The ATP-dependent Lon protease of Mus musculus is a DNA-binding protein that is functionally conserved between yeast and mammals

Abstract

The ATP-dependent Lon protease is a multi-functional enzyme that is conserved from archae to mammalian mitochondria, which not only degrades protein substrates but also binds DNA. As a starting point toward understanding Lon function in development, the mouse Lon cDNA was cloned and the encoded protein was characterized in cultured mammalian cells, in yeast and in vitro. Mouse Lon shows 87, 40 and 33% amino acid similarity with the human, yeast and bacterial homologs, respectively. Expression of a single mouse Lon transcript is detected in liver>heart>kidney>testis and is present during early embryonic development. Endogenous as well as transiently overexpressed mouse Lon co-localize with mitochondrial markers and have half-lives greater than 24 h as determined by pulse-chase studies. Enzymatically active mouse Lon that hydrolyses ATP and degrades protein and peptide substrates in an ATP-dependent manner also specifically binds to single-stranded but not to double-stranded DNA oligonucleotides. We propose that binding to TG-rich DNA sequences has been conserved between the mouse and human proteins. In addition, the evolutionary conservation of mitochondrial Lon function is demonstrated by the ability of mouse Lon to substitute for the yeast protein in vivo.

Introduction

The ATP-dependent Lon (La) protease is a highly conserved enzyme that is present in archae and eubacteria as well as in mitochondria of lower and higher eukaryotes (Gottesman et al., 1997a, Rep and Grivell, 1996, Suzuki et al., 1997, van Dyck and Langer, 1999). Interestingly, Lon is the only energy-dependent protease encoded by the minimal gene complement of Mycoplasma genitalium (580 kb) suggesting that its function is indispensable for a self-replicating organism. Mitochondrial Lon mediates the degradation of misfolded, unassembled or oxidatively damaged polypeptides (Bota and Davies, 2002, Suzuki et al., 1994, van Dijl et al., 1998, van Dyck et al., 1994, Wagner et al., 1997). Mildly oxidized, hydrophobic forms of aconitase are selectively degraded by mitochondrial Lon, while severely oxidized aconitase forms insoluble aggregates that are not degraded (Bota and Davies, 2002). One function of Lon may be to degrade damaged proteins before they aggregate and become protease-resistant. Abnormal proteins that are prone to aggregation are degraded by Lon in cooperation with the DnaK- and DnaJ-like chaperone system (Huang et al., 2001, Savel'ev et al., 1998, Sherman and Goldberg, 1992, Wagner et al., 1994). Molecular chaperones facilitate proteolysis by maintaining polypeptides in an unfolded conformation. The ATP-dependence of Lon likely serves a chaperone-like function itself, mechanistically similar to that demonstrated for other ATP-dependent proteases (Gottesman et al., 1997a, Gottesman et al., 1997b). Thus eukaryotic Lon fulfills a quality control function by selectively degrading abnormal proteins thereby preserving mitochondrial function.

In addition, mitochondrial Lon is required for the stability and expression of the mitochondrial genome. An intriguing phenotype of yeast strains lacking Lon is the presence of large deletions within mtDNA (Suzuki et al., 1994, van Dyck et al., 1994). This phenotype does not occur in the absence of the other mitochondrial ATP-dependent proteases Afg3p, Rca1p or Yme1p. The role of Lon in mtDNA maintenance and expression is not well understood. However, both recombinant human and bacterial Lon bind DNA oligonucleotides with sequence specificity in vitro (Fu and Markovitz, 1998, Fu et al., 1997). Human Lon binds to single-stranded DNA oligonucleotides corresponding to a TG-rich region that overlaps the heavy strand- and light strand- promoters of human mtDNA (Fu and Markovitz, 1998). Bacterial Lon specifically binds to the same sequences as human Lon, however, the DNA must be double-stranded. The physiological role of DNA-binding by Lon has not been determined. However, in vivo studies in bacteria and yeast suggest that Lon is involved in controlling gene expression, either by modulating the levels of factors required for transcription or by influencing the stability of mRNA transcripts (Gill et al., 1993, Schmidt et al., 1994, van Dyck et al., 1998). Interestingly, DNA binding has been shown to stimulate the ATPase activity as well as the ATP-dependent proteolysis of bacterial Lon (Charette et al., 1984, Chung and Goldberg, 1982, Gill et al., 1993, Schmidt et al., 1994, Sonezaki et al., 1995, van Dyck et al., 1998, Zehnbauer et al., 1981). Thus Lon may be uniquely poised to regulate both protein turnover and DNA function.