Figure 1.
The Pex3(1–45)-Pex4p fusion protein binds the soluble Pex22(C)p-fragment at peroxisomes.
(a) Schematic topological representations of full-length Pex4p and the applied chimeric and truncated versions of Pex4p, Pex3p and Pex22p. The luminal domains are shown in orange, the trans-membrane domains (TMD) in red, the cytosolic parts in blue and GFP (green fluorescent protein) in green. The membrane protein targeting signal (mPTS), the ubiquitin-conjugating domain (UBC) as well as numbers of important amino acid positions are denoted. Note: While the domains of Pex22p, Pex4p and Pex3p are depicted at the same scale, the size of GFP (green fluorescent protein) is reduced in this model. (b) Peroxisomal targeting of a chimeric Pex3(1–45)-Pex4p. Intracellular localization of chimeric Pex4p in wild-type and indicated pex-mutant cells is monitored by fluorescence microscopy. Pex4p is usually anchored to peroxisomes via binding to the C-terminal part of the peroxisomal membrane protein Pex22p. Colocalization of the GFP-tagged chimeric Pex3p(1–45)-Pex4p with the DsRed-tagged peroxisomal membrane marker Ant1p indicates that it localized to peroxisomes in all strains.
Figure 2.
The function of Pex22(C)p is essential for peroxisome biogenesis, while Pex22(N)p is dispensable.
(a) Peroxisomal function was tested by analyzing the capability of the plasmid-encoded constructs to complement the mutant growth phenotype of corresponding deletion strains. To this end, the Pex3(N)-Pex4p, Pex22(C)p and/or Pex22p were expressed in the pex4Δpex22Δ, pex4Δ or pex22Δ deletion strains, as indicated. The optical density at 600 nm (OD600) of cells grown in oleate medium was monitored (n = 3 experiments) and results are presented in relation to growth of the wild-type, which is set as 100% (+/−standard error of the mean). The corresponding OD600 at the end of the growth period is depicted. While Pex3(N)-Pex4p can complement the growth defect of the pex4Δ strain, it is only capable to regain functionality in the pex4Δpex22Δ background, when Pex22(C)p is present. (b) Peroxisomal function was tested by analyzing the capability of the plasmid-encoded constructs to complement the peroxisomal protein import defect of corresponding deletion strains. Plasmid-encoded DsRed-PTS1 served as marker for peroxisomal protein import. Transformed cells were grown on oleic acid plates for two days and examined by fluorescence microscopy. The wild-type cells as well as the pex4Δ strain carrying the Pex3(N)-Pex4p plasmid displayed a punctate pattern, indicating that DsRed-PTS1 cargo is imported into peroxisomes and which is the typical appearance of cells with intact PTS1-dependent matrix protein import. The non-transformed pex4Δ strain as well as the pex4Δpex22Δ strain with Pex3(N)-Pex4p displayed a cytosolic staining of the DsRed-signal, demonstrating that the marker is mislocalized to the cytosol due to a block of import. The pex4Δpex22Δ strain expressing both Pex3(N)-Pex4p and Pex22(C)p displayed a heterogenous phenotype of a partial cytosolic mislocalization but also detectable import of DsRed-PTS1 into peroxisomes. (c) The lysates of oleate-induced cells were analyzed for the level of Pex22(C)p. The detected level of Pex22(C)p was the same in both the strain with partial functional complementation (pex4Δpex22Δ+Pex3(N)-Pex4p+Pex22(C)p) and the strain without functional complementation (pex22Δ+Pex22(C)p). The asterisk denotes a cross-reaction signal.
Figure 3.
Pex22(C)p enhances autoubiquitination of Pex4p and Pex4p/Pex12p-catalyzed ubiquitination of the PTS1-receptor in vitro.
(a) Autoubiquitination of GST-Pex4p. The effect of Pex22(C)p on the autoubiquitination of GST-Pex4p was examined in vitro using heterologously expressed and purified proteins as indicated. Autoubiquitination of GST-Pex4p depends on the presence of E1 and ubiquitin (Ub). As shown by immunoblot analysis with antibodies against GST and Ub, the autoubiquitination of GST-Pex4p is significantly enhanced in the presence of His-Pex22(C)p. (b) Ubiquitination of the PTS1-receptor Pex5p. Recombinant proteins were used for in vitro ubiquitination of Pex5p, which depends on the presence of E1, GST-Pex4p, Ub as well as the RING-domain of the E3 enzyme Pex12p and was significantly enhanced in presence of His-Pex22(C)p.
Figure 4.
Pex22(C)p reduces polyubiquitination and stimulates monoubiquitination of the PTS1-receptor Pex5p in vivo.
(a) It is known that the inhibition of the export of Pex5p by the deletion of PEX4 and/or PEX22 induces polyubiquitination and proteasomal degradation of Pex5p. The PEX4/PEX22-deletion induced polyubiquitination of Pex5p can be significantly reduced when the cells express Pex3(N)-Pex4p in combination with Pex22(C)p. (b) The Pex3(N)-Pex4p chimera is capable to monoubiquitinate Pex5p in vivo. The level of monoubiquitinated Pex5p is reduced in the absence of Pex22p, but is restored in the presence of Pex22(C)p.
Figure 5.
The stimulating effect of Pex22(C)p on Pex5p ubiquitination and peroxisomal function cannot be enforced or replaced by Pex22(N)p.
(a) Schematic representation of Pex22(N)-Pex4p. The Pex22(aa1–35)-fragment contains the transmembrane domain (TMD) in red and the intraperoxisomal part in orange fused to full-length Pex4p (blue) as well as green fluorescent protein (green). (b) Pex22(N)-Pex4p-GFP is targeted to peroxisomes. The Pex22(N)-Pex4p-GFP construct was introduced in wild-type, pex4Δ and pex4Δpex22Δ cells as indicated and co-localization of the chimeric protein with the peroxisomal membrane marker DsRed-Ant1p was analyzed under by fluorescence microscopy. (c) The functionality of indicated constructs was tested by analysis of their capability to complement the growth defect of depicted strains. To this end, the Pex3(N)-Pex4p and/or Pex22(C)p or Pex22p encoding information was expressed in the pex4Δpex22Δ, pex4Δ or pex22Δ deletion strains, as indicated. The optical density (OD600) of cells grown in oleate medium was monitored (n = 3 experiments) and the results are presented in % in comparison to the wild-type (+/−standard error of the mean). While Pex22(N)-Pex4p complements the growth defect of the pex4Δ strain, it is only capable to regain functionality in the pex4Δpex22Δ background, when Pex22(C)p is present. (d) The Pex22(N)-Pex4p fusion protein is capable to monoubiquitinate Pex5p in vivo. In the absence of full-length Pex22p, the level of monoubiquitinated Pex5p is significantly reduced, which is compensated in the presence of Pex22(C)p.