Dual localization of human DNA topoisomerase IIIα to mitochondria and nucleus

Abstract

The human TOP3␣ gene encoding DNA topoisomerase III␣ (hTop3␣) has two potential start codons for the synthesis of proteins 1,001 and 976 aa residues in length. The sequence of the N-terminal region of the 1,001-residue form resembles signal peptide sequences for mitochondrial import, and fluorescence microscopy shows that the addition of as few as the first 34 aa of the 1,001-residue form of hTop3␣ to a green fluorescent protein can direct the chimeric protein to mitochondria. Biochemical analyses of subcellular fractions of HeLa cells further demonstrate that a distinctive fraction of hTop3␣ is present inside mitochondria, as evidenced by its resistance to proteinase K. This fraction constitutes several percent of the enzyme in the nuclear fraction, suggesting that the distribution of the mitochondrial and nuclear forms of hTop3␣ is roughly in proportion to the DNA contents of these cellular compartments. The presence of a type IA DNA topoisomerase in the mitochondria of other eukaryotes is supported by an examination of the amino acid sequences of mouse and Drosophila DNA topoisomerase III␣ and Schizosaccharomyces pombe DNA topoisomerase III. Given the presence of at least one type IA DNA topoisomerase in all forms of life examined to date, the finding of a type IA enzyme in mitochondria further supports the notion of a key role of such enzymes in DNA transactions.

An Upstream Open Reading Frame and the Context of the Two AUG Codons Affect the Abundance of Mitochondrial and Nuclear RNase H1

“…The Top3␣ and Trnt1 genes have conserved uORFs and two in-frame AUGs in at least 11 mammalian genomes (see Tables S2 and S3 in the supplemental material). Top3␣ encodes DNA topoisomerase III␣, which has been shown to be present in mitochondria and nuclei (34). Trnt1 encodes tRNA-nucleotidyltransferase 1, which is an enzyme that adds the CCA triplet to the 3Ј end of tRNAs.…”

Drosophila topo IIIα is required for the maintenance of mitochondrial genome and male germ-line stem cells

“…Drosophila, human, and mouse topo IIIα all have a putative, unconserved mitochondrial import sequence at the N terminus of the protein, sandwiched between two tandem initiating methionines (4,19). topo IIIα of metazoans is thus characterized with the presence of dual subcellular localization sequences: a mitochondrial import sequence at the N-terminus, and a nuclear localization signal in the C-terminal domain following the catalytic core domain.…”

“…Biochemical analysis using subcellular fractionation provides evidence for the presence of a truncated form of topo IIβ in bovine heart (18). There is also clear evidence to demonstrate the presence of a mitochondia-targeting form of a type IA enzyme using an alternate translational initiation of top3α gene in human cells (19). However, whether these two enzymes are essential for mitochondrial DNA maintenance remains to be determined.…”

“…The aminoterminal sequence of eukaryotic topo IIIα contains a mitochondial targeting signal, and human topo IIIα contains this sequence can localize to the mitochondria in the cultured cells (19). The N-terminal sequence of metazoan topo IIIα, including that of Drosophila, also contains such a mitochondrial import sequence (4,19). Drosophila oogenesis and spermatogenesis are favorable systems to examine the mitochondrial subcellular localization and its functional importance.…”

Dual Sites of Protein Initiation Control the Localization and Myristoylation of Methionine Sulfoxide Reductase A

“…Mammalian MsrA joins the list of dually localized proteins that are differentially targeted by the presence of two functional sites of protein initiation. These include proteins present in both the nucleus and mitochondria (35)(36)(37), in the cytosol and mitochondria (38 -43), and in other pairs of compartments (44 -46). The relative distribution of many of these proteins is regulated by the comparative efficiency of initiation of the two sites, which was also the case for MsrA.…”