cDNA sequence, in vitro synthesis, and intramitochondrial lipoylation of H-protein of the glycine cleavage system.
H-protein is a component of the glycine cleavage system loosely associated with the mitochondrial inner membrane and has lipoic acid as a prosthetic group. cDNA clones encoding H-protein were isolated from a bovine liver cDNA library with an oligonucleotide probe from the amino acid sequence of the NH2-terminal region. DNA sequence analysis and deduced amino acid sequence showed that the cDNAs encoded an H-protein precursor of 173 amino acids including a 48-amino acid presequence. Calculated molecular mass of the precursor and mature protein without lipoic acid were 18,790 and 13,846, respectively. Northern blot analysis indicated a major mRNA component of 1.1 kilobases and a minor component of 0.6 kilobase. The result is consistent with the presence of two adenylation signals in the 3'-untranslated region of the cDNA. In vitro transcription and translation of the H-protein cDNA produced a 19-kDa protein recognized by antibody raised to chicken H-protein. Bovine liver H-protein precursor has no lipoic acid prosthetic group. When incubated with isolated bovine liver mitochondria, the precursor was imported into mitochondria, processed to its mature form with a molecular mass of 14 kDa, and lipoylated at lysine 59. These results indicate that lipoylation is not required for the import of H-protein precursor into mitochondria and H-protein is lipoylated in mitochondria which probably contain the physiologically active form of lipoic acid as well as the enzyme(s) responsible for the attachment.
Fujiwara
K
K
Institute for Enzyme Research, University of Tokushima, Japan.
Okamura-Ikeda
K
K
Motokawa
Y
Y
D000595:Amino Acid Sequence; D050960:Aminomethyltransferase; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002645:Chickens; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015723:Gene Library; D019877:Hydroxymethyl and Formyl Transferases; D008099:Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D008063:Thioctic Acid / Q000378:metabolism*; D014158:Transcription, Genetic; D014166:Transferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D015345:Oligonucleotide Probes; D008063:Thioctic Acid; D004247:DNA; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; D050960:Aminomethyltransferase