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Fig. 3. Targeting of PEX26 is dependent on its PEX19-binding site. (A) Identification of an mPTS in PEX26. Human fibroblasts were transiently transfected with plasmids designed to express GFP fusions of the indicated PEX26 fragments. Thereafter, cells were processed for indirect immunofluorescence using polyclonal anti-PEX14 antibodies. Merged images reveal eventual colocalization of the GFP fusion proteins with peroxisomal PEX14 (Px). (B) The C-terminal PEX19-binding site of PEX26 functions as a PMP-targeting motif. A GFP fusion of a chimera of PEX26_275-305 and ALDP_87-164 was similarly analyzed for subcellular location. In this chimera, the targeting motif is contributed by the PEX26 fragment, whereas the transmembrane segment that is required for efficient membrane insertion is contributed by the ALDP fragment. GFP fusions of the individual fragments (PEX26_275-305 and ALDP_87-164) served as controls. (C) Function of the TMD-containing PEX19-binding site of PEX26 as a PMP targeting signal. A GFP fusion of PEX26_245-274 was inspected for colocalization with PEX14. This transmembrane segment with PEX19 binding capability suffices for peroxisomal targeting. Bar, 10 µm.

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