Pex18p and Pex21p interact with Pex7p in the yeast two-hybrid assay. Host strain PJ69-4A was transformed with two plasmids, one encoding the Gal4p transactivation domain (TA) alone (−) or fused to either residues 108–283 of Pex18p (Pex18), residues 155-288 of Pex21p (Pex21), or full-length thiolase (Thio), and the other encoding the DNA binding domain (BD) of Gal4p fused to full-length Pex7p (Pex7), full-length thiolase (Thio) or thiolase lacking the NH₂-terminal 16 amino acids (ThioΔ1-16). Transformants were tested for growth on synthetic complete medium with (+Ade +His) or without adenine (−Ade) or histidine (−His). Total cell homogenates were also prepared, and assayed for β-galactosidase activity (β-Gal), which is expressed in nmol/min/mg protein. − = <0.5 units of activity. Some experiments were performed using PJ69-4A from which the PEX7, POT1, PEX13, PEX14, or PEX18/PEX21 genes had been deleted (PJ69-4AΔpex7, PJ69-4AΔpot1, PJ69-4AΔpex13, PJ69-4AΔpex14, and PJ69-4AΔpex18Δpex21, respectively). PJ69-4AΔpex7 and PJ69-4AΔpot1 transformants were not tested for activation of the GAL1-HIS3 marker, since these strains had been converted to His⁺ during gene disruption.

Pex18p and Pex21p interact with Pex7p in vitro. Bacterial extracts from E. coli BL21 (DE3) cells overexpressing His₆-tagged Pex18p (Pex18), Pex21p (Pex21), or neither (−) were first applied to His₆-affinity columns, which were then loaded with glass bead homogenates of yeast expressing epitope tagged Pex7p (plasmid pBXNPEB1-HA3 in Zhang and Lazarow [1994], now referred to as pBXNPEX7-HA3). The unbound yeast proteins were recovered and, after extensive washing, the bound proteins were eluted together with the His₆-tagged Pex18p/Pex21p. Aliquots of the unbound and bound fractions were resolved in duplicate by SDS PAGE, and subjected to (A) Coomassie blue staining, which detected the eluted Pex18p (which migrates as a dimer) and Pex21p, and (B) immunoblotting with monoclonal antibody 12CA5 to detect the epitope-tagged Pex7p. An abundant unidentified yeast protein that adheres to the columns even in the absence of His₆-tagged bait is marked by an asterisk.

Bestfit alignment of the protein sequences of Pex18p and Pex21p. The arrowheads indicate the fusion junctions between Gal4p transactivation domain and COOH-terminal regions of Pex18p and Pex21p of the clones isolated from two-hybrid libraries with Pex7p bait.

Growth on oleic acid and packaging of thiolase into peroxisomes are abolished in cells lacking both Pex18p and Pex21p, but are supported fully by Pex18p alone, and partially by Pex21p alone. (A) Growth on plates. Cells were grown to mid-log phase in liquid synthetic complete medium, and then applied at various dilutions (10⁵, 10⁴, or 10³ cells per spot) to plates containing synthetic complete medium with either oleic acid (SC-OLEATE) or dextrose (SC-DEXTROSE) as sole carbon source, and grown at 30°C. The bottom two panels show the double knockout strain transformed with centromeric plasmids encoding epitope tagged Pex18p (pYcp-PEX18) or Pex21p (pYcp-PEX21). (B) Growth in liquid oleate media. Cells were precultured to mid-log phase in SCD medium and then inoculated at a density of 10⁵ cells/ml into liquid SCO. (C) Fluorescence and cell fractionation. Wild-type cells, and cells lacking Pex18p and/ or Pex21p, were grown for peroxisome induction, and analyzed by immunofluorescence with antibodies against thiolase (top row) and catalase A (third row). These same strains were transformed with plasmids expressing chloramphenicol acetyl transferase (ChAT) appended with the PTS1 −KANL (Purdue and Lazarow, 1996), or a fusion between the thiolase PTS2 and GFP (PTS2-GFP; Huang and Lazarow, 1996), or a fusion between acyl-CoA oxidase and GFP (AOX-GFP), and analyzed with anti-ChAT antibodies (fourth row), or by GFP autofluorescence (second and fifth rows). Induction was with YPGO in all cases except for PTS2-GFP fluorescence, when SCEO was used. For fractionation, cells were grown for peroxisome induction in YPGO, and fractionated into postnuclear pellet (P) and supernatant (S) fractions, which were run in equivalent proportions on SDS-polyacrylamide gels, and immunoblotted to detect thiolase and the β-oxidation multifunctional protein (MFP). All the strains are in a W303 genetic background.

