Leishmania donovani Peroxin 14 Undergoes a Marked Conformational Change following Association with Peroxin 5*

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 (Kd) of ∼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.

Leishmania and Trypanosoma protozoan parasites represent organisms that branched off early from the eukaryotic cell lineage (1)(2)(3). Consequently, these organisms have retained a myriad of unique metabolic, biochemical, and structural features that are distinctive from other eukaryotic cells. Prominent among these features is the glycosome, an organelle that is dis-tantly related to the peroxisomes in mammalian, yeast, fungi, and plant cells (4 -6). The glycosome compartmentalizes a multitude of indispensable metabolic and biosynthetic pathways that include glycolysis, purine salvage, pyrimidine and ether-lipid biosynthesis, and ␤-oxidation of fatty acids (6,7). Glycosomal function is essential for parasite viability as mistargeting of glycolytic enzymes to the cytosol or disruption of glycosome biogenesis leads to a lethal phenotype (8 -12) making the glycosome and glycosome biogenesis machinery attractive chemotherapeutic targets (13,14).
Glycosomal and peroxisomal matrix proteins are post-translationally trafficked from cytosolic ribosomes to these microbodies by utilizing primarily one of two topogenic signals termed peroxisomal targeting signal 1 and 2 (PTS1 and PTS2) located at the C or N termini of proteins, respectively (15)(16)(17)(18)(19)(20). 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). This latter protein-protein interaction is paramount for the translocation of proteins across the glycosomal and peroxisomal membrane and for the biogenesis of these organelles. In ⌬pex14 yeast and mammalian mutant cell lines that lack a functional PEX14, the matrix PTS1 and PTS2 proteins are mis-targeted into the cytosol (21)(22)(23)(24). In trypanosomes knockdown of PEX14 using RNA interference caused mis-targeting of glycosomal matrix proteins to the cytosol and resulted in a lethal phenotype when parasites were cultivated in media containing glucose or glycerol (9,12,25).
PEX14 in fungi and mammals interacts with PEX13 forming a subcomplex known as the importomer (26 -31); whether a comparable importomer complex exists in the protozoa Leishmania is not clear because a PEX13 homolog has not yet been identified in this group of organisms. Numerous studies in phylogenetically diverse organisms have demonstrated that PEX14 is a membrane-associated protein; however, the nature of this interaction and the topology of PEX14 in the peroxisomal and glycosomal membranes, as assessed by physiochemical tech-niques, seem to vary. In mammalian cells, Hansenula polymorpha, Pichia pastoris, and Trypanosoma brucei (24,25,(32)(33)(34)(35)(36) PEX14 is reported to be an integral protein, whereas in Saccharomyces cerevisiae PEX14 association with the peroxisomal membrane is more plastic and has been reported to behave either as a peripheral or integral membrane protein (21,37,38). Using similar biochemical approaches, the Leishmania PEX14 has been shown to be a peripheral membrane protein that associates tightly with the cytosolic surface of the glycosomal membrane (39).
Interestingly, 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 AX 2 FLX 8 PX 6 FLXKGX 5 IX 2 A that contains a PEX5-binding motif (19,40), a hydrophobic region, and a coiled-coil motif (21,36,37,39,41). The hydrophobic region and the coiled-coil motif are believed to be involved in the formation of PEX14 homomeric structures (24,35,42). However, little is known about the structural changes induced in PEX14 upon binding its receptor PEX5. Here 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.

EXPERIMENTAL PROCEDURES
Material-All restriction endonucleases and DNA-modifying enzymes were purchased from Invitrogen or New England Biolabs (Beverly, MA). Horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit IgG were purchased from Sigma. All other reagents were of the highest quality commercially available.
E. coli ER2566 cells transformed with pTYB12-LdPEX5, pTYB12-ldpex5-(203-391), or pTYB12-ldpex5-(203-391) W3F (45) were grown to an A 600 of 1.2 at 37°C then shifted to 20°C for protein expression. Protein expression was induced for 4 h with 0.5 mM isopropyl thiogalactoside. Cell pellets were resuspended in 20 ml of 40 mM Tris-HCl, pH 8.0, containing an EDTA-free protease inhibitor mixture (Roche Applied Science) and lysed by a French press. Lysates were clarified by centrifugation, and the supernatant was loaded onto a chitin column (1 ϫ 3 cm), and the column was washed with 100 ml of 0.5 M NaCl in TB buffer. LdPEX5 and ldpex5-(203-391) were cleaved by incubating the column matrix with 5 ml of 50 mM dithiothreitol in TB buffer for 40 h at 4°C. Recombinant LdPEX14/ ldpex14 proteins were overexpressed in E. coli ER2566 strain and purified as described previously (39).
