Membrane Interactions of the Peroxisomal Proteins PEX5 and PEX14

Abstract

Human PEX5 and PEX14 are essential components of the peroxisomal translocon, which mediates import of cargo enzymes into peroxisomes. PEX5 is a soluble receptor for cargo enzymes comprised of an N-terminal intrinsically disordered domain (NTD) and a C-terminal tetratricopeptide (TPR) domain, which recognizes peroxisomal targeting signal 1 (PTS1) peptide motif in cargo proteins. The PEX5 NTD harbors multiple WF peptide motifs (WxxxF/Y or related motifs) that are recognized by a small globular domain in the NTD of the membrane-associated protein PEX14. How the PEX5 or PEX14 NTDs bind to the peroxisomal membrane and how the interaction between the two proteins is modulated at the membrane is unknown. Here, we characterize the membrane interactions of the PEX5 NTD and PEX14 NTD in vitro by membrane mimicking bicelles and nanodiscs using NMR spectroscopy and isothermal titration calorimetry. The PEX14 NTD weakly interacts with membrane mimicking bicelles with a surface that partially overlaps with the WxxxF/Y binding site. The PEX5 NTD harbors multiple interaction sites with the membrane that involve a number of amphipathic α-helical regions, which include some of the WxxxF/Y-motifs. The partially formed α-helical conformation of these regions is stabilized in the presence of bicelles. Notably, ITC data show that the interaction between the PEX5 and PEX14 NTDs is largely unaffected by the presence of the membrane. The PEX5/PEX14 interaction exhibits similar free binding enthalpies, where reduced binding enthalpy in the presence of bicelles is compensated by a reduced entropy loss. This demonstrates that docking of PEX5 to PEX14 at the membrane does not reduce the overall binding affinity between the two proteins, providing insights into the initial phase of PEX5-PEX14 docking in the assembly of the peroxisome translocon.

Insights into the Structure and Function of the Pex1/Pex6 AAA-ATPase in Peroxisome Homeostasis

“…Most matrix proteins use a C-terminal peroxisometargeting signal (PTS1) that is defined by the consensus sequence [S/A/C] [K/R/H] [L/H] and is sufficient to artificially target cytosolic proteins to the peroxisome [55,56]. PTS1 proteins are recognized by the soluble cytosolic receptor Pex5, which contains a Cterminal PTS1-binding domain and an intrinsically disordered N-terminal domain [57][58][59][60][61] (Figure 1B). Some cargo proteins lacking a PTS1 tag bind Pex5's N-terminal domain; for these proteins, the N-terminal half of Pex5 is sufficient for import [62][63][64].…”

“…Although the membrane topology of Pex13 and Pex14 has been controversial [68,[78][79][80], recent evidence suggests that Pex13's N-terminus and Pex14's C-terminus are in the peroxisome lumen, and the opposite termini are cytosolic [81,82]. Multiple copies of Pex5 oligomerize in the pore and these are thought to be integral structural components of the DTM [65], although the nature and relevance of any direct contacts between Pex5 and the lipid bilayer are unclear [61,67,83]. Pex proteins involved in Pex5 export-Pex8, Pex2, Pex10, Pex12, Pex15, Pex1, and Pex6-are also associated with the DTM [84].…”

Mechanism of PEX5-mediated protein import into peroxisomes

“…The N-terminal region additionally includes four predicted amphipathic helices (designated AH1-AH4 in Fig. 3A) of unknown significance (Gaussmann et al, 2021). AH1 resides downstream of Cys11 and has a hydrophobic surface conserved across eukaryotes (Fig.…”

Determinants of Peroxisome Membrane Dynamics

“…PEX5 cycles between a cytosolic and membrane-associated state and interacts with the PMP PEX14 to deliver matrix proteins to the peroxisome for their import across the membrane. Using NMR, it was recently demonstrated that the disordered N-terminal domain of human PEX5 weakly interacts with membrane-mimicking lipid bicelles, which stabilises transient amphipathic helices in this region of the protein (Gaussmann et al, 2021). Similarly, the globular N-terminal domain of human PEX14 was also shown to interact weakly with peroxisome-like lipid bilayers, although this did not lead to any overall structural changes and was independent of its transmembrane domain.…”

“…Similarly, the globular N-terminal domain of human PEX14 was also shown to interact weakly with peroxisome-like lipid bilayers, although this did not lead to any overall structural changes and was independent of its transmembrane domain. The association of PEX5 with the membrane does not significantly alter its binding affinity for PEX14 and the interaction of PEX14 with the membrane is readily outcompeted by its binding to PEX5, which occurs at partially overlapping sites (Gaussmann et al, 2021). Together, this suggests a possible model whereby the PEX14-phospholipid interaction partially hides the PEX5 binding site, preventing low-affinity non-specific interactions with other cytosolic proteins in the absence of PEX5, but being easily displaced when PEX5 is present to permit docking.…”