Structural insights into cargo recognition by the yeast PTS1 receptor

Area of Science:

• Cell Biology
• Molecular Biology
• Protein Trafficking

Background:

• Peroxisomal matrix protein import relies on import receptors that shuttle between the cytosol and peroxisomal membrane.
• The import receptor Pex5p specifically mediates the import of proteins containing a peroxisomal targeting signal type 1 (PTS1).

Purpose of the Study:

• To elucidate the structural basis of receptor-cargo recognition between Pex5p and its PTS1-containing cargo, Pcs60p.
• To investigate the role of the PTS1 signal and potential secondary binding sites in this interaction.

Main Methods:

• In vitro site-directed photo-cross-linking to identify Pex5p binding regions on Pcs60p.
• High-resolution mass spectrometry to pinpoint cross-linked sites on Pex5p.
• Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to analyze binding kinetics and modes.

Main Results:

• Pex5p and Pcs60p form a dimeric complex in vitro (KD = 0.19 μm).
• Cross-linking identified TPR6, TPR7, and the 7C-loop of Pex5p as key binding regions.
• A bivalent interaction mode was observed, with a secondary contact site on Pcs60p beyond the PTS1.
• Truncated Pcs60p lacking the PTS1 still bound Pex5p, albeit with lower affinity (KD = 7.7 μm) and a monovalent mode.

Conclusions:

• Pcs60p interacts with its receptor Pex5p via a bivalent binding mode, involving both the PTS1 and an ancillary binding region.
• This bivalent interaction likely enhances the efficiency and stability of peroxisomal protein import.
• A two-step model is proposed: initial cargo recognition and tethering via PTS1, followed by a stabilizing 'lock-in' through the secondary site.