Analysis of the Leishmania peroxin 7 interactions with peroxin 5, peroxin 14 and PTS2 ligands

Abstract

LPEX7 (Leishmania peroxin 7) is essential for targeting newly synthesized proteins with a PTS2 (peroxisome-targeting signal type 2) import signal into the glycosome. In the present paper, we describe the biophysical characterization of a functional LPEX7 isolated from Escherichia coli inclusion bodies. Pull-down assays showed that LPEX7 binds the interacting partners LdPEX5 (Leishmania donovani peroxin 5) and LdPEX14, but, more importantly, this receptor can specifically bind PTS2 cargo proteins in the monomeric and dimeric states. However, in the absence of interacting partners, LPEX7 preferentially adopts a tetrameric structure. Mapping studies localized the LdPEX5- and LdPEX14-binding sites to the N-terminal portion of LPEX7. Deletion of the first 52 residues abolished LdPEX14 association without altering the LdPEX5 interaction. Intrinsic fluorescence techniques suggested that each LPEX7 subunit has a single unique binding site for each of the respective interacting partners LdPEX5, LdPEX14 and PTS2 cargo proteins. Extrinsic fluorescence studies with ANS (8-anilinonaphthalene-1-sulfonic acid) demonstrated that LPEX7 contains a surface-exposed hydrophobic region(s) that was not altered by the binding of a PTS2 protein or LdPEX5. However, in the presence of these ligands, the accessibility of the hydrophobic domain was dramatically restricted, suggesting that both ligands are necessary to induce notable conformational changes in LPEX7. In contrast, binding of LdPEX14 did not alter the hydrophobic domain on LPEX7. It is possible that the hydrophobic surfaces on LPEX7 may be a crucial characteristic for the shuttling of this receptor in and out of the glycosome.

The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations

“…Moreover, as occurs in peroxisomes, glycosomal proteins are targeted for import to and location in glycosomes by the presence of the peroxisomal targeting signals (PTSs) PTS1 and PTS2. Two essential proteins for targeting newly synthesized proteins, with a PTS2 import signal, into the glycosome are peroxins (PEXs) 5 and 7 [56]. Remarkably, both proteins, PEX5 (LINF_350019100) and PEX7 (LINF_290012400), were identified in the experimental proteome of L. infantum promastigotes.…”

The Leishmania donovani peroxin 14 binding domain accommodates a high degeneracy in the pentapeptide motifs present on peroxin 5

“…Indeed, the binding to PEX14 has been implicated as vital step for promoting the release of the PTS1 cargo from PEX5 [33,44,45]. It is possible that the additional pentapeptide motifs on LdPEX5 may become accessible to PEX14 or a PEX13 homolog when LdPEX5 binds LdPEX7 or LdPEX5-PTS1 complex docks to LdPEX14 anchored on the glycosomal membrane [16,22,28,30,46,47].…”

The Experimental Proteome of Leishmania infantum promastigote and Its Usefulness for Improving Gene Annotations

“…Moreover, as occurs in peroxisomes, glycosomal proteins are targeted for import to and location in glycosomes by the presence of the peroxisomal targeting signals PTS1 and PTS2. Two essential proteins for targeting newly synthesized proteins, with a PTS2 import signal, into the glycosome are peroxins (PEXs) 5 and 7 [56]. Remarkably, both proteins, PEX5 (LINF_350019100) and PEX7 (LINF_290012400) have been identified in the experimental proteome of L. infantum promastigotes.…”