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First published online 22 July 2003
doi: 10.1242/jcs.00678

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Pex10p links the ubiquitin conjugating enzyme Pex4p to the protein import machinery of the peroxisome

¹ Ruhr-Universität Bochum, Institut für Physiologische Chemie, Medizinische Fakultät, 44780 Bochum, Germany
² Institut für Toxikologie und Genetik, Forschungszentrum Karlsruhe, Postfach 3640, 76021 Karlsruhe, Germany

View larger version (32K): [in a new window]   Fig. 1. (A) The split-ubiquitin technique and its application to the analysis of the membrane-associated peroxins. Cub-RUra3p was linked to the C-terminus of peroxin Y and Nub was linked to Peroxin X. Provided that Pex X and Y interact, the complex brings Nub and Cub 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) Nub and Cub fusions. Upper panel: Nub (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. Cub (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: Nub was fused to the N-terminus of Snc1p, Guk1p, Vam3p, Tom22p, Sec22p, Vam3p, and Ste14p (Wittke et al., 1999). These Nub fusions were used as controls for the specificity of the assay and to verify the correct localization of the Cub fusions of the peroxins. ER, endoplasmic reticulum; Mitoch., mitochondrium; PM, plasma membrane; Vac, vacuole.

View larger version (78K): [in a new window]   Fig. 2. Binding partners of Pex12p. Pex12-Cub-RUra3p containing cells expressing the indicated Nub fusion proteins were grown on SD-media and diluted in water. 4 µl of an OD600 of 1, and of tenfold serial dilutions were spotted onto media either lacking uracil (A) or containing both uracil and 5'-FOA (B) or containing only uracil (C). (D) as in (A) but with cells lacking the gene encoding the recognition component of the N-end rule UBR1. All plates contained 100 µM CuSO4 to induce the PCUP1-promotor and were lacking tryptophan and histidine to select for the plasmids. Cells were incubated at 30°C for 48 hours. Interactions between the Nub and Cub fusions are indicated in the UBR1 cells by the growth of the construct containing cells on 5-FOA and by the non-growth on SD-ura.

View larger version (62K): [in a new window]   Fig. 3. Removal of the C-terminal domain abolishes most of the interactions of Pex12p. Cells containing Pex12C-Cub-RUra3p and expressing the Nub constructs of Pex3p, Pex4p, Pex10p, Pex11p, Pex12p, Pex13p, Pex14p, Pex15p, Pex19p and Pex22p were diluted to an OD600 of 1, and 4 µl of this and of tenfold serial dilutions were spotted onto media containing 100 µM CuSO4 and either lacking uracil (A) or containing both uracil and 5-FOA (B). Both media were lacking tryptophan and histidine to select for the plasmids. Cells were incubated at 30°C for 48 hours.

View larger version (45K): [in a new window]   Fig. 4. Binding partners of Pex10p. (A,B) Cells containing Pex10-Cub-RUra3p and expressing the indicated Nub fusion proteins were diluted to an OD600 of 1, and 4 µl of this and of tenfold serial dilutions were spotted onto media containing 100 µM CuSO4 and either lacking uracil (A) or containing both uracil and 5-FOA (B). Both media were lacking tryptophan and histidine to select for the plasmids. Cells were incubated at 30°C for 48 hours. (C) Yeast cells co-expressing Pex10p tagged with a six-fold repeated ha-epitope (Pex10-6ha) together with either Pex4p, Pex10p, Pex12p, Pex11p or Pex22p, all bearing a ninefold repeated myc-epitope at their C-termini, were subjected to a co-precipitation using the anti-ha-antibody to precipitate and the anti-MYC antibody to detect the co-precipitated protein after SDS-PAGE and transfer to nitrocellulose (C). (D) as in (C) but using anti-myc antibody for precipitation and anti-ha antibody for the detection of the co-precipitated Pex10-6ha. The asterisk indicates the heavy chain of the antibody used for the precipitation.

View larger version (70K): [in a new window]   Fig. 5. Binding partners of Pex2p and Pex11p. Yeast cells containing Pex2-Cub-RUra3p (A,B) or Pex11-Cub-RUra3p (C,D) and expressing the indicated Nub-fusion proteins were diluted to an OD600 of 1, and 4 µl of this and of tenfold serial dilutions were spotted onto media containing 100 µM CuSO4 and either lacking uracil (A,C) or containing both uracil and 5-FOA (B,D). Both media were lacking tryptophan and histidine to select for the plasmids. Cells were incubated at 30°C for 48 hours.

View larger version (13K): [in a new window]   Fig. 8. Summary of the found peroxin interactions. The numbers represent the corresponding peroxins. T22 stands for Tom22p. The bars connecting the circles indicate interactions between the proteins in the split-Ub assay. A plus indicates an interaction that is detected only in the presence of the gene; a minus indicates an interaction that is only detected in the absence of the gene. The bars enclose the multiple interactions of a single peroxin. The numbers accompanying the bars specify the interactions in a certain pex deletion strain. Brackets enclosing a number indicate that the interaction occurs independently of the presence of the respective PEX gene. *Except Pex4p, Pex5p; **except Pex3p, Pex4p; ***measured only for Pex10p, Pex12p.

View larger version (35K): [in a new window]   Fig. 6 . The interaction between Pex10p and Pex4p depends on the presence of Pex22p. (A) PEX5/PEX22 cells, (B) pex5/PEX22 cells and (C) PEX5/pex22 cells containing Pex10-Cub-RUra3p and expressing the Nub constructs of Pex4p, Pex10p, Pex13p, Pex15p, Pex19p, Tom22p, Guk1p and the empty plasmid were diluted to an OD600 of 1, and 4 µl of this and of tenfold serial dilutions were spotted onto media containing 100 µM CuSO4 and 5-FOA. The media lacked tryptophan and histidine to select for the plasmids. Cells were incubated at 30°C for 48 hours. (D) as (C) but cells contained Pex4-Cub-RUra3p and expressed Nub-Pex4p or the empty vector in wild-type or pex22 cells. (E) The Pex4p-Pex4p interaction is specific. Cells co-expressing Pex4-Cub-RUra3p and the Nub-constructs of Pex4p, Ubc6p, Guk1p or the empty vector were analyzed as in (A-C). (F) Yeast cells co-expressing Pex4-6ha together with either Pex4-9myc or Pex11-9myc were subjected to anti-ha precipitation after DSP crosslinking. The co-precipitated proteins were detected with anti-myc antibody after reverting the cross-link, 12.5% SDS-PAGE, and transfer onto nitrocellulose. The blot was stripped and re-probed with anti-ha-antibody to ensure that similar quantities of Pex4-6ha had been loaded (boxed).

View larger version (48K): [in a new window]   Fig. 7. Analysis of peroxin interactions in the absence of peroxisomal membranes. pex3 cells (A,B,E) containing Pex12-Cub-RUra3p (A), Pex10-Cub-RUra3p (B) or Pex11-Cub-RUra3p (E) and expressing the indicated Nub-fusions or the empty plasmid were diluted to an OD600 of 1, and 4 µl of this and of tenfold serial dilution were spotted onto media containing 100 µM CuSO4 and 5-FOA. The media lacked tryptophan and histidine to select for the plasmids. The cells were incubated at 30°C for 48 hours. (C) and (D) as in (E) but with wild-type cells (C) or pex11 cells (D).

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