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

• Type: Article in International Scientific Journal
• Date: 1990

Authors

videira, a (Author) ICBAS; tropschug, m (Author) Other; wachter, e (Author) Other; schneider, h (Author) Other; werner, s (Author) Other

Journal

Title: Journal of Biological Chemistry

Vol. 265

Pages: 13060-13065

ISSN: 0021-9258

Publisher: Elsevier

Indexing

ISI Web of Knowledge

Scopus - 23 Citations

Other information

Authenticus ID: P-008-NKY

Abstract (EN): A ¿ 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 6