De novo synthesis of peroxisomes upon mitochondrial targeting of Pex3p.

Peroxisomes can form either by growth and division of pre-existing peroxisomes or by de novo synthesis from the endoplasmic reticulum. