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9.A.17 The Integral Membrane Peroxisomal Protein Importer-2 (PPI2) Family

The PPI2 family members import proteins from the cytoplasm into the membrane of the peroxisome. Three proteins, Pex19 (farnesylated peroxin 19), Pex16 (3 isoforms in humans) and Pex3 (peroxin 3; peroxisomal biogenesis protein) mediate this process in both yeast and humans. Pex3 and Pex16 are present in the organelle membrane, while Pex19 displays a dual subcellular distribution (peroxisomal and cytosolic). Pex19 proteins interact with most peroxisomal intrinsic membrane proteins. Pex19 proteins are cytoplasmic receptor/carrier proteins that bind integral membrane hydrophobic proteins. The bound complexes traverse the peroxisomal membrane, inserting the substrate proteins. The Pex19 proteins exhibit semihydrophobic hydropathy profiles with about 10 amphipathic peaks and 9 hydrophobic peaks. The Pex3 proteins may serve as the channel through which Pex19 and its cargo pass. These proteins exhibit two strongly hydrophobic peaks (residues 18-39 and 136-152 in the S. cerevisiae homologue, residues 17-33 and 117-141 in the human homologue). However, they are otherwise hydrophilic. The hydrophilic regions show striking peaks of amphipathicity when the angle is set at either 100° or 180°. In humans, two substrate proteins, Pex16 (4-5 putative TMSs; Q9Y5Y5) and Pex26 (1 TMS; Q7Z412) have been identified (Matsuzono et al., 2006). There is some debate as to whether the import process is ATP dependent.

The long-standing paradigm that all peroxisomal proteins are imported post-translationally into pre- existing peroxisomes has been challenged by the detection of peroxisomal membrane proteins (PMPs) inside the endoplasmic reticulum (ER). The human PMP, PEX3, inserts co-translationally into the mammalian ER via the Sec61 translocon. The N-terminal transmembrane segment (TMS) of ribosome-bound PEX3 is recognized by the signal recognition particle (SRP). Binding to SRP is a prerequisite for targeting of the PEX3-containing ribosome nascent chain complex (RNC) to the translocon, where an ordered multistep pathway integrates the nascent chain into the membrane adjacent to translocon proteins, Sec61alpha and TRAM. This insertion of PEX3 into the ER is physiologically relevant because PEX3 then exits the ER via budding vesicles in an ATP-dependent process (Mayerhofer et al. 2016).

Import of matrix proteins can be broken down into four steps: targeting signal recognition by the cycling import receptors; receptor-cargo docking at the peroxisome membrane; translocation and cargo unloading; and receptor recycling. Import is mediated by a set of evolutionarily conserved proteins called peroxins that have been identified primarily via genetic screens (Brown and Baker, 2008). Insertion of a reporter protein into the peroxisomal membrane is Pex3-dependent and does not require ATP/GTP hydrolysis (Pinto et al., 2006). The system can be programmed with recombinant versions of Pex19p, showing that Pex19p-cargo protein complexes formed in the absence of peroxisomes are the substrates for the peroxisomal docking/insertion machinery. Data suggest that cargo-loaded Pex19p displays a much higher affinity for Pex3p than Pex19p alone. These results suggest that soluble Pex19p participates in the targeting of newly synthesized peroxisomal membrane proteins to the organelle membrane and support the existence of a cargo-induced peroxisomal targeting mechanism for Pex19p (Pinto et al., 2006).

Tail-anchored (TA) proteins are anchored into cellular membranes by a single TMS close to the C  terminus. Targeting of TA proteins to peroxisomes is dependent on PEX19. Yagita et al. (2013) showed that PEX19 forms a complex with the peroxisomal TA protein PEX26 in the cytosol and  translocates it directly to peroxisomes by interacting with the peroxisomal membrane protein PEX3. Unlike in yeast, the adenosine triphosphatase TRC40, which delivers TA proteins to the endoplasmic reticulum, was dispensable for peroxisomal targeting of PEX26. Basic amino acids within the luminal domain of PEX26 were essential for binding to PEX19 and therefore for peroxisomal targeting. A TMS that escapes capture by TRC40 and is followed by a highly basic luminal domain directs TA proteins to peroxisomes via the PEX19-dependent route.

Leishmania proteins containing a peroxisomal targeting signal sequence 2 (PTS2) are selectively trafficked to the glycosome by associating with the peroxin 7 receptor protein (PEX7). The L. major PEX7 (LmPEX7) encodes a 41kDa protein that exhibits limited sequence identity with PEX7 homologues from other eukaryotic organisms. Functional characterization of LmPEX7 revealed that this receptor bound the PTS2 protein fructose-1,6-bisphosphate aldolase. Moreover, LmPEX7 also formed a tight association with the Leishmania PEX5, the cytosolic PTS1 receptor, and PEX14, a glycosomal peripheral membrane protein required for protein import into the glycosome. Leishmania PEX7 has a dual distribution within the cytosolic compartment and glycosomal lumen (Pilar et al., 2007).

The reaction catalyzed by PPI2 is:

protein (cytosol) → protein (peroxisomal membrane)

References associated with 9.A.17 family:

Brown, L.A. and A. Baker. (2008). Shuttles and cycles: transport of proteins into the peroxisome matrix (review). Mol. Membr. Biol. 25: 363-375. 18651315
Hadden, D.A., B.A. Phillipson, K.A. Johnston, L.A. Brown, I.W. Manfield, M. El-Shami, I.A. Sparkes, and A. Baker. (2006). Arabidopsis PEX19 is a dimeric protein that binds the peroxin PEX10. Mol. Membr. Biol. 23: 325-336. 16923726
Karnik, S.K. and R.N. Trelease. (2005). Arabidopsis peroxin 16 coexists at steady state in peroxisomes and endoplasmic reticulum. Plant Physiol. 138: 1967-1981. 16040658
Matsuzono, Y. and Y. Fujiki. (2006). In vitro transport of membrane proteins to peroxisomes by shuttling receptor Pex19p. J. Biol. Chem. 281: 36-42. 16280322
Mayerhofer, P.U., M. Bañó-Polo, I. Mingarro, and A.E. Johnson. (2016). Human Peroxin PEX3 Is Co-translationally Integrated into the ER and Exits the ER in Budding Vesicles. Traffic 17: 117-130. 26572236
Pilar, A.V., K.P. Madrid, and A. Jardim. (2007). Interaction of Leishmania PTS2 receptor peroxin 7 with the glycosomal protein import machinery. Mol. Biochem. Parasitol. 158: 72-81. 18178267
Pinto, M.P., C.P. Grou, I.S. Alencastre, M.E. Oliveira, C. Sá-Miranda, M. Fransen, and J.E. Azevedo. (2006). The import competence of a peroxisomal membrane protein is determined by Pex19p before the docking step. J. Biol. Chem. 281: 34492-34502. 16980692
Yagita, Y., T. Hiromasa, and Y. Fujiki. (2013). Tail-anchored PEX26 targets peroxisomes via a PEX19-dependent and TRC40-independent class I pathway. J. Cell Biol. 200: 651-666. 23460677
Zimmermann, R., S. Lang, M. Lerner, F. Förster, D. Nguyen, V. Helms, and B. Schrul. (2021). Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER. Int J Mol Sci 22:. 34884833