For analytical centrifugation and tryptic digest experiments the LdPEX5-LdPEX14 complex was isolated from E. coli ER2566 cells co-transformed with the pTYB12-LdPEX5 and pET30b-His 6 /S-LdPEX14. Clarified cells lysates were applied onto a Ni 2ϩ -NTA column (1 ϫ 5 cm), and the bound proteins were eluted with 250 mM imidazole in 50 mM phosphate, pH 7.5, 150 mM NaCl (PBS). The eluates were then applied to a chitin column (1 ϫ 3 cm) to capture complexes containing the chitin-LdPEX5 fusion protein. The column was washed with 50 ml of 40 mM Tris-HCl, pH 8.0, 0.5 M NaCl, and the LdPEX5-LdPEX14 complex was eluted by incubating the column matrix with 5.0 ml of 50 mM dithiothreitol in 40 mM Tris-HCl, pH 8.0, for 40 h at 4°C. All recombinant proteins were concentrated, and the buffer was exchanged for 40 mM Tris-HCl, pH 8.0, 150 mM NaCl using a Biomax 5K NMWL centrifugal filter unit (Millipore, Bedford, MA). Protein concentrations were determined spectrophotometrically (48).
Quaternary Structure Analysis-Size exclusion chromatography (SEC) was performed on a Beckman Alternatively, the syringe was loaded with 397 M solution of ldpex5-(203-391) and was titrated into a 145 M ldpex14-(1-120) solution in the ITC cell. All reactions were performed at a constant temperature of 303 K, and protein solutions were dialyzed against the same batch of 40 mM sodium phosphate, 120 mM NaCl, pH 7.5, 2 mM ␤-mercaptoethanol buffer to minimize heat of dilution effects. For all experiments, the contents of the cell were mixed at 300 rpm, and an equilibration time of 6 min between injections was used. The first injection used a 2-l aliquot, and the 20 -30 subsequent injections were performed using 10 l volumes over a duration of 10 s. The experimental titration curves were corrected for the heat of dilution, and the initial 2-l injections were typically omitted from the data set, and the curve fitting was performed using the Microcal Origin software 7.0 assuming a one-site model. The binding constants (K d ϭ 1/K a ) and the enthalpy (⌬H) were determined from the isotherm and the Gibbs free energy (⌬G), and entropy (⌬S) was calculated using the equation ⌬G ϭ ⌬H Ϫ T⌬S ϭ ϪRT lnK a .
Analytical Ultracentrifugation-Sedimentation velocity experiments were performed in 40 mM Tris, pH 7.5, at 20°C in a Beckman Optima XL-I (Fullerton, CA) analytical ultracentrifuge using an An 55 AL aluminum rotor. Samples containing 250 g/ml LdPEX14 or LdPEX5-LdPEX14 (molar ratio of 1:4) were loaded into double-sector cell with aluminum-filled Epon centerpieces. LdPEX14 and LdPEX5-LdPEX14 complex and ldpex14 deletion mutants were analyzed at rotor speeds of 26,000 and 30,000 rpm, respectively. UV scans were obtained at 230/280 nm and analyzed by the van Holde-Weischet method (49). The G/g(S) integral distribution attained with this method was determined using the XL-I UltraScan II version 9.7 sedimentation data analysis software (B. Demeler, University of Texas Health Science Center, San Antonio, TX).
Fluorescence Spectroscopy-Fluorescent measurements were performed on a Varian Cary Eclipse spectrofluorometer (Palo Alto, CA) at 25°C using an excitation wavelength of 295 nm. Emission spectra were recorded from 305 to 400 nm using a scan rate of 120 nm/min with excitation and emission slit Circular Dichroism (CD)-Purified proteins were exhaustively dialyzed at 4°C against 10 mM phosphate buffer, pH 7.6, and the protein concentration was measured by the method of Pace et al. (48). CD measurements were performed on a Jasco 810 spectropolarimeter at 20°C, using a cuvette with a 0.1-cm path length at a scan rate of 50 nm/min. Five spectra were collected and averaged per sample. For all samples, data were collected at wavelengths between 250 and 190 nm. LdPEX14 was diluted in dialysis buffer to a concentration of 3.1 M, and ldpex5-(203-391) was added to a final concentration of 0.8 M to obtain a 4:1 molar ratio of LdPEX14:ldpex5-(203-391).
