Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting
Metadatos
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MDPI
Materia
Alanine: glyoxylate aminotransferase Pyridoxal phosphate Peroxisomes Peroxisomal import Dimerization Protein folding Fluorescence recovery after photobleaching
Fecha
2021Referencia bibliográfica
Dindo, M.; Ambrosini, G.; Oppici, E.; Pey, A.L.; O’Toole, P.J.; Marrison, J.L.; Morrison, I.E.G.; Butturini, E.; Grottelli, S.; Costantini, C.; et al. Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting. J. Pers. Med. 2021, 11, 273. https://doi.org/ 10.3390/jpm11040273
Patrocinador
Italian Ministry of University and Research (SIR project RBSI148BK3 to B.C.); Oxalosis and Hyperoxaluria Foundation (OHF2016, to B.C.). A.L.P; ERDF/Spanish Ministry of Science, Innovation and Universities— State Research Agency (Grant RTI2018-096246-B-I00); Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía (Grant P18-RT-2413).Resumen
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 50
-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 membranepermeable 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.