Peroxisomal protein import

The deubiquitinating enzyme USP9X hydrolyzes the thioester bond between the carboxyl terminus of ubiquitin and cysteine-11 of PEX5S (inferred from the large isoform of PEX5L in Grou et al. 2012). The thioester bond is unstable and appears also to be spontaneously disrupted by nucleophilic attack of small metabolites such as reduced glutathione (Grou et al. 2009).

The ensuing step in which the cargo protein is translocated across the membrane is not completely understood. During translocation, PEX5 and PEX7 become inserted into the membrane (Wiemer et al. 1995, Dodt et al. 1995, Oliveira et al. 2003 and expose a portion of their polypeptide chains to the organellar matrix (Rodrigues et al. 2015). One current model envisages PEX5 as a plunger that inserts into a transmembrane barrel formed by PEX14, PEX13, PEX2, PEX10, and PEX12 (the Docking-Translocation Module) (Francisco et al. 2017).
After delivering cargo to the matrix, PEX5 and PEX7 are recycled back to the cytosol by a process requiring mono-ubiquitination of PEX5 and ATP hydrolysis (Imanaka et al. 1987, Thoms and Erdmann 2006). PEX7 is not ubiquitinated but its recycling requires PEX5 mono-ubiquitination. A subcomplex of the Docking-Translocation Module comprising the RING-finger proteins PEX2, PEX10, and PEX12 conjugates a single ubiquitin to a cysteine residue of PEX5 (Carvalho et al. 2007, reviewed in Platta et al. 2016. The mono-ubiquitinated PEX5 and associated PEX7 are then extracted by the exportomer complex consisting of PEX1, PEX6, PEX26, and ZFAND6 (inferred from rat homologs in . PEX1 and PEX6 are members of the ATPases Associated with diverse cellular Activities (AAA) family, a group of proteins that use the energy of ATP hydrolysis to remodel molecular complexes. PEX1 and PEX6 form a hetero-hexameric ring, best described as a trimer of PEX1/PEX6 dimers (inferred from yeast in Platta et al. 2005, yeast homologs reviewed in Schwerter et al. 2017. Data on the yeast PEX1:PEX6 complex suggest that these ATPases use a substrate-threading mechanism to disrupt protein-protein interactions (Gardner et al. 2018). PEX7 is also then returned to the cytosol (Rodrigues et al. 2014). Once in the cytosol, ubiquitinated PEX5 is enzymatically deubiquitinated by USP9X and may also be non-enzymatically deubiquitinated by nucleophilic attack of the thioester bond between ubiquitin and the cysteine residue of PEX5 by small metabolites such as glutathione .

Literature references
A current hypothesis suggests alternative mRNA splicing. Both isoforms can bind the peroxisome targeting signal 1 (PTS1) located at the C-terminus of most of the proteins that are targeted to the peroxisomal matrix (Dodt et al. 1995, Fransen et al. 1995, Wiemer et al. 1995, Gatto et al. 2000, Brocard et al. 2003, Gatto et al. 2003, Harper et al. 2003, Ghosh and Berg 2010, Freitas et al. 2011, Okumoto et al. 2011. PTS1 typically contains Ser-Lys-Leu (SKL) at the C-terminus but substantial variation in sequences and affinities for PEX5 are observed and upstream residues can modulate binding to PEX5 (Lametschwandtner et al. 1998, Ghosh and Berg 2010, reviewed in Brocard and Hartig 2006.
A minority of peroxisomal matrix proteins contain PTS2. While the PEX5S isoform binds proteins containing PTS1, the PEX5L isoform binds either proteins containing PTS1 or PEX7 bound to proteins containing PTS2 (Braverman et al. 1998). Some proteins appear to be imported as oligomers, however this is rather inefficient as PEX5 appears to have a preference for monomeric substrates (Otera and Fujiki 2012, Freitas et al. 2011, Freitas et al. 2015, also inferred from mouse homologs). Mutations in PEX5 cause defects in peroxisomal import and comprise complementation group 2 of peroxisomal biogenesis disorders (also called Zellweger spectrum disorders) (Dodt et al. 1995, Wiemer et al. 1995
The thiol ester bond between ubiquitin and the cysteine residue of PEX5 is unusual among ubiquitin substrates, which usually have isopeptide bonds between ubiquitin and a lysine residue. Monoubiquitination of PEX5 at cysteine-11 is an integral and mandatory step in the PEX5-mediated peroxisomal protein transport pathway; in its absence, PEX5 cannot be extracted from the peroxisomal membrane docking/translocation machinery (the peroxisomal protein translocon), and thus transport of newly synthesized peroxisomal matrix proteins to the organelle matrix stops ). In addition to monoubiquitinating PEX5 during peroxisomal protein import, the PEX2:PEX10:PEX12 complex has also been implicated in pexophagy, a type of selective autophagy targeting peroxisomes. Pexophagy seems to be triggered mainly by ubiquitination of PEX5, which, in this case, can occur either at its cysteine-11 or lysine-209 residues, but ubiquitination of ABCD3 (also known as PMP70) and other peroxisomal membrane proteins may also be involved ( Literature references Gould, SJ., Dodt, G. (1996). Multiple PEX genes are required for proper subcellular distribution and stability of Pex5p, the PTS1 receptor: evidence that PTS1 protein import is mediated by a cycling receptor.  The deubiquitinating enzyme USP9X binds cytosolic ubiquitinated PEX5S (Ub:PEX5S) and then hydrolyzes the thioester bond between the carboxyl terminus of ubiquitin and cysteine-11 of PEX5S (inferred from the large isoform of PEX5 in .  The deubiquitinating enzyme USP9X hydrolyzes the thioester bond between the carboxyl terminus of ubiquitin and cysteine-11 of PEX5S (inferred from the large isoform of PEX5L in . The thioester bond is unstable and appears also to be spontaneously disrupted by nucleophilic attack of small metabolites such as reduced glutathione ).   Am. J. Hum. Genet., 70, 612-24. ↗ Gould, SJ., Steel, G., Braverman, N., Moser, A., Obie, C., Moser, H. et al. (1997). Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic chondrodysplasia punctata. Nat. Genet., 15, 369-76. ↗ Schmid, JA., Maurer-Stroh, S., Malkani, N., Berger, J., Wiesinger, C., Kunze, M. (2015). Mechanistic insights into PTS2-mediated peroxisomal protein import: the co-receptor PEX5L drastically increases the interaction strength between the cargo protein and the receptor PEX7. J. Biol. Chem., 290, 4928-40. ↗ Steel, G., Braverman, N., Moser, A., Moser, H., Valle, D., Lin, P. (2000). PEX7 gene structure, alternative transcripts, and evidence for a founder haplotype for the frequent RCDP allele, L292ter. The long isoform of PEX5, PEX5L, binds PEX7 that is already bound to a PTS2-containing cargo protein (Braverman et al. 1998, Dodt et al. 2001, Kunze et al. 2015, Rodrigues et al. 2015