Membrane binding and pore forming insertion of PEX5 into horizontal lipid bilayer

Abstract

The assembly of the peroxisomal translocon involves the transition of a soluble form of the peroxisomal targeting receptor PEX5 into a membrane-bound form, which becomes an integral membrane component of the import pore for peroxisomal matrix proteins. How this transition occurs is still a mystery. We addressed this question using a artificial horizontal bilayer in combination with fluorescence time-correlated single photon counting (TCSPC) and electrophysiological channel recording. Purified human isoform PEX5L and truncated PEX5L(1–335) lacking the cargo binding domain were selectively labeled with thiol-reactive Atto-dyes. Diffusion coefficients of labeled protein in solution show that PEX5L is monomeric with a rather compact spherical conformation, while the truncated protein appeared in a more extended conformation. Labeled PEX5L and the truncated PEX5L(1–335) bind stably to horizontal bilayer thereby accumulating around 100-fold. The diffusion coefficients of the membrane-bound PEX5L forms are 3–4 times lower than in solution, indicating the formation of larger complexes. Electrophysiological single channel recording shows that membrane-bound labeled and non-labeled PEX5L, but not the truncated PEX5L(1–335), can form ion conducting membrane channels. The data suggest that PEX5L is the pore-forming component of the oligomeric peroxisomal translocon and that spontaneous PEX5L membrane surface binding might be an important step in its assembly.

High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

“…In addition, the Ca 2+ -calmodulin-activated phosphatase calcineurin A ( Ppp3ca gene), the G-protein-activated inward rectifier potassium channels 1 and 2 (girk1 and girk2, also named Kir3.1 ( Kcnj3 gene) and Kir3.2 ( Kcnj6 gene)), and the adenylate cyclase 1 ( Adcy1 gene) were identified. Unexpectedly, we also found a highly significant interaction of α 1 -Cav1.2 subunit with Pex5, a protein that associates type 1 peroxisomal targeting signal (PTS1)-containing proteins [ 38 ] with peroxisome membranes.…”

“…subunit with Pex5, a protein that associates type 1 peroxisomal targeting signal (PTS1)containing proteins [38] with peroxisome membranes. Proteins co-purifying with Cav1.3 channel subunits are shown on the right panel of Figure 4A.…”

The peroxisome: an update on mysteries 3.0

“…Most of the mammalian cargo proteins contain a PTS1 at the very C-terminus, explaining why the proteins need to be imported post-translationally. The “transient pore” model postulates that the PEX5-cargo complex inserts into the peroxisomal membrane and initiates the formation of a large pore for cargo translation, which is however short-lived in nature (Erdmann and Schliebs 2005 ; Meinecke et al 2010 ; Walter and Erdmann 2019 ; Blum et al 2023 ; Rudowitz and Erdmann 2023 ) (Fig. 1 top).…”