Molecular cloning of subunits of complex I from Neurospora crassa. Primary structure and in vitro expression of a 22-kDa polypeptide

• Published in: The Journal of biological chemistry 1990-08, Vol.265 (22), p.13060-13065
• Main Authors: Videira, A. (Universitat Muchen, Munchen, Federal Republic of Germany), Tropschug, M, Wachter, E, Schneider, H, Werner, S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies - isolation & purification; Base Sequence; Biological and medical sciences; CADENA RESPIRATORIA; Cell structures and functions; CHAINE RESPIRATOIRE; CLONACION; CLONAGE; CLONING; Cloning, Molecular - methods; DNA, Fungal - genetics; DNA, Fungal - isolation & purification; ELECTRON TRANSPORT CHAIN; EMBL/JO5559; ENZIMAS; ENZYME; ENZYME COMPLEX; ENZYMES; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; GENBANK/JO5559; Gene Expression; Gene Library; genes; Genes, Fungal; Immunoblotting; iron-sulfur protein; Macromolecular Substances; Mercaptoethanol - pharmacology; Mitochondria and cell respiration; Molecular and cellular biology; MOLECULAR SEQUENCE DATA; Molecular Weight; NAD(P)H Dehydrogenase (Quinone); NEUROSPORA; Neurospora - genetics; Neurospora crassa - enzymology; Neurospora crassa - genetics; NUCLEOTIDE; NUCLEOTIDES; NUCLEOTIDOS; PEPTIDE; PEPTIDES; PEPTIDOS; Plasmids; Quinone Reductases - genetics; RESPIRATORY CHAIN; RNA, Messenger - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)38267-5

A lambda gt11 cDNA expression library was screened with antibodies directed against individual subunits of complex I from Neurospora crassa. Clones encoding cytoplasmically synthesized polypeptides with apparent molecular masses of 22, 29, 31, and 33 kDa were isolated. Northern blot analysis revealed that the corresponding genes are transcribed into mRNA species of about 0.85, 0.95, 1.3, and 1.4 kilobases, respectively. Further characterization of clones encoding the 22-kDa subunit was performed. A cDNA insert of 755 base pairs containing the complete coding sequence was used to express the polypeptide in vitro. A precursor of the protein is synthesized on cytoplasmic ribosomes without a cleavable signal sequence. Our data indicate that after import into the organelle and before assembly into complex I, the 22-kDa polypeptide forms intramolecular disulfide bridge(s). Nucleotide sequencing revealed an open reading frame coding for a protein of 183 amino acids. A molecular mass of 20,828 daltons was calculated. The polypeptide is hydrophilic and contains no obvious membrane-spanning domains. Eight cysteine residues arranged in a regular pattern are found in the primary structure of the protein. Therefore, this subunit is a good candidate to bind at least one of the iron-sulfur centers present in complex I of the respiratory chain.