The cytosolic domain of Pex22p stimulates the Pex4p-dependent ubiquitination of the PTS1-receptor

Peroxisomal biogenesis is an ubiquitin-dependent process because the receptors required for the import of peroxisomal matrix proteins are controlled via their ubiquitination status. A key step is the monoubiquitination of the import receptor Pex5p by the ubiquitin-conjugating enzyme (E2) Pex4p. This monoubiquitination is supposed to take place after Pex5p has released the cargo into the peroxisomal matrix and primes Pex5p for the extraction from the membrane by the mechano-enzymes Pex1p/Pex6p. These two AAA-type ATPases export Pex5p back to the cytosol for further rounds of matrix protein import. Recently, it has been reported that the soluble Pex4p requires the interaction to its peroxisomal membrane-anchor Pex22p to display full activity. Here we demonstrate that the soluble C-terminal domain of Pex22p harbours its biological activity and that this activity is independent from its function as membrane-anchor of Pex4p. We show that Pex4p can be functionally fused to the trans-membrane segment of the membrane protein Pex3p, which is not directly involved in Pex5p-ubiquitination and matrix protein import. However, this Pex3(N)-Pex4p chimera can only complement the double-deletion strain pex4Δ/pex22Δ and ensure optimal Pex5p-ubiquitination when the C-terminal part of Pex22p is additionally expressed in the cell. Thus, while the membrane-bound portion Pex22(N)p is not required when Pex4p is fused to Pex3(N)p, the soluble Pex22(C)p is essential for peroxisomal biogenesis and efficient monoubiquitination of the import receptor Pex5p by the E3-ligase Pex12p in vivo and in vitro. The results merge into a picture of an ubiquitin-conjugating complex at the peroxisomal membrane consisting of three domains: the ubiquitin-conjugating domain (Pex4p), a membrane-anchor domain (Pex22(N)p) and an enhancing domain (Pex22(C)p), with the membrane-anchor domain being mutually exchangeable, while the Ubc- and enhancer-domains are essential

The Peroxisomal PTS1-Import Defect of PEX1- Deficient Cells Is Independent of Pexophagy in Saccharomyces cerevisiae

The important physiologic role of peroxisomes is shown by the occurrence of peroxisomal biogenesis disorders (PBDs) in humans. This spectrum of autosomal recessive metabolic disorders is characterized by defective peroxisome assembly and impaired peroxisomal functions. PBDs are caused by mutations in the peroxisomal biogenesis factors, which are required for the correct compartmentalization of peroxisomal matrix enzymes. Recent work from patient cells that contain the Pex1(G843D) point mutant suggested that the inhibition of the lysosome, and therefore the block of pexophagy, was beneficial for peroxisomal function. The resulting working model proposed that Pex1 may not be essential for matrix protein import at all, but rather for the prevention of pexophagy. Thus, the observed matrix protein import defect would not be caused by a lack of Pex1 activity, but rather by enhanced removal of peroxisomal membranes via pexophagy. In the present study, we can show that the specific block of PEX1 deletion-induced pexophagy does not restore peroxisomal matrix protein import or the peroxisomal function in beta-oxidation in yeast. Therefore, we conclude that Pex1 is directly and essentially involved in peroxisomal matrix protein import, and that the PEX1 deletion-induced pexophagy is not responsible for the defect in peroxisomal function. In order to point out the conserved mechanism, we discuss our findings in the context of the working models of peroxisomal biogenesis and pexophagy in yeasts and mammals

Regulation of the Tumor-Suppressor BECLIN 1 by Distinct Ubiquitination Cascades

Autophagy contributes to cellular homeostasis through the degradation of various intracellular targets such as proteins, organelles and microbes. This relates autophagy to various diseases such as infections, neurodegenerative diseases and cancer. A central component of the autophagy machinery is the class III phosphatidylinositol 3-kinase (PI3K-III) complex, which generates the signaling lipid phosphatidylinositol 3-phosphate (PtdIns3P). The catalytic subunit of this complex is the lipid-kinase VPS34, which associates with the membrane-targeting factor VPS15 as well as the multivalent adaptor protein BECLIN 1. A growing list of regulatory proteins binds to BECLIN 1 and modulates the activity of the PI3K-III complex. Here we discuss the regulation of BECLIN 1 by several different types of ubiquitination, resulting in distinct polyubiquitin chain linkages catalyzed by a set of E3 ligases. This contribution is part of the Special Issue “Ubiquitin System”