Cross-linking Studies-L. donovani promastigotes (5 ϫ 10 8 cells/ml PBS) were permeabilized for 5 min at 20°C with 15 g/ml digitonin, and aliquots (100 l) were incubated with increasing concentrations of glutaraldehyde (0 -1.5 mM) for 20 min at 20°C. Cells were washed three times with 1.0 ml of PBS to remove excess cross-linking agent, and the cell pellet was resuspended in 100 l of 2ϫ SDS-PAGE sample buffer containing 6 M urea. Purified recombinant LdPEX14 (2 g/50 l PBS) was subjected to glutaraldehyde cross-linking using the above conditions. The reaction was terminated by adding Tris-HCl to a final concentration of 50 mM, and the mixtures were resolved on a 5-10% gradient SDS-PAGE, and the proteins were transferred to a PVDF membrane. Western blots were probed with rabbit anti-LdPEX14 (1:10,000) (39) or rabbit anti-L. donovani adenine phosphoribosyltransferase (LdAPRT) (1:1,000) antibodies. For cross-linking reactions with ldpex5-(203-391) and ldpex14-(1-120), proteins were reduced for 3 h at 20°C with 5 mM tris(2-carboxyethyl)phosphine to reduce potential disulfide bonds prior to cross-linking with glutaraldehyde (0 -3.2 mM). Cross-linked complexes were characterized by Western blots probed with anti-LdPEX5 and anti-LdPEX14 antisera.

RESULTS
LdPEX14 Quaternary Structure-Native LdPEX14 extracted from glycosomes was previously demonstrated to migrate on a sucrose density gradient predominantly as a macromolecular structure of ϳ800 kDa. 4 To further validate oligomeric structure of LdPEX14 on the glycosome surface, chemical crosslinking was used to trap these complexes. Western blot analysis of digitonin-permeabilized L. donovani promastigotes treated with glutaraldehyde showed a concentration-dependent accumulation of a cross-linked complex that SDS-PAGE was estimated to have an apparent mass of Ͼ250 kDa (Fig. 1). A comparable complex was also observed when the cross-linking reaction was performed with zero length cross-linking agent ethyl dimethylaminopropyl carbodiimide (data not shown). Western blots of glutaraldehyde cross-linked reactions probed with antisera against adenine phosphoribosyltransferase, a 26-kDa cytosolic protein (50,51), revealed a single immunoreactive band indicating that nonspecific cross-linking was minimal ( Fig. 1B). Similar complexes were also detected with purified glycosomes (39) or recombinant LdPEX14 treated with glutaraldehyde ( Fig. 1C).
Analysis of several of these mutant proteins by analytical ultracentrifugation confirmed the formation of large complexes of differing sizes (Fig. 3D). In low ionic strength buffers LdPEX14 assembled into structures that varied in size from ϳ10 to 70 S, whereas ldpex14-(⌬149 -179) appeared to form smaller structures ranging in size from 10 to 30 S (Fig. 3D). Surprisingly, in low ionic strength buffer ldpex14-(1-254) migrated as a relatively homogeneous complex of ϳ2-10 S. However, when the sedimentation analysis of this protein was performed in buffer containing 150 mM NaCl, a marked increase in oligomerization occurred resulting in structures with a size distribution of 2-90 S (data not shown). Similarly, sedimentation velocity analysis of ldpex14-(1-200) in low ionic strength buffers showed that this protein was also heterodis-  The black and gray shaded boxes represent the hexahistidine and S-protein tags, respectively, that were derived from the pET30b(ϩ) expression vector. These tags were used for the affinity purification of the recombinant proteins on Ni 2ϩ -NTA or S-protein affinity resins. The box labels denoted with an H or LZ correspond to the predicted hydrophobic domain (residues 149 -179) and the coiled-coiled motif (residues 270 -321) present in LdPEX14 (39). Finally, the hatched box corresponds to the PEX14 signature motif that is highly conserved among PEX14 homologs. The theoretical molecular weights for the constructs are also indicated. B, sequence encompasses residues 149 -173, the predicted hydrophobic region for LdPEX14. The underlined sequences GXXXA and SXXS represent putative helix-helix interaction motifs (63,64). Lowercase letters correspond to the secondary structure prediction using the Hierarchical Neural Network algorithm. e denotes residues in an extended strand; h denotes residues favoring helix structure, and c represents residues favoring a random coil structure. The double underline denotes the single tryptophan residue present in LdPEX14. NOVEMBER 14, 2008 • VOLUME 283 • NUMBER 46 perse and sedimented with S values ranging from 10 to 170 S (Fig. 3D).

