We used Thermo Excalibur ProMass to calculate the molecular excess weight of this fragment in the presence and absence of Pex15 after deuteration for 0, 15, and 600?s?(Supplementary Table 2). For experiments shown in Fig.?3a and Supplementary Number?7, we incubated 30?M Pex15 1-309 with 2?M Pex1/Pex6?hexamer or buffer for Lumicitabine 60?s at 30?C with 30?mM ATP. its C-terminal disordered region engages with the pore loops of the motor, which then processively threads Pex15 through the central pore. Furthermore, Pex15 directly binds the cargo receptor Pex5, linking Pex1/Pex6 to additional components of the peroxisomal import machinery. Our results therefore support a role of Pex1/Pex6 in mechanical unfolding of peroxins or their extraction from your peroxisomal membrane during matrix-protein import. Intro Peroxisomes are membrane-bound organelles that perform specialized metabolic reactions, including the -oxidation of very long chain fatty acids1. The function, size, and quantity of peroxisomes can be modified by eukaryotic cells relating to metabolic needs2. Peroxisomes are created by either division of existing peroxisomes or by de novo biogenesis, a process that depends on ~35 dedicated peroxin (Pex) proteins3. Disruption of peroxisome function through mutations of Pex genes in humans causes a spectrum of developmental disorders called peroxisome biogenesis disorders Rabbit polyclonal to NR1D1 (PBDs). The severity of these disorders depends on how the mutation affects the formation of practical peroxisomes4. Current models of de novo peroxisome biogenesis posit the peroxisomal membrane proteins traffic through the endoplasmic reticulum or mitochondria, and consequently bud into pre-peroxisomal vesicles that fuse to form an empty membrane-bound compartment, the peroxisome ghost5,6. Peroxisomal matrix proteins are made in the cytosol and targeted to peroxisomes by one of two peroxisomal targeting signals: PTS1, a C-terminal tripeptide, or PTS2, an N-terminal nonapeptide7,8. Distinct receptor proteins identify the targeting signals (Pex5 for PTS1, Pex7 for PTS2), shuttle the matrix proteins to the peroxisomal membrane, and Lumicitabine then interact with the docking complex Pex13/149C11. By an unfamiliar mechanism, the cargo receptors mediate the import of the fully folded matrix protein into the peroxisome. During this import, the cargo receptors presume a protease-protected state, likely inlayed in the peroxisomal membrane12,13. A transmembrane complex of RING domain-containing E3 ubiquitin ligases, Pex2/10/12, then ubiquitinates an N-terminal cysteine of the cargo receptors and therefore primes them for membrane extraction and subsequent rounds of import14C17. Pex1 and Pex6 form a single, heterohexameric Type-2 AAA-ATPase engine with Lumicitabine an architecture much like Cdc48/p97 and NSF, but with alternating subunits18C20. Akin to the functions of NSF in the recycling of SNARE proteins and Cdc48/p97 in ER-associated degradation (ERAD), Pex1/Pex6 has been proposed to have functions both in peroxisome vesicle fusion5,21 and in extraction of the ubiquitinated cargo receptors from peroxisomal membranes17,22C24. Closer examination of cells lacking practical Pex1 revealed vacant peroxisome membrane compartments25 and continuing delivery of peroxisome membrane proteins26, indicating that Pex1/Pex6s main role is in peroxisomal matrix-protein import. Without practical Pex1 or Pex6, ubiquitinated Pex5 accumulates on peroxisomes, which are then targeted for specific autophagy22,27,28, suggesting that Pex1/Pex6 is also important for peroxisome stability. While inhibiting autophagy can stabilize peroxisomes and allow peroxisomal matrix-protein import in cells with hypomorphic Pex1, it could not recover peroxisome function in Pex1-null cells28. It consequently appears that Pex1/Pex6 has a main part in peroxisomal matrix-protein import, whose impairment prospects to peroxisome-specific autophagy. Like additional Type-2 AAA-ATPases, the Pex1/Pex6 engine contains two stacked rings of ATPase domains, termed D1 and D2, which are preceded by N-terminal domains that are typically involved in binding of substrates or cofactors29. Pex1 and Pex6 each have two N-terminal domains that are structurally related to the singular N-terminal domains on Cdc48/p97 and NSF20,30. The nucleotide-binding pouches of AAA-ATPases are located in the interfaces between neighboring subunits, with the Walker A (WA) and Walker B (WB) motifs contributed by one subunit, and the arginine-finger/package VII motif provided by the clockwise-next neighbor. In the candida W303 and cloned into bacterial and candida plasmids using primers outlined in Supplementary Table?4. Pex1/Pex6 purification Pex1-FLAG and His-Pex6 crazy type and mutant.
We used Thermo Excalibur ProMass to calculate the molecular excess weight of this fragment in the presence and absence of Pex15 after deuteration for 0, 15, and 600?s?(Supplementary Table 2)