[Identification of the rat ceruloplasmin mRNA isoform putative coded of a protein localized in mitochondria]

Abstract

Alternative expression of ceruloplasmin (Cp) gene, whose product, blue multicopper ferroxidase, is a neuron survival factor, was studied in the current work. Computer analysis showed that Cp-mRNA isoform, coding for 109 kDa polypeptide, can be formed as a result of the transcription from the alternative promoter in 3'-region of intron 2 of rat Cp gene. Alternative Cp form starts with 25 amino acid residues sequence, coded with intron 2 region. It is followed by amino acid sequence of the main Cp isoform starting from Gly 113. In silico data were experimentally confirmed using RT-PCR. It was demonstrated that the predicted mRNA was generally localized in liver and brain cells of adult rats. Direct sequencing of the obtained PCR-product showed the entire coincidence of the real and predicted mRNAs. It was in vitro showed that approximately 110 kDa Cp-like protein was completed and accumulated in the absence of mitochondria. This protein is transferred into the isolated mitochondria in the reconstructed system. Transport is energy-dependent, it is not accompanied with the shortening of Cp polypeptide length and needs the presence of cytosolic factors. Probably import is determined by the inner protein mitochondria import signal with amino acid sequence KVVYREFTDSTFRE, located in 756-769 region of mature Cp. Possible role of Cp in iron metabolism in mitochondria is under discussion.