Leishmania PEX14
In contrast, truncation mutants lacking the hydrophobic region, which include ldpex14-(1-148), ldpex14-(1-120), and ldpex14-(1-75), migrated on the Superdex 200 column with masses of ϳ50, 42, and 20 kDa, respectively, consistent with these proteins forming a dimeric structure (Fig. 2 and Fig. 3B). For ldpex14- (1-75), the dimer appears to be particularly stable as indicated by the significant population of the dimeric species (ϳ22 kDa) by SDS-PAGE (Fig. 3C). It should be noted that the wild type and mutant LdPEX14 proteins all migrated with anomalously higher molecular weight than theoretically predicted (39). Stabilization of the LdPEX14 homomeric structure probably involves multiple protein-protein contacts because the mutants ldpex14-(⌬149 -179) and ldpex14-(⌬270 -321), which lack the hydrophobic domain or the coiled-coil motif, still formed large complexes that were not disrupted by singly deleting either of the elements (Fig. 3, A and D).
Isothermal Titration Calorimetry-Isothermal titration calorimetry (ITC) was employed to determine the binding stoichiometry (N), binding affinity (K d ), enthalpy (⌬H), entropy (T⌬S), and the Gibbs free energy (⌬G) for the LdPEX5-LdPEX14 and ldpex5-(203-391)-ldpex14-(1-120) interactions (54,55). Fig. 4 illustrates the isothermograms for representative reactions. Titration of LdPEX14 into a solution of LdPEX5 showed that the LdPEX5-LdPEX14 interaction resulted in an exothermic heat release that diminished as the level of free LdPEX5 in the ITC cell decreased (Fig. 4A). Integration heat release for each injection was fit using the Microcal Origin 7.0 nonlinear regression software to a one-site model that had a binding stoichiometry of 4.2:1 (LdPEX14:LdPEX5). The K d for the LdPEX5-LdPEX14 interaction was 512 nM (or 128 nM per LdPEX14 subunit). Similar titration experiments performed with ldpex14-(1-120) and ldpex5-(203-391), fragments known to a form stable interactions (44), gave a binding stoichiometry of 4.1:1 (ldpex14-(1-120):ldpex5-(203-391)) and a K d of 625 nM (156 nM per ldpex14-(1-120) subunit) (Fig. 4B). The ⌬G (ϳϪ36 kJ/mol) for the binding reaction was derived from favorable negative ⌬H and positive T⌬S components (Table 1). In ligand binding reactions, hydrogen bonding and van der Waal interactions are proposed to be the major sources contributing to a negative ⌬H, whereas desolvation of hydrophobic surfaces, an event that increases the randomness of the system, favors a positive ⌬S (54,56,57). Collectively, this suggests that the LdPEX5-LdPEX14 interaction is stabilized by a combination of hydrogen bonding and hydrophobic interactions.
Reverse titration experiments in which LdPEX5 or ldpex5-(203-391) was titrated into a solution of LdPEX14 or ldpex14-(1-120), respectively, in both cases gave a binding stoichiometry ϳ0.25:1 (LdPEX5:LdPEX14 or ldpex5-(203-391):ldpex14-(1-120)) (Fig. 4, C and D; Table 1). These titrations, like the forward reaction, were exothermic throughout and had a ⌬G of approximately Ϫ40 kJ/mol (Table 1). However, the favorable ⌬G was primarily derived from a large negative ⌬H term (Ϫ90 to Ϫ99 kJ/mol), which offset the negative entropy change (T⌬S) ( Table 1). The unfavorable T⌬S suggests that interaction of LdPEX5 or ldpex5-(203-391) with LdPEX14 or ldpex14-(1-120) likely leads to the solvation of a hydrophobic surface or a conformational change that decreases the degrees of freedom in the system (54,56). Comparable K d values for the LdPEX5-LdPEX14 and ldpex5-(203-391)-ldpex14-(1-120) interactions were also obtained for these reactions ( Table 1). The solution phase binding affinities for the LdPEX5-LdPEX14 interactions, measured by ITC, are somewhat higher than values previously reported using an ELISA-based assay (44). This discrepancy is likely because of the cooperative nature or avidity effects asso-  ciated with interactions that may occur at liquid-solid interphase (58). SEC analysis of the ITC reaction mixtures revealed that the LdPEX5-LdPEX14 complex eluted in the column void volume suggesting that LdPEX5 was recruited to the LdPEX14 complex (Fig. 5A). The presence of LdPEX5 in the void volume was confirmed by SDS-PAGE (Fig. 5A). Previous studies demonstrated that LdPEX5 alone migrated on an SEC column with an apparent molecular mass of ϳ270 kDa (45), and indeed a small population of LdPEX5 alone was detected in the ITC reaction mixtures (fractions 31-36 (Fig. 5A)). SEC analysis of the ldpex5-(203-391)-ldpex14-(1-120) ITC reaction mixture revealed a peak eluting with a mass of ϳ95 kDa that by SDS-PAGE was found to contain both ldpex5-(203-391) and ldpex14-(1-120) (Fig. 5B) and is consistent with a hetero-oligomeric complex containing one ldpex5-(203-391) and four ldpex14-(1-120), as predicted by ITC.
