Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting.

Mirco Dindo,Angel L Pey,Peter J O’Toole,Elisa Oppici,Elena Butturini,Silvia Grottelli,Giulia Ambrosini,Claudio Costantini,Joanne L Marrison,Ian E G Morrison,Barbara Cellini

doi:10.3390/jpm11040273

Abstract

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5′-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.

Highlights

Introduction distributed under the terms andAbout one third of all known proteins display an oligomeric structure, and most of them are homodimeric enzymes [1]
Several factors other than the intrinsic physico-chemical properties of the polypeptide chain influence the energy landscape inside a cell, including the crowded environment, the vectorial synthesis, and the presence of molecular chaperones, and the possibility to be imported in a subcellular organelle [3,4]
Both monomers were catalytically inactive ([44] and Figure S2b), in line with AGT being a functionally-obligate dimer. They differed from the corresponding dimeric forms for (i) the higher content of hydrophobic surfaces, indicated by their increased fluorescence emission in the presence of the probe ANS (Figure S2c) that binds exposed hydrophobic patches, as previously observed under native and denatured conditions [32,45] and (ii) the lower pyridoxal -phosphate (PLP)-binding affinity (Table 1) due to the fact that the coenzyme is held in place by residues coming from both subunits [44]

Summary

Introduction

Introduction distributed under the terms andAbout one third of all known proteins display an oligomeric structure, and most of them are homodimeric enzymes [1]. Several factors other than the intrinsic physico-chemical properties of the polypeptide chain influence the energy landscape inside a cell, including the crowded environment, the vectorial synthesis, and the presence of molecular chaperones, and the possibility to be imported in a subcellular organelle [3,4]. In the latter case, folding and targeting are strictly intertwined processes. Proteins destined for the nucleus or peroxisomes are imported in a fully-folded state and dimerization/oligomerization may occur before or after the import [7]. While the monomer/dimer equilibrium has been shown to influence the import and the functional properties of nuclear proteins [8,9,10], the impact of the quaternary assembly on the import of peroxisomal proteins is still debated [7,11,12]

Methods

Results

Conclusion