Regulation of the tumor-suppressor BECLIN 1 by distinct ubiquitination cascades

Autophagy contributes to cellular homeostasis through the degradation of various intracellular targets such as proteins, organelles and microbes. This relates autophagy to various diseases such as infections, neurodegenerative diseases and cancer. A central component of the autophagy machinery is the class III phosphatidylinositol 3-kinase (PI3K-III) complex, which generates the signaling lipid phosphatidylinositol 3-phosphate (PtdIns3P). The catalytic subunit of this complex is the lipid-kinase VPS34, which associates with the membrane-targeting factor VPS15 as well as the multivalent adaptor protein BECLIN 1. A growing list of regulatory proteins binds to BECLIN 1 and modulates the activity of the PI3K-III complex. Here we discuss the regulation of BECLIN 1 by several different types of ubiquitination, resulting in distinct polyubiquitin chain linkages catalyzed by a set of E3 ligases. This contribution is part of the Special Issue "Ubiquitin System"

Vac8 controls vacuolar membrane dynamics during different autophagy pathways in Saccharomyces cerevisiae\textit {Saccharomyces cerevisiae}

The yeast vacuole is a vital organelle, which is required for the degradation of aberrant intracellular or extracellular substrates and the recycling of the resulting nutrients as newly available building blocks for the cellular metabolism. Like the plant vacuole or the mammalian lysosome, the yeast vacuole is the destination of biosynthetic trafficking pathways that transport the vacuolar enzymes required for its functions. Moreover, substrates destined for degradation, like extracellular endocytosed cargoes that are transported by endosomes/multivesicular bodies as well as intracellular substrates that are transported via different forms of autophagosomes, have the vacuole as destination. We found that non-selective bulk autophagy of cytosolic proteins as well as the selective autophagic degradation of peroxisomes (pexophagy) and ribosomes (ribophagy) was dependent on the armadillo repeat protein Vac8 in $\textit {Saccharomyces cerevisiae}$. Moreover, we showed that pexophagy and ribophagy depended on the palmitoylation of Vac8. In contrast, we described that Vac8 was not involved in the acidification of the vacuole nor in the targeting and maturation of certain biosynthetic cargoes, like the aspartyl-protease Pep4 (PrA) and the carboxy-peptidase Y (CPY), indicating a role of Vac8 in the uptake of selected cargoes. In addition, we found that the hallmark phenotype of the $\it vac\Delta$ strain, namely the characteristic appearance of fragmented and clustered vacuoles, depended on the growth conditions. This fusion defect observed in standard glucose medium can be complemented by the replacement with oleic acid or glycerol medium. This complementation of vacuolar morphology also partially restores the degradation of peroxisomes. In summary, we found that Vac8 controlled vacuolar morphology and activity in a context- and cargo-dependent manner

The stimulating effect of Pex22(C)p on Pex5p ubiquitination and peroxisomal function cannot be enforced or replaced by Pex22(N)p.

<p>(<b>a</b>) Schematic representation of Pex22(N)-Pex4p. The Pex22(aa1–35)-fragment contains the transmembrane domain (TMD) in <i>red</i> and the intraperoxisomal part in <i>orange</i> fused to full-length Pex4p (<i>blue</i>) as well as green fluorescent protein (<i>green</i>). (<b>b</b>) Pex22(N)-Pex4p-GFP is targeted to peroxisomes. The Pex22(N)-Pex4p-GFP construct was introduced in wild-type, <i>pex4</i>Δ and <i>pex4</i>Δ<i>pex22</i>Δ cells as indicated and co-localization of the chimeric protein with the peroxisomal membrane marker DsRed-Ant1p was analyzed under by fluorescence microscopy. (<b>c</b>) 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 <i>pex4</i>Δ<i>pex22</i>Δ, <i>pex4</i>Δ <i>or pex22</i>Δ 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 <i>pex4</i>Δ strain, it is only capable to regain functionality in the <i>pex4</i>Δ<i>pex22</i>Δ background, when Pex22(C)p is present. (<b>d</b>) The Pex22(N)-Pex4p fusion protein is capable to monoubiquitinate Pex5p <i>in vivo</i>. 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.</p

The function of Pex22(C)p is essential for peroxisome biogenesis, while Pex22(N)p is dispensable.