Fluorescence Measurements-A striking feature of the LdPEX5-LdPEX14 ITC isotherm (Fig. 4C) was the large ⌬H change that occurred with low levels of LdPEX5. The absence of this dramatic heat loss when LdPEX14 was added to LdPEX5 (Fig. 4A) suggested that this heat loss may be associated with a conformational change in the LdPEX14 oligomeric complex triggered by binding of LdPEX5. Secondary structure analysis of LdPEX14 using the HNN algorithm revealed that the single tryptophan residue (Trp-152) in LdPEX14 mapped to the N terminus of a putative ␣-helix formed by the hydrophobic domain (Fig. 2B). We exploited the intrinsic fluorescence of Trp-152 to follow structural changes induced in LdPEX14 on binding LdPEX5. To eliminate fluorescence contributions from LdPEX5, ldpex5-(203-391) W3F, a mutant fragment in which the three tryptophan residues found within this region were mutated to phenylalanines was used in these studies (59). Pulldown assays indicated that ldpex5-(203-391) and ldpex5- (203-391) W3F exhibited similar LdPEX14 binding characteristics. Excitation of LdPEX14 at 295 nm revealed that Trp-152 had an emission maximum ( max ) at 332 nm, a wavelength diagnostic of a tryptophan located in a nonpolar environment (Fig.  6A). Addition of ldpex5-(203-391) W3F to LdPEX14, however, induced a concentration-dependent increase in the fluorescence intensity and a red-shift in the emission max to 341 nm, an alteration consistent with Trp-152 shifting to a more exposed polar environment (Fig. 6A). Correlating the wavelength change with the molar ratio of ldpex5-(203-391) W3F revealed a plateau in the shift of the Trp-152 emission max at an ldpex5-(203-391) W3F:LdPEX14 mole ratio of 0.25:1. This binding stoichiometry is in agreement with the results obtained by ITC. Denaturation of LdPEX14 with 4.5 M guanidinium hydrochloride resulted in a shift in the Trp-152 max from 332 to 360 nm, indicating that this tryptophan residue in native LdPEX14 was located in nonpolar environment (Fig. 6B) (60). Stern-Volmer plots revealed a linear response with Stern-Volmer constant of 5.4 M Ϫ1 for LdPEX14-ldpex5-(203-391) W3F indicating that Trp-152 underwent dynamic quenching with acrylamide (Fig. 6C). A lower Stern-Volmer constant of 3.8 M Ϫ1 was measured for LdPEX14 alone, indicating that in the absence of ldpex5-(203-391) W3F, Trp-152 was less accessible to the quenching agent (Fig. 6C) (60).
CD Analysis-The effect of ldpex5-(203-391)-LdPEX14 interaction on the secondary structure was examined by the method of Greenfield (61). The far-UV CD difference spectrum generated by subtracting the calculated spectra generated from the unmixed ldpex5-(203-391) and LdPEX14 spectra from the CD spectrum obtained for the LdPEX14:ldpex5-(203-391) (4:1) complex revealed a maxima at ϳ215 nm consistent with an increase in the random coil content of the complex (Fig. 6D,  inset). Interestingly, ldpex5-(203-391) did not show a prominent circular dichroism signal suggesting that this fragment has a relatively flexible conformation (Fig. 6D).
