Based on peroxin protein 5 (Pex5p) homology searches in the expressed sequence tag database and sequencing of large full-length cDNA inserts, three novel and related human cDNAs were identified. The brain-derived cDNAs coded for two related proteins that differ only slightly at their N-terminus, and exhibit 39.8% identity to human PEX5p. The shorter liver-derived cDNA coded for the C-terminal tetratricopeptide repeat-containing domain of the brain cDNA-encoded proteins. Since these three proteins specifically bind to various C-terminal peroxisome-targeting signals in a manner indistinguishable from Pex5p and effectively compete with Pex5p in an in vitro peroxisome-targeting signal 1 (PTS1)-binding assay, we refer to them as ‘Pex5p-related proteins’ (Pex5Rp). In contrast to Pex5p, however, human PEX5Rp did not bind to Pex14p or to the RING finger motif of Pex12p, and could not restore PTS1 protein import in Pex5−/− mouse fibroblasts. Immunofluorescence analysis of epitope-tagged PEX5Rp in Chinese hamster ovary cells suggested an exclusively cytosolic localization. Northern-blot analysis showed that the PEX5R gene, which is localized to chromosome 3q26.2–3q27, is expressed preferentially in brain. Mouse PEX5Rp was also delineated. In addition, experimental evidence established that the closest-related yeast homologue, YMR018wp, did not bind PTS1. Based on its subcellular localization and binding properties, Pex5Rp may function as a regulator in an early step of the PTS1 protein import process.
Abbreviations used: CHO, Chinese hamster ovary; DsRed, red fluorescent protein of a Discosoma species; EGFP, enhanced green fluorescent protein; EST, expressed sequence tag; GST, glutathione S-transferase; HSA, human serum albumin; Gal4ad, Gal4 activation domain; Gal4bd, Gal4 DNA-binding domain; Pex5p, peroxin protein 5; Pex5Rp, Pex5p-related proteins; PTS1, peroxisome-targeting signal 1; TPR, tetratricopeptide repeat.
The nucleotide sequence data will appear in the Genbank® and EMBL Nucleotide Sequence Databases under the accession numbers AJ245503 (short human PEX5R isoform, HsPEX5RS), AB032592 (long human PEX5R isoform A, HsPXR2A; renamed as HsPEX5RA), AB032593 (long human PEX5R isoform B, HsPXR2B; renamed as HsPEX5RB), and AB032591 (mouse PEX5R, MmPXR2; renamed as MmPEX5R).