Fig 1.
Prediction of PEX coding genes.
A. Predicted PEXs in E. histolytica and related Evosea species. B. Scheme of the peroxisomal machinery identified in Entamoeba species except for E. invadens. The white color indicates PEXs that were not identified. Ub, ubiquitin; PMP, peroxisomal membrane protein.
Fig 2.
Domain structures of E. histolytica and Homo sapiens PEXs.
PFAM domain identifiers (in brackets) and probability percentages are indicated. TMD, transmembrane domain; TPR, tetratricopeptide domain; stars indicate Pex14 amino acid residues that are involved in Pex5 interactions [30].
Fig 3.
Phylogeny of Pex1, Pex6 and other proteins with AAA domains.
The maximum likelihood tree was inferred with IQ-TREE using 51 protein sequences and 522 positions. Numbers at nodes of the tree indicate statistical support in the form of an ultrafast bootstrap of the IQ-Tree and posterior probability of the PhyloBayes analysis.
Fig 4.
Expression of tagged PEXs in E. histolytica.
A. Western blot analysis of cell lysate (L), organelle (P) and cytosolic soluble (S) fractions. Fe-superoxide dismutase (FeSOD) was used as the cytosolic marker. B. Western blot analysis of Pex5 and Pex11 in seven fractions isolated by differential centrifugation using 0.05% Tween-20 to limit protein aggregation.
Fig 5.
Cellular localization of PEXs in E. histolytica.
A. Immunofluorescence microscopy of His-tagged PEXs and the mitosomal marker APSK. B. Structured illumination microscopy of Pex11, Pex14, and APSK. BF, bright field. Scale bar: 10 μm.
Fig 6.
Confocal microscopy of vesicles labeled with Pex14, ER marker BiP1 and lysosomal marker Atg8.
BF, bright field. Scale bar: 10 μm.
Fig 7.
Immunoelectron microscopy detection of Pex14 and Pex16 in E. histolytica.
Pex14 (A,B) was detected using Ni-NTA conjugated with 5 nm gold particles (A) or anti-V5 antibody (B). Pex16 (C,D) was detected using rabbit (C) and mouse (B) anti-His antibodies and the corresponding antibody conjugated with 5 nm gold particles. Scale bar: 100 nm.
Fig 8.
Affinity purification of peroxisomes.
A. Lysate of cells (L) expressing His-tagged Pex14 was used for differential centrifugation at 50 000 and 150 000 x g, and pellet (P) and soluble (S) fractions were analyzed by western blotting. In the last step, peroxisomes were purified using anti-His antibody-conjugated beads (His-beads) that were used for mass spectrometry. Antibodies against Cpn60 were used as mitosomal markers. B. Relative protein enrichment was calculated with densitometry of Pex14-His and Cpn60 signals.
Fig 9.
Localization of PTS1-containing candidate proteins in yeast peroxisomes.
Fluorescence microscopy of mCherry-tagged myo-IDH and two hypothetical proteins (EHI_051440 and EHI_045060) (red) expressed in yeast. Pox1 fused with GFP was used as a peroxisomal marker (green). Signal colocalization analysis (white) was performed by ImarisColoc software.
Fig 10.
Localization of IDH in E. histolytica.
A. Detection of myo-IDH (green)and APSK (mitosomal marker protein, red) in wild- type cells. Bar = 10 μm. B. Detection of native myo-IDH (red) in cells expressing C-terminally His-tagged PEXs (green). Signal colocalization analysis (white) was performed by ImarisColoc software. Bar = 10 μm. C. Immunoelectron microscopy. Myo-IDH was detected using a rabbit polyclonal anti-myo-IDH antibody and anti-rabbit IgG conjugated to 15 nm gold particles (white arrows, a-f), Pex16 was detected using a mouse monoclonal anti-poly-His antibody, and anti-mouse IgG conjugated to 5 nm gold nanoparticles (black arrows, d-f). Empty arrows indicate membranes. Bars 100 μm.
Fig 11.
Effect of myo-IDH PTS1 tripeptide deletion.
A. Detection of N-terminally V5-tagged full-length myo-IDH (IDH-V5) and its C-terminal truncated version (ΔIDH-V5) in the cell lysate (L), organelle (P) and cytosolic (S) fractions using western blot analysis. FeSOD was used as the cytosolic marker. B. Relative expression of IDH-V5 and ΔIDH-V5 using RT-qPCR. The level of native IDH was determined in wild-type cells and used for standardization.
Table 1.
Kinetic parameters of E. histolytica myo-IDH.
Fig 12.
The maximum likelihood tree was inferred with IQ-TREE using 103 protein sequences and 289 positions. Bootstrap support is given at nodes. Sequences with known crystal structures are highlighted in red. Numbers in triangles indicate the number of included sequences. PTS1 triplet is given in brackets.