Analysis of the LdPEX14 and LdPEX5-LdPEX14 Complex-Sedimentation velocity analytical ultracentrifugation analysis of the LdPEX14 and LdPEX5-LdPEX14 macromolecular complexes by the method of van Holde-Weischet (49), which correlates the sedimentation coefficient of a protein species with its abundances at the moving boundary, gave rise to curves with a positive deflection (Fig. 7). This relationship is diagnostic of a To estimate the conformation change associated with the protein-protein interaction the spectra for LdPEX14 alone and ldpex5-(203-391) alone were summed and compared with the recorded spectrum of the LdPEX14:ldpex5-(203-391) complex (61). The difference CD spectrum generated from the measured and calculated spectra for the LdPEX14:ldpex5-(203-391) complex is shown as an inset.
protein forming heterogeneous oligomeric structures (49). As demonstrated previously (Fig. 3D), LdPEX14 formed disperse structures with sedimentation coefficients ranging from 17 to 52 S. Under similar conditions, the LdPEX5-LdPEX14 complexes also exhibited a heterogeneous behavior, but the complexes had a more compact architecture with sedimentation coefficients ranging from ϳ6 to 19 S. These results imply that binding LdPEX5 induced a conformational change in LdPEX14 leading to the formation of a more ordered LdPEX5-LdPEX14 hetero-oligomeric complex (Fig. 7) and support the ITC finding that docking of LdPEX5 to LdPEX14 induced a striking conformational change.
Limited Proteolysis-The LdPEX14 conformational changes were next examined using trypsin limited proteolysis. Treatment of recombinant LdPEX14 alone with trypsin resulted in cleavage of this protein to an N-terminal ϳ40-kDa fragment that was more resistant to further proteolysis even after a prolonged incubation (3 h at 20°C) (Fig. 8, A and B). Proteaseresistant fragments have been reported for mammalian and S. cerevisiae PEX14; however, these fragments were associated with insertion of PEX14 into the peroxisomal membrane (21,35,36). Western blot analysis of the digests with Ni 2ϩ -NTAconjugated horseradish peroxidase (Ni 2ϩ -NTA-HRP or anti-LdPEX14 antisera (44)) established that this ϳ40-kDa proteolytic product corresponds to an N-terminal fragment (Fig. 8, B and C) exhibiting an electrophoretic mobility similar to ldpex14-(1-254) (Fig. 3C). Smaller N-terminal fragments ranging from ϳ19 to 40 kDa were also observed within 15 min of digestion (Fig. 8C).
Formation of the LdPEX5-LdPEX14 (1:4 molar ratio) dramatically altered the susceptibility of LdPEX14 to proteolytic degradation, as shown by the Coomassie Blue-stained gels (Fig.  8A). Notably, no reactivity was detected with Ni 2ϩ -NTA-HRP, suggesting that the proximal N-terminal region of LdPEX14 became exposed resulting in rapid degradation of the hexahis-tidine tag. Moreover, Western blots probed with anti-LdPEX14 antibodies confirmed that binding of LdPEX5 caused a conformational change resulting in ϳ80% of LdPEX14 degradation to an ϳ19-kDa fragment within 2 min. This fragment was further degraded, albeit at a significantly much slower rate (Fig. 8C). Interestingly, the C terminus of LdPEX14 was very prone to protease digestion, in the presence and absence of LdPEX5 suggesting that this region may not be tightly folded, possibly because of the high content of proline residues, which appears to be particular to LdPEX14 (39). The ϳ60-kDa protein that accumulates in Fig. 8A corresponds to a proteolytic fragment of LdPEX5 that because of its abundance weakly reacts with the HRP-conjugated secondary antibody accounting for the 60-kDa immunoreactive species in Fig. 8C. That this protein fragment was derived from LdPEX5 was confirmed by immunostaining with anti-LdPEX5 antibodies.
To evaluate if LdPEX5 triggered similar conformational changes in LdPEX14 anchored to the glycosomal membrane, purified glycosomes in the presence of an excess of recombinant LdPEX5 or the control protein bovine serum albumin (BSA) together with the PTS1 protein LdXPRT (62) were treated with trypsin. Digest mixtures containing BSA revealed that native LdPEX14 was resistant to proteolytic degradation, and ϳ50% of the parent protein remained intact even after a 3-h incubation. Only an ϳ55-kDa anti-LdPEX14 immunoreactive product was observed to accumulate (Fig. 8D). This 55-kDa anti-LdPEX14 immunoreactive fragment is likely because of the partial proteolytic degradation of LdPEX14 that occurred during glycosome purification because only low levels of protease inhibitors were used in these preparations (Fig. 8D). A comparable sized fragment was also observed with the recombinant LdPEX14 (Fig. 8C). In the presence of an LdPEX5-LdXPRT complex, however, Ͼ70% of the native LdPEX14 was degraded to an ϳ19-kDa fragment within 15 min (Fig. 8D). Because the anti-LdPEX14 antisera recognize an epitope between residues 23 and 63 (44), it is clear that association with LdPEX5 renders the C terminus of LdPEX14 extremely susceptible to proteolysis. A similar observation has been reported for mammalian PEX14 (35). Interestingly, the digest of native LdPEX14, anchored to the glycosomal membrane, closely mimicked the limited proteolysis patterns observed with the recombinant LdPEX14.