Peroxisomal targeting of Pex7p is somewhat decreased in cells lacking Pex18p, and almost abolished in cells lacking both Pex18p and Pex21p. (A) Strains overexpressing epitope tagged Pex7p (plasmid pBXNPEB1-HA3 in Zhang and Lazarow [1994], now referred to as pBXNPEX7-HA3) were fractionated into postnuclear pellet (P) and supernatant (S) fractions, which were then analyzed by SDS-polyacrylamide gels and immunoblotting with monoclonal antibody 12CA5 (to detect the tagged Pex7p) and antibodies against the β-oxidation multifunctional protein (MFP). (B) Immunoprecipitation of epitope tagged Pex7p expressed at wild-type levels (plasmid YipPEB1-HA3 in Zhang and Lazarow [1994], now referred to as YipPEX7-HA3). Total cell extracts (T) and organellar pellets (P) from YPGO-induced cultures of W303, W303-expressing tagged Pex7p, and W303Δpex18Δpex21-expressing tagged Pex7p were separated on SDS-polyacrylamide gels, and immunoblotted with monoclonal antibody 12CA5. (C) Strains overexpressing epitope tagged Pex7p were analyzed by immunofluorescence with 12CA5.

Pex18p and Pex21p are functional when COOH terminally tagged with HA epitopes, and localize primarily to the cytosol. Pex18p is partially peroxisomal. (A) Cells lacking both Pex18p and Pex21p (Δpex18Δpex21)before and after transformation with low copy (pYcp-PEX18, pYcp-PEX21) or high copy (pYep-PEX18, pYep-PEX21) plasmids encoding HA epitope-tagged Pex18p or Pex21p were analyzed by immunofluorescence with anti-thiolase (top panels) and 12CA5 (detects the HA-tagged proteins; bottom panels). (B) Wild-type cells expressing epitope-tagged Pex18p from low copy (pYcp-PEX18) and high copy (pYep-PEX18) plasmids were fractionated into organellar pellet (P) and supernatant (S) fractions, which were run in equivalent proportions on SDS-polyacrylamide gels, and immunoblotted with monoclonal 12CA5 to detect Pex18p (top), and anti-thiolase (bottom). (C) The organellar pellet from wild-type W303 cells transformed with plasmid pYep-PEX18 was further fractionated by Nycodenz gradient centrifugation. Fractions were run on a polyacrylamide gel and immunoblotted to detect Pex18p (monoclonal 12CA5), and to detect thiolase, ADP/ATP carrier and Kar2p, which are markers for the peroxisomes, mitochondria and endoplasmic reticulum, respectively. The fractions are labeled from top to bottom of the gradient.

Protease protection analysis shows that peroxisomal Pex18p is primarily located on the external face of the peroxisomal membrane. An organellar fraction of wild-type cells overexpressing HA-tagged Pex18p was digested with a final concentration of 5 μg/ml Proteinase K-agarose in the presence and absence of 0.1% Triton X-100. Reactions were stopped with 1 mM PMSF after various times, and the samples were separated by SDS-PAGE, and immunoblotted to detect thiolase, a known matrix protein, and the tagged Pex18p. A proteolytic thiolase digestion product is indicated by an asterisk.