Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes

Abstract

Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (Saccharomyces cerevisiae and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane. In this report, we show that the specificity of peroxisomal membrane protein binding for Trypanosoma brucei PEX19 is very similar to those previously identified for human and yeast PEX19. Our studies show that trafficking is conserved across these distant phyla and that both a PEX19 binding site and a transmembrane domain are required for the insertion of two test proteins into the glycosomal membrane. However, in contrast to T. brucei PEX10 and PEX12, T. brucei PEX14 does not traffic to human peroxisomes, indicating that it is not recognized by the human PEX14 import mechanism.

FIG. 1.

PEX19 is essential to blood form T. brucei. (A) Growth analysis of the TbPEX19 RNAi cell line. Bloodstream stage cells containing an RNAi construct targeting PEX19 RNA were induced with Tet in triplicate and counted by Coulter counter daily. The cumulative number of particles (cells plus any dead cells of similar size) is indicated on the y axis. Individual measurements are shown. Because the data were very reproducible, some time points appear to have fewer replicates. The inset shows results from Northern analysis of the RNAi line with (+) or without (−) Tet to induce RNAi. (B) IFA of cells induced (+) for RNAi after 48 h or uninduced controls (−). Cells were stained with anti-glycosome antibody which detects several matrix proteins. The induced cells were heterogeneous (see the text). A cell exhibiting punctate staining and a cell from the same culture exhibiting diffuse staining are shown. Bar, 2 μm.

FIG. 2.

Targeting of HsALDP domain fusions in T. brucei. GFP-HsALDP fusion proteins containing the TMDs (aa 87 to 164), the PEX19 binding site (aa 66 to 87), or both (aa 66 to 164) were expressed in T. brucei procyclic cells. (A) Immunoblot analysis of GFP fusion proteins showing migration appropriate to the predicted sizes of ∼37 kDa for GFP-HsALDP(87-164), ∼31 kDa for GFP-HsALDP(66-87), and ∼40 kDa for GFP-HsALDP(66-164). The migration of molecular mass markers is indicated. Tet induces the expression of the fusion proteins (+Tet). (B) Localization of fusion proteins expressed in T. brucei by fluorescence microscopy and cellular fractionation. GFP was visualized by intrinsic fluorescence, while glycosomes were revealed with rabbit anti-glycosome antibody followed by anti-rabbit immunoglobulin G coupled to Texas Red. Bar, 2 μm. For immunoblots, the fractions were analyzed using anti-GFP antibody to detect the fusion protein and anti-phosphoglycerate kinase (PGK) antibody, which detects both a cytosolic (C) and a glycosomal (G) isoform. Lanes are loaded by cell equivalents. T, total lysate; DS, digitonin supernatant containing cytosolic proteins; CS, carbonate supernatant containing organellar matrix and peripheral membrane proteins; CP, carbonate pellet containing integral membrane proteins.

FIG. 3.

Substitution analysis of the PEX19 binding site in ALDP. Replicate peptide arrays harboring the 13-amino-acid PEX19 binding peptide of HsALDP (FLQRLLWLLRLLF) as well as peptides with single amino acid substitutions thereof were tested for interaction with GST fused to human PEX19 (upper panel) or T. brucei PEX19 (middle panel) or with GST alone (bottom panel). The first column has replicate peptides with no substitutions. Subsequent columns have peptides in which each residue is replaced individually by the amino acid indicated at the top of the column (e.g., column 2 has peptides with Ala substitutions). Bound PEX19 was visualized by monoclonal anti-GST antibodies in combination with the enhanced chemiluminescence reaction system. Spots of reduced intensity reflect peptides with a decreased binding affinity for PEX19. Similar binding patterns were observed for both PEX19 proteins.

FIG. 4.

Identification of PEX19 binding sites in PMPs from trypanosomes. (A) Peptide scans for predicted PEX19 binding sites. The regions predicted to contain PEX19 binding sites were synthesized as stretches of nine overlapping 15-mer peptides with two amino acid shifts between neighboring peptides and tested for interaction with a GST fusion of TbPEX19. The central peptide represents the actual peak scoring peptide as listed in Table 1. (B) Binding site prediction for TbPEX10. The hits obtained with the PEX19 binding site prediction matrix for TbPEX10 are presented as dotted vertical lines. Two sites with a high probability score (in arbitrary units [a.u.]) are visible, with the more C-terminal site showing a cluster of hits around a peak scoring peptide, the position and amino acid sequence of which is indicated. (C) Peptide scan for TbPEX10. Peptides (15-mers) with three amino acid shifts between neighboring peptides (rather than the two amino acid shifts for Fig. 4A) and covering the entire protein were synthesized on a nitrocellulose membrane and tested for interaction with GST fusions of PEX19 from T. brucei (upper panel) and Homo sapiens (lower panel). For TbPEX19, two binding sites were identified (indicated by the arrows), one of which was also recognized by HsPEX19. Grid intersections mark the center region for each separate peptide on the array.

FIG. 5.

Evolutionarily conserved PEX19 binding site-dependent targeting of TbPEX10. Full-length GFP-TbPEX10 and various truncations (cartooned at left of panels) were expressed in human or T. brucei procyclic cells to assess the function of the TbPEX10 targeting signal. Immunoblot analysis of the T. brucei transfectants are shown for each construct, either uninduced (−) or induced (+) for expression of the fusion protein. These blots demonstrate migration to the expected molecular masses of ∼59 kDa for GFP-TbPEX10(Δ173-190), ∼42 kDa for GFP-TbPEX10(1-124), ∼48 kDa for GFP-TbPEX10(113-299), and ∼45 kDa for GFP-TbPEX10(140-299). The full-length construct in T. brucei was previously verified (45). (Left three panels) GFP fusion proteins were expressed in human fibroblasts by transient transfection with appropriate expression plasmids. Cells were processed for the IFA using polyclonal anti-PEX14 antibodies. Merged images reveal predominant colocalization of the GFP fusion proteins with peroxisomal PEX14 (Px). Bar, 10 μm. (Right three panels) GFP fusion constructs were transfected into T. brucei procyclic cells to create stable cell lines. Induction with Tet was followed by the IFA at 24 h, as described for Fig. 2. Bar, 2 μm.

FIG. 6.

Targeting of TbPEX12 and TbPEX14 in human fibroblasts. (A) Conserved targeting of TbPEX12. Human fibroblasts were transiently transfected with a plasmid designed to express a GFP fusion of TbPEX12. Thereafter, cells were processed for indirect immunofluorescence using polyclonal anti-PEX14 antibodies. The merged image reveals colocalization of the GFP-TbPEX12 with endogenous peroxisomal PEX14 (Px). Bar, 10 μm. The three panels on the right show T. brucei procyclic forms expressing the same GFP fusion protein, analyzed as in Fig. 2, at 24 h postinduction. Bar, 2 μm. (B) Localization of GFP-TbPEX14 was similarly analyzed in human and trypanosome cells, except that peroxisomes were visualized with anti-PMP70 antibodies. No colocalization of GFP-TbPEX14 and PMP70 is observed in the merged image, although the fusion protein is glycosomal in T. brucei.