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Primary structure of the nuclear-encoded 10.5 kDa subunit of complex I from Neurospora crassa
327
Biochimica et Biophysica Acta, 1172 (1993) 327-328 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00
Short Sequence-Paper
BBAEXP 90484
Primary structure of the nuclear-encoded 10.5 kDa subunit of complex I from Neurospora crassa Margarida Duarte, Jos6 A. Belo and Arnaldo Videira Instituto de Ci~ncias Biom~dicas de Abel Salazar, Universidade do Porto, Porto (Portugal) (Received 8 January 1993)
Key words: Mitochondrion; NADH : ubiquinone oxidoreductase; Complex I; cDNA; (N. crassa)
We have isolated a cDNA clone for the nuclear encoded 10.5 kDa subunit of complex I from N. crassa. DNA sequencing revealed an open reading frame corresponding to a polypeptide with 94 amino acids and a calculated molecular mass of 10531 Da. The protein is synthesized without a cleavable mitochondrial targeting sequence. The N. crassa polypeptide is the fungal equivalent of subunit B8 of bovine complex I. -120 CAAAGGTGCCCCTGCCCGTAGGACAACGACGACCACTCAAT -60 C G A T A C C C C C A T C T G C C A A T T C T T G C G C C T C T G T A A T T C G A G A T T G C C T T C A T C G C A A G C 1 ATGTCAGGCAGATTTGCTTTCAACAAGGGCTTGAAGGAGGTCCGCTTCCTGTTCTGCCAG 1 M S G R F A F N K G L K E V R F L F C Q 61 21
ACGGGCGAGCACAGCGCTGCCACAAGATCATTCCTCCTTCGAAACTACCCTGCCATGAAG T G E H S A A T R S F L L R N Y P A M
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121 41
AAGGATAACCCGGCGACACCGATTTTGATTCGAGATGCGTCCGGCACGCTACCCAAGGTC K D N P A T P I L I R D A S G T L P K
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CAAATCGAGGAGACAGTATCATCCCTCGTCAAGAACAATTCTTGAGCTGGGAAGTGAGAG Q I E E T V S S L V K N N S
301 361
GAGCCGTGAAGCATGTCTATAACACCAACATCCTTGACGCTACATACAACCCGCTGTGGT GGCATAATGAAACCCGCAACCCCGTCGACGCAACCGATGGATGGGTACGATTGTT
Fig. l. Nucleotidesequenceof~ll-lengthcDNA a n d d e d u c e d p r i m a r y s t r u c t u r e o f t h e l 0 . 5 k D a p r o t e i n .
Respiratory chain N A D H dehydrogenase or complex I (EC 1.6.99.3) is one of the components involved in energy transduction in the inner mitochondrial membrane. It is assembled from probably more than 30 polypeptides, seven of which are encoded and synthesized in mitochondria. In N e u r o s p o r a , complex I can be split into extrinsic and intrinsic membrane domains (the peripheral and hydrophobic arms, respectively), which seem also to be assembled independently. Several human diseases have been associated with defects in complex I, raising considerable interest in the enzyme (for a recent review see Ref. 1). Only a few
Correspondence to: A. Videira, Instituto de Ci~ncias Biom6dicas de Abel Salazar, Largo Prof. Abel Salazar 2, 4000 Porto, Portugal. The sequence data reported in this paper have been submitted to the EMBL Data Library under the accession number X69929.
polypeptides have been shown to participate directly in electron transfer, leaving the possibility that complex I may perform other biological functions. Indeed, recent experiments showed that complex I contains an awlcarrier protein and may be involved in lipid biosynthesis [2]. We are cloning subunits of the enzyme to understand in more detail their structure and function and as tools to investigate the biogenesis of complex I. In this report we present the primary structure of the 10.5 kDa subunit of complex I from N . c r a s s a (NUO10.5), the equivalent of subunit B8 of complex I from beef heart [3]. The methods for immunoaffinity purification of IgG from rabbit antisera, screening a N . c r a s s a cDNA expression library in phage Agtll, subcloning and sequencing of cDNA inserts have been detailed before [4]. An antiserum against NUO-10.5 has been previously obtained and characterized [5].
