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Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

ABSTRACT: The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.

SUBMITTER: Noguchi T

PROVIDER: S-EPMC1162877 | biostudies-other | 1981 Apr

REPOSITORIES: biostudies-other

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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.

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.

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Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

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