after a short period o f illumination. compared to it6 low level in the dark. Here we present results chat confirm the photoinduction of the al-3 m N A , brt on the Contrary its level in the dark is undetectable by Northern blotting analysis. This differenceis due LO the fact chat the previous Northern blots were probad with a DNA fragment chat contained the al-3 geneplus another gene. This gene is clo801y linked t o al-3 and encodes a messenqer RNA of almost identical sire Iabouc 1700 nucleotides); the linked gene is transcribed in the opposite direction and expressed in both light and dark regimes 4 . Fig. 4A shows chat the longer probe a [Pig. 11 that had been used in previous work (1) detected b t h mRNAs. chat of a l - 3 plus the mRNA of che closely-linked but unrelated gene. Fig. 3 demnacracad that when a probe was used which contains only the al-3 gene [probe b). che mNA for 01-3 was detected only in light-gram mycelia and was undetectable in dark-grown mycelia. Probe E detected Only the W A of the linked gens; this m A comigrated with the al-3 mRNA. brc was not phhotoregulated. These results have been confirmed with more sensitive 51 nuclease experiments (Pig. 4 ) . Probes 1 and 2 were totally degraded when Local RNA from dark-qrown mycelia was used. while they were Cmpletely protected using total 01: poly(A)* R N A from light-induced mycelia (see below).

4- A. Carattoli unpublished results

2057

A

L D

R C L D L D

Fiq.3: Northern hybridizations. Total mNA was prepared from light-induced ILI and from dark-grom ID) mycelia. samples ( 4 pgl of each RNA were sewrated On a 1.28 agarose-€.\ f o m l d e y d e gel. transferred LO w b n d - N membrane. and hybridized with probes a, b and c (Fig. 2). Results are sham. respectively. in panels A. B and c

Neurospora crassa Geranylgeranyl Pyrophosphate Synthetase Gene

D

5859