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

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.

SUBMITTER: Dindo M

PROVIDER: S-EPMC8067440 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Unlike most organellar proteins, some peroxisomal proteins are often found in significant amounts in the cytosol. Such apparent import inefficiency is very marked in guinea pig (Cavia porcellus) hepatocytes in which the cytosolic levels of two peroxisomal proteins, catalase and alanine:glyoxylate aminotransferase (AGT), are much higher than those found in human (Homo sapiens) hepatocytes, for example. In an attempt to provide an explanation for this phenomenon, we have cloned the guinea pig CpPEX5 gene, which encodes the peroxisomal targeting sequence type 1 (PTS1) import receptor Pex5p, and functionally compared it with its human homologue, HsPex5p. Our results showed the following: (1) CpPEX5, like its human homologue, encodes two splice variants differing by the presence or absence of an internal region of 37 amino acids; (2) both variants were expressed in all guinea pig tissues studied; (3) both variants were equally able to complement peroxisomal import of PTS1 proteins in microinjected Deltapex5 human fibroblasts; (4) CpPex5p was as efficient as HsPex5p in mediating the peroxisomal import of proteins possessing the consensus PTS1, Ser-Lys-Leu, but much less efficient in mediating the import of proteins possessing non-consensus PTS1s (i.e. Lys-Lys-Leu of human AGT and Ala-Asn-Leu of human catalase); (5) reporter proteins with the consensus PTS1, Ser-Lys-Leu, inhibited the peroxisomal import of endogenous catalase, whereas AGT with the non-consensus Lys-Lys-Leu did not; (6) high concentrations of HsPex5p, but not CpPex5p, markedly inhibited the import of AGT, but not catalase or proteins ending in Ser-Lys-Leu; and (7) in the yeast two-hybrid system, AGT-Ser-Lys-Leu interacted with the tetratricopeptide repeat domain of HsPex5p, but AGT-Lys-Lys-Leu did not. In addition, AGT-Ser-Lys-Leu was targeted to peroxisomes in Saccharomyces cerevisiae, whereas AGT-Lys-Lys-Leu was not. These data suggest that the inefficient peroxisomal import of AGT and catalase in guinea pig cells is due to the inefficiency with which CpPex5p mediates the peroxisomal import of proteins containing non-consensus PTS1s. They also suggest that the non-consensus PTS1 of human AGT might interact with HsPex5p very differently compared with the consensus PTS1, Ser-Lys-Leu.

Project description:The role of ?-oxidation and the glyoxylate cycle in fungal pathogenesis is well documented. However, an ambiguity still remains over their interaction in peroxisomes to facilitate fungal pathogenicity and virulence. In this report, we characterize a gene encoding an alanine, glyoxylate aminotransferase 1 (AGT1) in Magnaporthe oryzae, the causative agent of rice blast disease, and demonstrate that AGT1 is required for pathogenicity of M. oryzae. Targeted deletion of AGT1 resulted in the failure of penetration via appressoria; therefore, mutants lacking the gene were unable to induce blast symptoms on the hosts rice and barley. This penetration failure may be associated with a disruption in lipid mobilization during conidial germination as turgor generation in the appressorium requires mobilization of lipid reserves from the conidium. Analysis of enhanced green fluorescent protein expression using the transcriptional and translational fusion with the AGT1 promoter and open reading frame, respectively, revealed that AGT1 expressed constitutively in all in vitro grown cell types and during in planta colonization, and localized in peroxisomes. Peroxisomal localization was further confirmed by colocalization with red fluorescent protein fused with the peroxisomal targeting signal 1. Surprisingly, conidia produced by the ?agt1 mutant were unable to form appressoria on artificial inductive surfaces, even after prolonged incubation. When supplemented with nicotinamide adenine dinucleotide (NAD(+))+pyruvate, appressorium formation was restored on an artificial inductive surface. Taken together, our data indicate that AGT1-dependent pyruvate formation by transferring an amino group of alanine to glyoxylate, an intermediate of the glyoxylate cycle is required for lipid mobilization and utilization. This pyruvate can be converted to non-fermentable carbon sources, which may require reoxidation of NADH generated by the ?-oxidation of fatty acids to NAD(+) in peroxisomes. Therefore, it may provide a means to maintain redox homeostasis in appressoria.

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

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