scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis

Abstract

A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counter-parts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high degree of identity to the beta-succinyl-CoA synthetase of Escherichia coli (46%), the mitochondrial beta-succinyl-CoA synthetase from pig (48%) and the hydrogenosomal beta-succinyl-CoA synthetase from Trichomonas vaginalis (49%). The G + C content of the succinyl-CoA synthetase coding sequence (43.8%) was considerably higher than that of the 5' (14.8%) and 3' (13.3%) non-translated flanking sequences, as has been observed for other genes from N. frontalis. The codon usage pattern was biased, with only 34 codons used and a strong preference for a pyrimidine (T) in the third positions of the codons. The coding sequence of the beta-succinyl-CoA synthetase cDNA was cloned in an E. coli expression vector encoding a 6(His) tag. The recombinant protein was purified by affinity binding and used to produce polyclonal antibodies. The anti-succinyl-CoA synthetase serum recognized a 45 kDa protein from a N. frontalis fraction enriched for hydrogenosomes and similar polypeptides in two related anaerobic fungi, Piromyces rhizinflata (45 kDa) and Caecomyces communis (47 kDa). Immunocytochemical experiments suggest that succinyl-CoA synthetase is located in the hydrogenosomal matrix. Staining for SCS activity in native electrophoretic gels revealed a band with an apparent molecular weight of approximately 330 kDa. The C-terminus of the succinyl-CoA synthetase sequence was devoid of the typical targeting signals identified so far in microbody proteins, indicating that N. frontalis uses a different signal for sorting SCSB into hydrogenosomes. Based on comparisons with other proteins we propose a putative N-terminal targeting signal for succinyl-CoA synthetase of N. frontalis that shows some of the features of mitochondrial targeting sequences.