Figure 2.

Functional complementation of PTS2-mediated peroxisomal protein import by PEX7 alleles. Ten different PEX7 alleles identified in the patients were coexpressed with PTS2-tagged GFP to test their ability to restore PTS2-mediated peroxisomal protein import in skin fibroblasts from a patient homozygous for the L292X mutation. A, Expression of control PEX7 resulted in punctate peroxisomal fluorescence in >90% of GFP-expressing cells, with 20%–40% of the cells showing cytosolic fluorescence in addition to punctate fluorescence. B, None of the eight alleles derived from patients with severe RCDP (see Subjects and Methods) were able to complement the PTS2-mediated protein import defect, and PTS2-tagged GFP fluorescence was invariably cytosolic (the L70W allele, which is representative for all other seven alleles, is shown). C and D, Expression of the 8-nt duplication PEX7 allele resulted in punctate peroxisomal fluorescence in 90% of the cells. In 40%–60% of the GFP-expressing cells, however, cytosolic fluorescence and punctate fluorescence were evident, indicating that complementation by this allele is less efficient than complementation by the control allele. E and F, Expression of the H285R PEX7 allele resulted in punctate peroxisomal fluorescence in 50%–70% of GFP-expressing cells, but this was always against a background of cytosolic fluorescence; no cells were found in which fluorescence was exclusively punctate.