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Fig. 2. Alignment of the amino acid sequences of the 10.5 kDa protein from N. crassa and protein B8 from Bos taurus. Identical residues and conservative substitutions are indicated by vertical lines and asterisks, respectively. Several positive phages were detected by immunoscreening of the N . c r a s s a c D N A library. Six phages were isolated and the encoded recombinant proteins were used to affinity purify IgG from the antiserum against NUO-10.5. The antibodies obtained with 4 of them were able to recognize NUO-10.5 in immunoblots of complex I subunits (data not shown), indicating that they encode the relevant protein. One of the obtained c D N A inserts was then used to rescreen the library by hybridization. The c D N A insert of one of the isolated phage was subcloned in a plasmid vector and both DNA strands were sequenced. The obtained nucleotide sequence (516 bp) is depicted in Fig. 1. It contains 101 bp and 133 bp of 5' and 3' untranslated regions, respectively. The 282 bp open reading frame found predicts a protein of 94 amino acids with a molecular mass of 10531 Da. Although the N-terminal region resembles a mitochondrial targeting sequence [6], the protein is not synthesized as an extended precursor form [5]. The polypeptide is rather hydrophilic and does not contain potential membrane spanning domains (data not shown). A comparison with published sequences of subunits of complex I from beef heart [3], revealed that the 10.5 kDa fungal polypeptide is homologous with subunit B8 of the bovine enzyme (see Fig. 2). The two proteins display 38% overall identity (53% similarity). The B8 protein belongs to an extrinsic membrane fragment of bovine complex I, fragment Ih [7]. We could detect immunologically NUO-10.5 in a preparation of the so-called small isoform of N . c r a s s a complex I [1] (Videira, A. and Werner, S., unpublished data). Based on these facts and the hydropathy profile of NUO-10.5, we tend to speculate that it belongs to the peripheral
arm of complex I, but this assumption needs to be further clarified. For example, a recently cloned hydrophilic subunit of the N . c r a s s a complex I belongs to the hydrophobic arm of the enzyme [8]. The finding of more related subunits in complex I from N e u r o s p o r a and mammals strenghtens the idea that the enzymes are similar and that the fungus may be used as a general research model. No other proteins in release 23 of the Swiss-Prot Databank were found similar to NUO-10.5. Thus, the role of this polypeptide within complex I remains unclear. M.D. would like to thank Junta Nacional de Investiga~fio Cientifica e Tecnol6gica (JNICT) from Portugal, for financial support. We are grateful to Mrs. Natfilia Mota for excellent technical assistance. This research was supported by J N I C T (project SAU 48/90).
References
1 Weiss, H., Friedrich, T., Hofhaus, G. and Preis, D. (1991) Eur. J. Biochem. 197, 563-576. 2 Sackmann, U., Zensen, R., Rohlen, D. Jahnke, U. and Weiss, H. (1991) Eur. J. Biochem. 200, 463-469. 3 Walker, J.E., Arizmendi, J.M., Dupuis, A., Fearnley, I.M., Finel, M., Medd, S.M., Pilkington, S.J., Runswick, M.J. and Skehel, J.M. (1992) J. Mol. Biol. 226, 1051-1072. 4 Videira, A., Tropschug, M., Wachter, E., Schneider, H. and Werner, S. (1990) J. Biol. Chem. 265, 13060-13065. 5 Videira, A. and Werner, S. (1989) Eur. J. Biochem. 181,493-502. 6 Hartl, F.-U., Pfanner, N., Nicholson, D.W. and Neupert, W. (1989) Biochim. Biophys. Acta 988, 1-45. 7 Arizmendi, J.M., Skehel, J.M., Runswick, M.J., Fearnley, I.M. and Walker, J.E. (1992) FEBS Lett. 313, 80-84. 8 Videira, A., Azevedo, J.E., Werner, S. and Cabral, P. (1992) Biochem. J., in press.
