Interactions of Pex7p and Pex18p/Pex21p with the Peroxisomal Docking Machinery: Implications for the First Steps in PTS2 Protein Import

Abstract

Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13⌬ mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p-and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event.

Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals

“…Pcs60p served as control for a high salt-extractable peroxisomal protein. Plasmids designed to express GFP fusions of the indicated Pex13p fragments were transformed into wild-type strain yHPR251, which expresses the peroxisomal marker protein PTS2-DsRed from an integrated expression vector (Stein et al, 2002). The transformed strains were grown on ethanol plates for 2 days and examined for GFP-and DsRed-dependent fluorescence.…”

“…Expression of full-length Pex13p-GFP resulted in one or a few fluorescing spots that colocalized with PTS2-DsRed, indicating peroxisomal localization of the GFP fusion protein (Figure 6A). The design of subsequent Pex13p fragments took into account the position of the Pex19p-binding site as well as the dispensability of the N-terminal 151 amino acids of Pex13p for targeting (Stein et al, 2002). Even in the additional absence of the C-terminal SH3 domain and TMD2 (Pex13p 151-264 ), a largely peroxisomal localization was still observed ( Figure 6A).…”

“…For all yeast-two-hybrid assays, yeast strain PJ69-4A was used (P. James, Madison, WI). Localization of GFP-fusion proteins was analyzed in strain yHPR251, which expresses PTS2-DsRed from an integrated plasmid in strain UTL-7A (Stein et al, 2002). Standard media for the cultivation of yeast and bacterial strains were prepared as described (Erdmann et al, 1989;Sambrook et al, 1989).…”

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

“…Immunoprecipation of in vitro-imported PEX5L and PEX7 showed that PEX5 was predominantly associated with PEX14, whereas PEX7 was predominantly associated with PEX13 (Miyata et al, 2009). Although the PEX5L co-receptor is capable of independently and directly binding PEX14 and PEX13 (Otera et al, 2002), the yeast co-receptors Pex18/Pex21p require Pex7p to bind Pex14p and Pex13p (Stein et al, 2002). Pex13p can bind directly to Pex7p in S. cerevisiae (Girzalsky et al, 1999) and A. thaliana (Mano et al, 2006).…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…Yeast PEX13 interacts with PEX14 via a C-terminal Src homology 3 (SH3) domain and an intraperoxisomal sequence; this interaction is essential for matrix protein import (Schell-Steven et al, 2005). PEX13 also binds PEX5 and PEX7 in yeast (Douangamath et al, 2002;Stein et al, 2002;Pires et al, 2003) and mammals (Otera et al, 2002). Although Arabidopsis PEX13 does bind to PEX7 (Mano et al, 2006), Arabidopsis PEX13 lacks a recognizable SH3 domain (Boisson-Dernier et al, 2008), and PEX5-PEX13 interactions have not been reported in plants (Mano et al, 2006).…”