<p>(<b>a</b>) 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 <i>pex4</i>Δ<i>pex22</i>Δ, <i>pex4</i>Δ <i>or pex22</i>Δ 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 <i>pex4</i>Δ strain, it is only capable to regain functionality in the <i>pex4</i>Δ<i>pex22</i>Δ background, when Pex22(C)p is present. (<b>b</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 <i>pex4</i>Δ 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 <i>pex4</i>Δ strain as well as the <i>pex4</i>Δ<i>pex22</i>Δ 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 <i>pex4</i>Δ<i>pex22</i>Δ 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. (<b>c</b>) 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 (<i>pex4</i>Δ<i>pex22</i>Δ+Pex3(N)-Pex4p+Pex22(C)p) and the strain without functional complementation (<i>pex22</i>Δ+Pex22(C)p). The asterisk denotes a cross-reaction signal.</p

Pex22(C)p reduces polyubiquitination and stimulates monoubiquitination of the PTS1-receptor Pex5p <i>in vivo</i>.

<p>(<b>a</b>) 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>b</b>) The Pex3(N)-Pex4p chimera is capable to monoubiquitinate Pex5p <i>in vivo</i>. The level of monoubiquitinated Pex5p is reduced in the absence of Pex22p, but is restored in the presence of Pex22(C)p.</p

The Pex3(1–45)-Pex4p fusion protein binds the soluble Pex22(C)p-fragment at peroxisomes.

<p>(<b>a</b>) 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 <i>orange,</i> the trans-membrane domains (TMD) in <i>red</i>, the cytosolic parts in <i>blue</i> and GFP (green fluorescent protein) in <i>green</i>. 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>b</b>) Peroxisomal targeting of a chimeric Pex3(1–45)-Pex4p. Intracellular localization of chimeric Pex4p in wild-type and indicated <i>pex</i>-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.</p

The cytosolic domain of Pex22p stimulates the Pex4p-dependent ubiquitination of the PTS1-receptor

Peroxisomal biogenesis is an ubiquitin-dependent process because the receptors required for the import of peroxisomal matrix proteins are controlled via their ubiquitination status. A key step is the monoubiquitination of the import receptor Pex5p by the ubiquitin-conjugating enzyme (E2) Pex4p. This monoubiquitination is supposed to take place after Pex5p has released the cargo into the peroxisomal matrix and primes Pex5p for the extraction from the membrane by the mechano-enzymes Pex1p/Pex6p. These two AAA-type ATPases export Pex5p back to the cytosol for further rounds of matrix protein import. Recently, it has been reported that the soluble Pex4p requires the interaction to its peroxisomal membrane-anchor Pex22p to display full activity. Here we demonstrate that the soluble C-terminal domain of Pex22p harbours its biological activity and that this activity is independent from its function as membrane-anchor of Pex4p. We show that Pex4p can be functionally fused to the trans-membrane segment of the membrane protein Pex3p, which is not directly involved in Pex5p-ubiquitination and matrix protein import. However, this Pex3(N)-Pex4p chimera can only complement the double-deletion strain pex4$\Delta$/pex22$\Delta$ and ensure optimal Pex5p-ubiquitination when the C-terminal part of Pex22p is additionally expressed in the cell. Thus, while the membrane-bound portion Pex22(N)p is not required when Pex4p is fused to Pex3(N)p, the soluble Pex22(C)p is essential for peroxisomal biogenesis and efficient monoubiquitination of the import receptor Pex5p by the E3-ligase Pex12p $\textit {in vivo}$ and $\textit {in vitro}$. The results merge into a picture of an ubiquitin-conjugating complex at the peroxisomal membrane consisting of three domains: the ubiquitin-conjugating domain (Pex4p), a membrane-anchor domain (Pex22(N)p) and an enhancing domain (Pex22(C)p), with the membrane-anchor domain being mutually exchangeable, while the Ubc- and enhancer-domains are essential