It is postulated that the ϳ40-kDa trypsin-resistant fragment observed with LdPEX14 complex is protected from further degradation by forming a stable oligomeric complex, similar to the structures formed by ldpex14-(1-200) or ldpex14- . This contention is supported by the finding that LdPEX14 treated with trypsin retained a core structure that eluted on a Superdex 200 column with a mass Ͼ670 kDa (Fig. 8E). Western blot analysis of this core structure revealed that the ϳ45and ϳ19-kDa fragments cross-reacted with anti-LdPEX14 antibodies. It should be stressed that the resistance of the ϳ40-kDa fragment to tryptic degradation is attributed to protein folding because LdPEX14 contains 24 lysine and arginine residues, trypsin cleavage sites, within the first 250 residues.

DISCUSSION
The membrane-associated protein PEX14 is a crucial component required for the import of PTS1 and PTS2 proteins into glycosomes and peroxisomes (10,27,30,39,41,44,63,64). In glycosome biogenesis, the importance of PEX14 has been underscored by genetic experiments demonstrating that knockdown of PEX14 in the kinetoplastid parasite T. brucei results in a lethal phenotype (9,10,25). Although a considerable amount of knowledge regarding the protein complement involved in the assembly of these microbody organelles has been amassed, far less is known about the molecular dynamics associated with the docking of the PEX5 and PEX7 receptors to PEX14 and the subsequent translocation of folded nascent polypeptides across the lipid bilayer membrane.
In situ cross-linking studies demonstrate that on the glycosomal surface, LdPEX14 forms a homo-oligomeric complex that on sucrose density gradients migrates with a density of ϳ800 kDa. 4 Similar PEX14 oligomeric structures that constitute the importomer complex have also been reported in mammals and yeast (24,27,30,53). Recombinant LdPEX14 expressed in E. coli also formed comparable homomeric complex and suggested that oligomerization did not appear to be contingent on accessory proteins or chaperones unique to Leishmania. However, it is unclear how these complexes participate in the import of folded proteins across the glycosomal membrane.
Functional domain mapping indicated that the hydrophobic domain, which is predicted to adopt an ␣-helix configuration (Fig. 2B), is important for stabilizing the LdPEX14 homomeric complex. In LdPEX14, the hydrophobic domain contains centrally located GXXXA and SXXS motifs that by molecular modeling are predicted to be on the same face of the ␣-helix, an architecture that would promote helixhelix packing and oligomerization (63,64). Indeed, mutagenesis of the analogous GXXXG and AXXXA motifs in the mammalian PEX14 caused disruption of oligomerization (53). In contrast to the mammalian PEX14, where deletion of this hydrophobic segment abrogated oligomerization and resulted in cytosolic targeting of the mutant protein, elimination of the LdPEX14 hydrophobic domain alone did not disrupt glycosomal targeting or homo-oligomerization, although ldpex14-(⌬149 -179) was found to form smaller complexes. The association of ldpex14-(⌬149 -179) with the glycosome, in contrast to the mammalian PEX14 mutants lacking the analogous hydrophobic domain (52), was not surprising because recent studies demonstrated that residues 1-23 of LdPEX14 are the crucial elements required for glycosomal membrane attachment. 4 Whether ldpex14-(⌬149 -179) is biologically functional and capable of mediating protein import into the glycosome is not clear however, because in kin- after the addition of trypsin. Proteolysis was terminated by adding a large excess of a protease inhibitor mixture, and samples were resolved on a 10% SDS-PAGE. A, protein were transferred PVDF membrane stained with Coomassie Blue dye, and B, then probed with Ni 2ϩ -NTA-conjugated horseradish peroxidase (Ni 2ϩ -NTA-HRP) (1:2,000) in PBS. Membranes were stripped with 1% SDS, 5 mM EDTA to remove the Ni 2ϩ -NTA-HRP, and C, probed with anti-LdPEX14 antibodies. D, to evaluate the conformational changes induced LdPEX5, purified glycosomes were incubates with the control protein BSA or recombinant LdPEX5 in the presence of an excess of LdXPRT (59) and treated with trypsin as indicated above. Native LdPEX14 digest produces were analyzed by Western blot. E, chromatographic analysis of the LdPEX14 tryptic digest. LdPEX14 was digested for 4 h (LdPEX14:trypsin 200:1 molar ratio) and an aliquot was injected onto a Superdex 200 size exclusion column equilibrated with 50 mM phosphate, pH 7.5, 150 mM NaCl, 5 mM ␤-mercaptoethanol to assess the quaternary structure of trypsin-resistant LdPEX14 core. The gray trace is a 10-l injection of intact LdPEX14, and the black trace is a 10-l injection of the reaction mixture after a 4-h digest. Peak A for the black trace was collected, and the protein composition of this peak was examined by Coomassie Blue dye-stained SDS-PAGE. etoplastids PEX14 appears to be essential for parasite viability (9,25) and generation of Leishmania donovani ⌬ldpex14 mutant cell line required for these studies has been hampered by the absence of RNA interference machinery or tight regulatable expression systems (65). That the hydrophobic domain of LdPEX14 participates in a protein-protein or protein-membrane interaction may be inferred from the capacity of the ldpex14-(1-200) to form large heterodisperse oligomeric structures that are dependent on residues 149 -200. It should be noted that by analytical ultracentrifugation, ldpex14-(⌬149 -179) assembled into structures that were smaller than LdPEX14.