Biochimica et Biophysica Acta, 1172 (1993) 327-328 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00
Short Sequence-Paper
BBAEXP 90484
Primary structure of the nuclear-encoded 10.5 kDa subunit of complex I from Neurospora crassa Margarida Duarte, Jos6 A. Belo and Arnaldo Videira Instituto de Ci~ncias Biom~dicas de Abel Salazar, Universidade do Porto, Porto (Portugal) (Received 8 January 1993)
Key words: Mitochondrion; NADH : ubiquinone oxidoreductase; Complex I; cDNA; (N. crassa)
We have isolated a cDNA clone for the nuclear encoded 10.5 kDa subunit of complex I from N. crassa. DNA sequencing revealed an open reading frame corresponding to a polypeptide with 94 amino acids and a calculated molecular mass of 10531 Da. The protein is synthesized without a cleavable mitochondrial targeting sequence. The N. crassa polypeptide is the fungal equivalent of subunit B8 of bovine complex I. -120 CAAAGGTGCCCCTGCCCGTAGGACAACGACGACCACTCAAT -60 C G A T A C C C C C A T C T G C C A A T T C T T G C G C C T C T G T A A T T C G A G A T T G C C T T C A T C G C A A G C 1 ATGTCAGGCAGATTTGCTTTCAACAAGGGCTTGAAGGAGGTCCGCTTCCTGTTCTGCCAG 1 M S G R F A F N K G L K E V R F L F C Q 61 21
ACGGGCGAGCACAGCGCTGCCACAAGATCATTCCTCCTTCGAAACTACCCTGCCATGAAG T G E H S A A T R S F L L R N Y P A M
K
121 41
AAGGATAACCCGGCGACACCGATTTTGATTCGAGATGCGTCCGGCACGCTACCCAAGGTC K D N P A T P I L I R D A S G T L P K
V
181 61
TATGCGAGATTCGAGTTCGGTAAGGAGAAGAGTCAGTCGTTGGAAGGTCTGTCCGACCAG Y A R F E F G K E K S Q S L E G L S D
Q
241 81
CAAATCGAGGAGACAGTATCATCCCTCGTCAAGAACAATTCTTGAGCTGGGAAGTGAGAG Q I E E T V S S L V K N N S
301 361
GAGCCGTGAAGCATGTCTATAACACCAACATCCTTGACGCTACATACAACCCGCTGTGGT GGCATAATGAAACCCGCAACCCCGTCGACGCAACCGATGGATGGGTACGATTGTT
Fig. l. Nucleotidesequenceof~ll-lengthcDNA a n d d e d u c e d p r i m a r y s t r u c t u r e o f t h e l 0 . 5 k D a p r o t e i n .
Respiratory chain N A D H dehydrogenase or complex I (EC 1.6.99.3) is one of the components involved in energy transduction in the inner mitochondrial membrane. It is assembled from probably more than 30 polypeptides, seven of which are encoded and synthesized in mitochondria. In N e u r o s p o r a , complex I can be split into extrinsic and intrinsic membrane domains (the peripheral and hydrophobic arms, respectively), which seem also to be assembled independently. Several human diseases have been associated with defects in complex I, raising considerable interest in the enzyme (for a recent review see Ref. 1). Only a few
Correspondence to: A. Videira, Instituto de Ci~ncias Biom6dicas de Abel Salazar, Largo Prof. Abel Salazar 2, 4000 Porto, Portugal. The sequence data reported in this paper have been submitted to the EMBL Data Library under the accession number X69929.
polypeptides have been shown to participate directly in electron transfer, leaving the possibility that complex I may perform other biological functions. Indeed, recent experiments showed that complex I contains an awlcarrier protein and may be involved in lipid biosynthesis [2]. We are cloning subunits of the enzyme to understand in more detail their structure and function and as tools to investigate the biogenesis of complex I. In this report we present the primary structure of the 10.5 kDa subunit of complex I from N . c r a s s a (NUO10.5), the equivalent of subunit B8 of complex I from beef heart [3]. The methods for immunoaffinity purification of IgG from rabbit antisera, screening a N . c r a s s a cDNA expression library in phage Agtll, subcloning and sequencing of cDNA inserts have been detailed before [4]. An antiserum against NUO-10.5 has been previously obtained and characterized [5].
