Leaf peroxisomes are present in greening cotyledons and contain enzymes of the glycolate pathway that functions in photorespiration. However, only a few leaf peroxisomal proteins, that is hydroxypyruvate reductase (HPR), glycolate oxidase (GO) and alanine:glyoxylate aminotransferase 1 (AGT1), have been characterized, and other functions in leaf peroxisomes have not been solved. To better understand the functions of leaf peroxisomes, we established a method to isolate leaf peroxisomes of greening cotyledons. We analyzed 53 proteins by MALDI-TOF MS and then identified 29 proteins. Among them, five proteins are related to the glycolate pathway, four proteins function in scavenging of hydrogen peroxide and additionally 20 novel leaf peroxisomal proteins were identified. In particular, protein kinases and protein phosphatase were first identified as peroxisomal proteins suggesting that protein phosphorylation is one of the regulatory mechanisms in leaf peroxisomes. Novel leaf peroxisomal proteins contained five PTS1-like proteins that have sequences where one amino acid is substituted with another one in PTS1 sequences. The PTS1 motif was suggested to have novel PTS1 sequences.

In order to clarify the peroxisomal membrane proteins (PMPs), we characterized one of the major PMPs, PMP38. The deduced amino acid sequence for its cDNA in Arabidopsis thaliana contained polypeptides with 331 amino acids and had high similarity with those of Homo sapiens PMP34 and Candida boidinii PMP47 known as homologues of mitochondrial ATP/ADP carrier protein. We expected PMP38 to be localized on peroxisomal membranes, because it had the membrane peroxisomal targeting signal. Cell fractionation and immunocytochemical analysis using pumpkin cotyledons revealed that PMP38 is localized on peroxisomal membranes as an integral membrane protein. The amount of PMP38 in pumpkin cotyledons increased and reached the maximum protein level after 6 d in the dark but decreased thereafter. Illumination of the seedlings caused a significant decrease in the amount of the protein. These results clearly showed that the membrane protein PMP38 in glyoxysomes changes dramatically during the transformation of glyoxysomes to leaf peroxisomes, as do the other glyoxysomal enzymes, especially enzymes of the fatty acid beta-oxidation cycle, that are localized in the matrix of glyoxysomes.

No abstract