Pex5p binding affinities for canonical and noncanonical PTS1 peptides

Abstract

The majority of proteins targeted to the peroxisomal lumen contain a C-terminal peroxisomal targeting signal-1 (PTS1) that is bound by the peroxin Pex5p. The PTS1 is generally regarded as a C-terminal tripeptide that adheres to the consensus (S/A/C)(K/R/H)(L/M). Previously, we studied the binding affinity of peptides of the form YQX(-3)X(-2)X(-1) to the peptide-binding domain of human Pex5p (referred to as Pex5p-C). Optimal affinity was found for YQSKL, which bound with an affinity of 200 +/- 40 nM. To extend this work, we investigated the properties of a peptide containing the last 9 residues of acyl-CoA oxidase (RHYLKPLQSKL) and discovered that it binds to Pex5p-C with a dissociation constant of 1.4 +/- 0.4 nM, 180 times tighter than YQSKL. Further analysis revealed that the enhanced affinity is primarily due to the presence of leucine in the (-5) position. In addition, a peptide corresponding to the luciferase C-terminus (YKGGKSKL) was found to bind Pex5p-C about 20 times tighter than YQSKL. The majority of this effect results from having lysine in position (-4). Catalase contains a noncanonical PTS1 (-AREKANL). The affinity of YQANL was found to be 3600 +/- 400 nM. This relatively weak binding is consistent with previous unsuccessful attempts to direct chloramphenicol acetyltransferase to the peroxisome by fusing -ANL to its C-terminus (-GGA-ANL). The peptides YKANL, YEKANL, YREKANL, and YAREKANL all bound Pex5p-C with higher affinities than did YQANL, but the affinities are still lower than peptides that correspond to functional targeting signals in other contexts. Because both catalase and Pex5p are tetramers (as opposed to the monomeric Pex5p-C and the peptides used in our studies), multidentate effects on binding affinity between Pex5p and other oligomeric proteins should be considered. Our study provides direct thermodynamic data revealing that peptide binding to Pex5p-C binding is favored by lysine in the (-4) position and leucine in the (-5) position. Our results suggest that peptides or proteins with optimized residues in the (-4) and/or (-5) positions can bind to Pex5p with affinities that are at least two orders of magnitude greater than that of YQSKL, and that this stabilization can compensates for otherwise weakly binding PTS1s.

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…Alanine:glyoxylate aminotransferase is targeted by two signals (Huber et al, 2005); a C-terminal KKKL motif and an internal eightamino-acid sequence, both of which are predicted to be separately located on the surface of the protein (Ikeda et al, 2008). Sequences immediately upstream of the C-terminal tripeptide appear to contribute to specificity, especially when the match to the consensus tripeptide deviates (Maynard et al, 2004;Ma and Reumann, 2008).…”

“…The only interactions that are supported by quantitative data are for mammalian PEX5-PTS1 (K d ∼200 nM) (Gatto et al, 2000;Madrid et al, 2004;Maynard et al, 2004); and mammalian PEX5-PEX14 (K d ∼low nanomolar) (Schliebs et al, 1999;Saidowsky et al, 2001). One PEX5 molecule binds multiple PEX14 molecules simultaneously, as there are multiple binding sites for PEX14 on PEX5 (Schliebs et al, 1999).…”

Protein Quaternary Structure and Expression Levels Contribute to Peroxisomal-Targeting-Sequence-1-Mediated Peroxisomal Import of Human Soluble Epoxide Hydrolase

“…According to in vitro studies using fluorescence anisotropy, the SKM tripeptide has a lower binding affinity to the PTS1 receptor Pex5p than SKL, but is more potent than SKI (the PTS1 in rat and mouse sEH). 26,29 In order to compare the peroxisomal import efficiency of the three PTS1s in vivo, GFP-WT hsEH was expressed in CHO-K1 cells with SKL, SKM or SKI as PTS1. At all three time points during the 72 hour time-course of transient expression, more GFP-WT hsEH(SKL) was found in peroxisomes compared to GFP-WT hsEH(SKM) and GFP-WT hsEH(SKI) (Fig.…”

Peroxisomal Import of Human Alanine:glyoxylate Aminotransferase Requires Ancillary Targeting Information Remote from Its C Terminus

“…Whereas in the case of the latter, amino acids in close proximity (i.e. within 10 -20 residues) to the C-terminal tripeptide can markedly influence the properties of, and allowable residues within, the PTS1 (16,17,(32)(33)(34)(35). The importance of specific residues near the C terminus appears to be particularly important if the C-terminal tripeptide deviates from the conservative consensus PTS1 (16).…”