The Role of Conserved PEX3 Regions in PEX19‐Binding and Peroxisome Biogenesis

Abstract

The human peroxins PEX3 and PEX19 are essential for peroxisome biogenesis. They mediate the import of membrane proteins as well as the de novo formation of peroxisomes. PEX19 binds newly synthesized peroxisomal membrane proteins post-translationally and directs them to peroxisomes by engaging PEX3, a protein anchored in the peroxisomal membrane. After protein insertion into the lipid bilayer, PEX19 is released back to the cytosol. Crystallographic analysis provided detailed insights into the PEX3-PEX19 interaction and identified three highly conserved regions, the PEX19-binding region, a hydrophobic groove and an acidic cluster, on the surface of PEX3. Here, we used site-directed mutagenesis and biochemical and functional assays to determine the role of these regions in PEX19-binding and peroxisome biogenesis. Mutations in the PEX19-binding region reduce the affinity for PEX19 and destabilize PEX3. Furthermore, we provide evidence for a crucial function of the PEX3-PEX19 complex during de novo formation of peroxisomes in peroxisome-deficient cells, pointing to a dual function of the PEX3-PEX19 interaction in peroxisome biogenesis. The maturation of preperoxisomes appears to require the hydrophobic groove near the base of PEX3, presumably by its involvement in peroxisomal membrane protein insertion, while the acidic cluster does not appear to be functionally relevant.Key words: affinity measurements, de novo peroxisome formation, membrane protein import, peroxin, peroxisomal membrane biogenesis, peroxisomal membrane protein, preperoxisome, protein-protein interaction

Peroxisomal Pex3 Activates Selective Autophagy of Peroxisomes via Interaction with the Pexophagy Receptor Atg30

“…2B). Leu-320 and Asn-325 are found in a structural loop that is distinct from any defined protein-interacting domains of yeast Pex3 thus far (25,32).…”

“…Other highly conserved domains of PEX3 have also been described: the hydrophobic domain, which may play a role in PMP binding and peroxisome maturation, and a patch of acidic residues that do not have an assigned function (32). The Inp1-binding site on S. cerevisiae Pex3 was also determined by mutations affecting only inheritance while leaving biogenesis function intact (25).…”

PEX16 contributes to peroxisome maintenance by constantly trafficking PEX3 via the ER

“…Nevertheless, based on colocalization and in vitro targeting assays, others have argued that mammalian PMPs only target to peroxisomes via the group I pathway in cells without pre-existing peroxisomes, and that the ER does not contribute to the maintenance of mammalian peroxisomes (Matsuzaki and Fujiki, 2008; Huybrechts et al, 2009). Rather, it has been suggested that all PMPs in normal cells are targeted directly to peroxisomes without accessing the ER (Lazarow and Fujiki, 1985; Sacksteder et al, 2000; Matsuzaki and Fujiki, 2008; Huybrechts et al, 2009; Schmidt et al, 2012). …”

“…Rather than being completely absent from the ER, PMPs may be rapidly exported from the ER to peroxisomes resulting in their short time of residence in the ER (Nuttall et al, 2011; Schmidt et al, 2012). To test this hypothesis, we have developed a biophysical imaging technique to quantify the kinetics of PMP import into peroxisomes.…”

Hydrophobic handoff for direct delivery of peroxisome tail-anchored proteins

“…This interaction occurs through an amino-terminal segment of PEX19 (refs 29-31), which forms an amphipathic helix that intercalates a groove at the membrane distal end of PEX3 (refs 32,33). PEX3 also has a conserved membraneproximal hydrophobic surface 32 , and mutations in this region produce defects in peroxisome maturation 34 . How this region acts to promote PEX3 function remains unclear.…”