(A) Scheme of S. cerevisiae Pex5p. Pex5p contains an N-terminal domain (1–312) with interaction sites required for protein transport and a C-terminal domain (313–612) with six TPR domains (1–6) responsible for the binding of PTS1-proteins. The extreme N-terminus contains the target amino acids for mono- (Cys6) and polyubiquitination (Lys18 and Lys24) highlighted in red. (B) Indicated strains were spotted as a series of 10-fold dilutions on a medium with oleic acid as sole carbon source and incubated for 4 days at 30°C. Mutant pex5Δ and pex5Δ expressing Pex5pC6A were unable to grow on oleic acid medium. In contrast, the mutant growth phenotype was complemented upon expression of Pex5p, Pex5pK18R/K24R and Pex5pC6K, which display a growth behaviour similar to the wild-type. (C) Oleic acid-induced indicated strains were analysed for the subcellular localization of the transformed PTS1-marker DsRed-SKL by fluorescence microscopy. Mutant pex5Δ and pex5Δ expressing Pex5pC6A exhibited an overall cellular fluorescence, indicative for a mislocalization of the peroxisomal marker protein as consequence of a peroxisomal import defect. Mutant cells expressing wild-type Pex5p or mutant Pex5pK18R/K24R or Pex5pC6K imported PTS1-proteins properly as indicated by the punctate fluorescence pattern. Scale bar=5 μm