SCSB, A CDNA-ENCODING THE HYDROGENOSOMAL BETA-SUBUNIT OF SUCCINYL-COASYNTHETASE FROM THE ANAEROBIC FUNGUS NEOCALLIMASTIX FRONTALIS

A clone containing a Neocallimastix frontalis cDNA assumed to encode t he beta subunit of succinyl-CoA synthetase (SCSB) was identified by se quence homology with prokaryotic and eukaryotic counterparts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequenc e showed a high degree of identity to the beta-succinyl-CoA synthetase of Escherichia coli (46%), the mitochondrial beta-succinyl-CoA synthe tase from pig (48%) and the hydrogenosomal beta-succinyl-CoA synthetas e from Trichomonas vaginalis (49%). The G + C content of the succinyl- CoA synthetase coding sequence (43.8%) was considerably higher than th at 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 usa ge pattern was biased, with only 34 codons used and a strong preferenc e for a pyrimidine (T) in the third positions of the codons. The codin g sequence of the beta-succinyl-CoA synthetase cDNA was cloned in an E . coli expression vector encoding a 6(His) tag. The recombinant protei n was purified by affinity binding and used to produce polyclonal anti bodies. The anti-succinyl-CoA synthetase serum recognized a 45 kDa pro tein from a N. frontalis fraction enriched for hydrogenosomes and simi lar polypeptides in two related anaerobic fungi, Piromyces rhizinflata (45 kDa) and Caecomyces communis (47 kDa). Immunocytochemical experim ents suggest that succinyl-CoA synthetase is located in the hydrogenos omal 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 devoi d of the typical targeting signals identified so far in microbody prot eins, 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 synth etase of N. frontalis that shows some of the features of mitochondrial targeting sequences.