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Two Splice Variants of Human PEX19 Exhibit Distinct Functions in Peroxisomal Assembly

https://doi.org/10.1006/bbrc.2002.6568Get rights and content

Abstract

PEX19 has been shown to play a central role in the early steps of peroxisomal membrane synthesis. Computational database analysis of the PEX19 sequence revealed three different conserved domains: D1 (aa 1–87), D2 (aa 88–272), and D3 (aa 273–299). However, these domains have not yet been linked to specific biological functions. We elected to functionally characterize the proteins derived from two naturally occurring PEX19 splice variants: PEX19ΔE2 lacking the N-terminal domain D1 and PEX19ΔE8 lacking the domain D3. Both interact with peroxisomal ABC transporters (ALDP, ALDRP, PMP70) and with full-length PEX3 as shown by in vitro protein interaction studies. PEX19ΔE8 also interacts with a PEX3 protein lacking the peroxisomal targeting region located at the N-terminus (Δ66aaPEX3), whereas PEX19ΔE2 does not. Functional complementation studies in PEX19-deficient human fibroblasts revealed that transfection of PEX19ΔE8-cDNA leads to restoration of both peroxisomal membranes and of functional peroxisomes, whereas transfection of PEX19ΔE2-cDNA does not restore peroxisomal biogenesis. Human PEX19 is partly farnesylated in vitro and in vivo. The farnesylation consensus motif CLIM is located in the PEX19 domain D3. The finding that the protein derived from the splice variant lacking D3 is able to interact with several peroxisomal membrane proteins and to restore peroxisomal biogenesis challenges the previous assumption that farnesylation of PEX19 is essential for its biological functionality. The data presented demonstrate a considerable functional diversity of the proteins encoded by two PEX19 splice variants and thereby provide first experimental evidence for specific biological functions of the different predicted domains of the PEX19 protein.

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      In contrast to pex19Δ cells, the mutants still contained peroxisomes, indicating that the non-farnesylated Pex19p still maintained part of its function in peroxisome biogenesis. The PEX19-defective human cell line PBD399 represents complementation group 14 (complementation group J in Japan) and expresses a truncated version of Pex19p with a 44-amino acid deletion at the C terminus (11, 15, 26). This underscores the importance of the farnesylation motif, although it is unclear whether the truncated Pex19p is stably expressed in this patient cell line.

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    Abbreviations used: ABC, half transporter ATP binding cassette transporter; ALDP, adrenoleukodystrophy protein; ALDRP, adrenoleukodystrophy related protein; GFP, green fluorescent protein; GST, glutathione-S-transferase; PEX, peroxisomal assembly protein; PMP70, 70-kDa peroxisomal membrane protein; PMP(s), peroxisomal membrane protein(s).

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