Fig. 1. (A) The split-ubiquitin technique and its application to the analysis of the membrane-associated peroxins. C_ub-RUra3p was linked to the C-terminus of peroxin Y and N_ub was linked to Peroxin X. Provided that Pex X and Y interact, the complex brings N_ub and C_ub into close proximity and the Ub-halves will reconstitute the native-like Ub (1). The Ub-specific proteases will recognize the reconstituted Ub and cleave off the attached RUra3p. The released RUra3p is targeted for rapid destruction by the enzymes of the N-end rule (2) to yield cells that are uracil auxotrophs and 5-FOA resistant. (B) N_ub and C_ub fusions. Upper panel: N_ub (residues 1-36 of Ub) was fused to the N-terminus of Pex1p, Pex3p, Pex4p, Pex5p, Pex10p, Pex11p, Pex13p, Pex14p, Pex15p, Pex19p, the peroxisomal targeting sequence SKL and the control sequence SSS, to the N- and C-terminus of Pex12p and to the C-terminus of Pex22p. C_ub (residues 35-76 of Ub) was attached to the C-terminus of Pex2p, Pex3p, Pex4p, Pex5p, Pex10p, Pex11p, Pex12p, Pex13p, Pex14p, Pex17p, and Pex22p. Shown are the localization and assumed topologies of the different peroxins. Note that some of the shown topologies are not yet conclusively proven. During the course of the work and in accordance with the results of our assay, the N-terminus of Pex12p was shown to point into the peroxisomal matrix (Albertini et al., 2001). The assay requires that both halves of Ub had to point into the cytosol of the cell. Lower panel: N_ub was fused to the N-terminus of Snc1p, Guk1p, Vam3p, Tom22p, Sec22p, Vam3p, and Ste14p (Wittke et al., 1999). These N_ub fusions were used as controls for the specificity of the assay and to verify the correct localization of the C_ub fusions of the peroxins. ER, endoplasmic reticulum; Mitoch., mitochondrium; PM, plasma membrane; Vac, vacuole.