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Identification of PEX5p-related novel peroxisome-targeting signal 1 (PTS1)-binding proteins in mammals.

ABSTRACT: 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.

SUBMITTER: Amery L

PROVIDER: S-EPMC1221994 | biostudies-other | 2001 Aug

REPOSITORIES: biostudies-other

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Project description:BackgroundHigh-accuracy prediction tools are essential in the post-genomic era to define organellar proteomes in their full complexity. We recently applied a discriminative machine learning approach to predict plant proteins carrying peroxisome targeting signals (PTS) type 1 from genome sequences. For Arabidopsis thaliana 392 gene models were predicted to be peroxisome-targeted. The predictions were extensively tested in vivo, resulting in a high experimental verification rate of Arabidopsis proteins previously not known to be peroxisomal.ResultsIn this study, we experimentally validated the predictions in greater depth by focusing on the most challenging Arabidopsis proteins with unknown non-canonical PTS1 tripeptides and prediction scores close to the threshold. By in vivo subcellular targeting analysis, three novel PTS1 tripeptides (QRL>, SQM>, and SDL>) and two novel tripeptide residues (Q at position -3 and D at pos. -2) were identified. To understand why, among many Arabidopsis proteins carrying the same C-terminal tripeptides, these proteins were specifically predicted as peroxisomal, the residues upstream of the PTS1 tripeptide were computationally permuted and the changes in prediction scores were analyzed. The newly identified Arabidopsis proteins were found to contain four to five amino acid residues of high predicted targeting enhancing properties at position -4 to -12 in front of the non-canonical PTS1 tripeptide. The identity of the predicted targeting enhancing residues was unexpectedly diverse, comprising besides basic residues also proline, hydroxylated (Ser, Thr), hydrophobic (Ala, Val), and even acidic residues.ConclusionsOur computational and experimental analyses demonstrate that the plant PTS1 tripeptide motif is more diverse than previously thought, including an increasing number of non-canonical sequences and allowed residues. Specific targeting enhancing elements can be predicted for particular sequences of interest and are far more diverse in amino acid composition and positioning than previously assumed. Machine learning methods become indispensable to predict which specific proteins, among numerous candidate proteins carrying the same non-canonical PTS1 tripeptide, contain sufficient enhancer elements in terms of number, positioning and total strength to cause peroxisome targeting.

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Identification of PEX5p-related novel peroxisome-targeting signal 1 (PTS1)-binding proteins in mammals.

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