Effect of N-terminal α-helix formation on the dimerization and intracellular targeting of alanine:glyoxylate aminotransferase

Abstract

The unparalleled peroxisome-to-mitochondrion mistargeting of alanine:glyoxylate aminotransferase (AGT) in the hereditary disease primary hyperoxaluria type 1 is caused by the combined presence of a common Pro11 → Leu polymorphism and a disease-specific Gly170 → Arg mutation. The Pro11 → Leu replacement generates a functionally weak N-terminal mitochondrial targeting sequence (MTS), the efficiency of which is increased by the additional presence of the Gly170 → Arg replacement. AGT dimerization is inhibited in the combined presence of both replacements but not when each is present separately. In this paper we have attempted to identify the structural determinants of AGT dimerization and mitochondrial mistargeting. Unlike most MTSs, the polymorphic MTS of AGT has little tendency to adopt an α-helical conformation in vitro. Nevertheless, it is able to target efficiently a monomeric green fluorescent (GFP) fusion protein, but not dimeric AGT, to mitochondria in transfected COS-1 cells. Increasing the propensity of this MTS to fold into an α-helix, by making a double Pro11 → Leu + Pro10 → Leu replacement, enabled it to target both GFP and AGT efficiently to mitochondria. The double Pro11 → Leu + Pro10 → Leu replacement retarded AGT dimerization in vitro as did the disease- causing double Pro11 → Leu + Gly170 → Arg replacement. These data suggest that N-terminal α-helix formation is more important for maintaining AGT in a conformation (i.e. monometic) compatible with mitochondrial import than it is for the provision of mitochondrial targeting information. The parallel effects of the Pro10 → Leu and Gly170 → Arg replacements on the dimerization and intracellular targeting of polymorphic AGT (containing the Pro11 → Leu replacement) raise the possibility that they might achieve their effects by the same mechanism.