In addition to the hydrophobic region, it is postulated that contacts involving the N-terminal dimerization domain and the coiled-coil motif located in the C-terminal portion of LdPEX14 contribute to stabilization of the homomeric complex. This repeating geometry of intermolecular contacts illustrated in Fig. 9 would account for the heterodisperse nature of the LdPEX14 complexes observed in sedimentation velocity experiments and also for the oligomerization of the ldpex14 C-terminal truncation mutants (Fig. 9, B and C).
A pivotal step in the import of PTS1 proteins into the glycosome involves the docking of LdPEX5 to LdPEX14. Thermodynamic studies indicate that this is not a simple bimolecular interaction as four molecules of LdPEX14 bind to one LdPEX5. Complexes isolated from peroxisomes appeared to have comparable PEX5:PEX14 (1:5) binding stoichiometries (46,47). Because the N terminus of LdPEX14 forms dimers, it is unlikely that LdPEX14 monomers bind to LdPEX5 in a sequential fashion, but rather we argue that the initial association occurs at a LdPEX14 dimer that subsequently recruits a second LdPEX14 dimer to complete the interaction. This contention is supported by the glutaraldehyde cross-linking experiments showing 1:2 (ϳ80 kDa) and 1:4 (ϳ100 kDa) ldpex5-(203-391): ldpex14-(1-120) species (Fig. 5C). However, the possibility that two LdPEX14 dimers associate to form a complete LdPEX5binding site cannot be discounted. In contrast to PEX5-PEX14 complexes previously characterized from rat livers that had an apparent molecular mass of 250 kDa (46), the LdPEX5: LdPEX14 structures appear to be substantially larger with an apparent mass of Ͼ670 kDa.
Several lines of evidence suggest that LdPEX5 triggers marked conformational changes in LdPEX14. First, analytical ultracentrifugation revealed that binding of LdPEX5 caused a rearrangement of the LdPEX14 leading to the formation of more compact complexes. Second, limited proteolysis experiments intimate that binding of LdPEX5 or LdPEX5-PTS1 dramatically impacted the quaternary and tertiary structures of both the soluble recombinant LdPEX14 and native LdPEX14 anchored to the glycosome, a consequence that rendered these proteins highly susceptible to proteolytic degradation. Third, intrinsic fluorescence measurement using the single tryptophan residue, located immediately adjacent to the hydrophobic domain of LdPEX14, confirmed that association with LdPEX5 caused a conformational perturbation that shifted this tryptophan from a nonpolar buried environment to a more polar and solvent-exposed environment. It should be emphasized that in the absence of an LdPEX5-PTS1 complex, LdPEX14 is attached to the glycosomal membrane via an N-terminal domain that requires the first 23 amino acids. Given these structural changes, it is tempting to suggest that docking of the PTS1 receptor leads to the exposure of the hydrophobic domain, which would interact and possibly insert into the glycosomal membrane to form a potential pore-like structure that would facilitate the import of PTS1 proteins into the glycosomal lumen. This speculation is supported by the finding that the hydrophobic domain of mammalian PEX14 is required for membrane insertion (52). Moreover, the proposed architecture LdPEX14 would be consistent with this protein being directly involved in the formation of a potential structure that is analogous to a transient pore that has been advanced for peroxisomal protein translocation (21,28,30,31).