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.
Institute for Enzyme Research, University of Tokushima, Japan.
K
K
Fujiwara
K
K
Okamura-Ikeda
Y
Y
Motokawa
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