Arabidopsis PEX19 is a dimeric protein that binds the peroxin PEX10.

Peroxisomes are organelles found in all eukaryotic cells. Peroxisomes import integral membrane proteins post-translationally, and PEX19 is a predominantly cytosolic, farnesylated protein of mammalian and yeast cells that binds multiple peroxisome membrane proteins and is required for their correct targeting/insertion to the peroxisome membrane. We report the characterisation of the Arabidopsisthaliana homologue of PEX19 which is a predominantly cytosolic protein. AtPEX19 is encoded by two genes (designated AtPEX19-1 and AtPEX19-2) that are expressed in all tissues and at all developmental stages of the plant. Quantitative real time PCR shows that AtPEX19-1 and AtPEX19-2 have distinct expression profiles. Using in vitro translation and co-immunoprecipitation AtPEX19-1 was shown to bind to the Arabidopsis peroxisomal membrane protein PEX10. Additionally, bacterially expressed recombinant AtPEX19-1 was able to bind a fusion protein consisting of the C-terminus of PEX10 and glutathione S-transferase in pull-down assays, thereby demonstrating that non-farnesylated AtPEX19 can interact with the C-terminus of AtPEX10. Purified recombinant AtPEX19-1 was analysed by gel filtration chromatography and was found to have a molecular weight consistent with it forming a dimer and a dimer was detected in Arabidopsis cell extracts that was slightly destabilised in the presence of DTT. Moreover, cross-linking studies of native AtPEX19 suggest that in vivo it is the dimeric species of the protein that preferentially forms complexes with other proteins.

Characterization of the targeting signal of the Arabidopsis 22-kD integral peroxisomal membrane protein.

Using a combination of in vivo and in vitro assays, we characterized the sorting pathway and molecular targeting signal for the Arabidopsis 22-kD peroxisome membrane protein (PMP22), an integral component of the membrane of all peroxisomes in the mature plant. We show that nascent PMP22 is sorted directly from the cytosol to peroxisomes and that it is inserted into the peroxisomal boundary membrane with its N- and C-termini facing the cytosol. This direct sorting of PMP22 to peroxisomes contrasts with the indirect sorting reported previously for cottonseed (Gossypium hirsutum) ascorbate peroxidase, an integral PMP that sorts to peroxisomes via a subdomain of the endoplasmic reticulum. Thus, at least two different sorting pathways for PMPs exist in plant cells. At least four distinct regions within the N-terminal one-half of PMP22, including a positively charged domain present in most peroxisomal integral membrane-destined proteins, functions in a cooperative manner in efficient peroxisomal targeting and insertion. In addition, targeting with high fidelity to peroxisomes requires all four membrane-spanning domains in PMP22. Together, these results illustrate that the PMP22 membrane peroxisomal targeting signal is complex and that different elements within the signal may be responsible for mediating unique aspects of PMP22 biogenesis, including maintaining the solubility before membrane insertion, targeting to peroxisomes, and ensuring proper assembly in the peroxisomal boundary membrane.