FIGURE 1.

Pex21p and Pex18p control distinct cargo-specific PTS2 import pathways. A, time course of induction of Gpd1p-TPA, Pex21p-TPA, Fox3p-TPA, and Pex18p-TPA in different media. Yeast cells expressing genomically tagged protein A (TPA)-fusion proteins under control of the endogenous promoters were grown to the log phase and then shifted to YPD medium supplemented with 2% glucose or oleate-containing medium, each with or without 1 m NaCl as indicated. At each time point, samples of equal volumes were analyzed by immunoblotting with antibodies against protein A. Analysis of the glycolytic enzyme 3-phosphoglycerate kinase Pgk1p of YPD grown cells served as loading control. B, composition of Pex21p-TEV-protein A complexes. Yeast cells expressing Pex21p-TEV-protein A from their genome were grown for 16 h in YPD supplemented with 1 m NaCl. Pex21p complexes were affinity-purified from soluble and membrane fractions after 100,000 × g centrifugation of the cell lysate. Membranes were solubilized with digitonin before affinity purification of membrane complexes. Pex21p-associated proteins were eluted by TEV protease cleavage. SDS samples were subjected to immunoblot analyses using antibodies against Pex7p, Pex13p, Pex14p, Fox3p, and Gpd1p.