Abstract
The import of PTS1 proteins into the glycosome or peroxisome requires binding of a PTS1-laden PEX5 receptor to the membrane-associated protein PEX14 to facilitate translocation of PTS1 proteins into the lumen of these organelles. Quaternary structure analysis of protozoan parasite Leishmania donovani PEX14 (LdPEX14) revealed that this protein forms a homomeric complex with a size > 670 kDa. Moreover, deletion mapping indicated that disruption of LdPEX14 oligomerization correlated with the elimination of the hydrophobic region and coiled-coil motif present in LdPEX14. Analysis of the LdPEX5-LdPEX14 interaction by isothermal titration calorimetry revealed a molar binding stoichiometry of 1:4 (LdPEX5: LdPEX14) and an in-solution dissociation constant (K(d)) of approximately 74 nm. Calorimetry, circular dichroism, intrinsic fluorescence, and analytical ultracentrifugation experiments showed that binding of LdPEX5 resulted in a dramatic conformational change in the LdPEX14 oligomeric complex that involved the reorganization of the hydrophobic segment in LdPEX14. Finally, limited tryptic proteolysis assays established that in the presence of LdPEX5, LdPEX14 became more susceptible to proteolytic degradation consistent with this protein interaction triggering a significant conformational change in the recombinant and native LdPEX14 structures. These structural changes provide essential clues to how LdPEX14 functions in the translocation of folded proteins across the glycosomal membrane.
Highlights
Tantly related to the peroxisomes in mammalian, yeast, fungi, and plant cells (4 – 6)
In Leishmania newly synthesized proteins containing PTS1 or PTS2 signals are bound by the receptors peroxin 5 (LdPEX5)3 and peroxin 7 (LdPEX7), respectively, and these cargo-laden receptors traffic to the glycosome surface where they bind to the membrane-associated protein peroxin 14 (LdPEX14)
We reported the use of a number of biophysical techniques that include size exclusion chromatography, intrinsic fluorescence, Circular Dichroism (CD), analytical ultracentrifugation, isothermal titration calorimetry, and limited proteolysis to examine the Leishmania donovani PEX14 (LdPEX14) structural and conformational changes triggered in the LdPEX14 complex following LdPEX5 binding
Summary
LdPEX5, L. donovani peroxin 5; LdPEX14, L. donovani peroxin 14, SEC, size exclusion chromatography; HRP, horseradish peroxidase; Ni2ϩ-NTA, nickel-nitrilotriacetic acid; PVDF, polyvinylidene difluoride; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ITC, isothermal titration calorimetry. The Leishmania PEX14 has been shown to be a peripheral membrane protein that associates tightly with the cytosolic surface of the glycosomal membrane (39). Despite the fact that PEX14 proteins exhibit Ͻ10% sequence conservation across phylogeny (39), this family of proteins has retained three structural elements. These include an N-terminal 33-amino acid signature motif AX2FLX8PX6FLXKGX5IX2A that contains a PEX5-binding motif (19, 40), a hydrophobic region, and a coiled-coil motif (21, 36, 37, 39, 41). We reported the use of a number of biophysical techniques that include size exclusion chromatography, intrinsic fluorescence, CD, analytical ultracentrifugation, isothermal titration calorimetry, and limited proteolysis to examine the LdPEX14 structural and conformational changes triggered in the LdPEX14 complex following LdPEX5 binding
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