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Fig. 2. Alignment of the amino acid sequences of the 10.5 kDa protein from N. crassa and protein B8 from Bos taurus. Identical residues and conservative substitutions are indicated by vertical lines and asterisks, respectively. Several positive phages were detected by immunoscreening of the N . c r a s s a c D N A library. Six phages were isolated and the encoded recombinant proteins were used to affinity purify IgG from the antiserum against NUO-10.5. The antibodies obtained with 4 of them were able to recognize NUO-10.5 in immunoblots of complex I subunits (data not shown), indicating that they encode the relevant protein. One of the obtained c D N A inserts was then used to rescreen the library by hybridization. The c D N A insert of one of the isolated phage was subcloned in a plasmid vector and both DNA strands were sequenced. The obtained nucleotide sequence (516 bp) is depicted in Fig. 1. It contains 101 bp and 133 bp of 5' and 3' untranslated regions, respectively. The 282 bp open reading frame found predicts a protein of 94 amino acids with a molecular mass of 10531 Da. Although the N-terminal region resembles a mitochondrial targeting sequence [6], the protein is not synthesized as an extended precursor form [5]. The polypeptide is rather hydrophilic and does not contain potential membrane spanning domains (data not shown). A comparison with published sequences of subunits of complex I from beef heart [3], revealed that the 10.5 kDa fungal polypeptide is homologous with subunit B8 of the bovine enzyme (see Fig. 2). The two proteins display 38% overall identity (53% similarity). The B8 protein belongs to an extrinsic membrane fragment of bovine complex I, fragment Ih [7]. We could detect immunologically NUO-10.5 in a preparation of the so-called small isoform of N . c r a s s a complex I [1] (Videira, A. and Werner, S., unpublished data). Based on these facts and the hydropathy profile of NUO-10.5, we tend to speculate that it belongs to the peripheral
arm of complex I, but this assumption needs to be further clarified. For example, a recently cloned hydrophilic subunit of the N . c r a s s a complex I belongs to the hydrophobic arm of the enzyme [8]. The finding of more related subunits in complex I from N e u r o s p o r a and mammals strenghtens the idea that the enzymes are similar and that the fungus may be used as a general research model. No other proteins in release 23 of the Swiss-Prot Databank were found similar to NUO-10.5. Thus, the role of this polypeptide within complex I remains unclear. M.D. would like to thank Junta Nacional de Investiga~fio Cientifica e Tecnol6gica (JNICT) from Portugal, for financial support. We are grateful to Mrs. Natfilia Mota for excellent technical assistance. This research was supported by J N I C T (project SAU 48/90).
References
1 Weiss, H., Friedrich, T., Hofhaus, G. and Preis, D. (1991) Eur. J. Biochem. 197, 563-576. 2 Sackmann, U., Zensen, R., Rohlen, D. Jahnke, U. and Weiss, H. (1991) Eur. J. Biochem. 200, 463-469. 3 Walker, J.E., Arizmendi, J.M., Dupuis, A., Fearnley, I.M., Finel, M., Medd, S.M., Pilkington, S.J., Runswick, M.J. and Skehel, J.M. (1992) J. Mol. Biol. 226, 1051-1072. 4 Videira, A., Tropschug, M., Wachter, E., Schneider, H. and Werner, S. (1990) J. Biol. Chem. 265, 13060-13065. 5 Videira, A. and Werner, S. (1989) Eur. J. Biochem. 181,493-502. 6 Hartl, F.-U., Pfanner, N., Nicholson, D.W. and Neupert, W. (1989) Biochim. Biophys. Acta 988, 1-45. 7 Arizmendi, J.M., Skehel, J.M., Runswick, M.J., Fearnley, I.M. and Walker, J.E. (1992) FEBS Lett. 313, 80-84. 8 Videira, A., Azevedo, J.E., Werner, S. and Cabral, P. (1992) Biochem. J., in press.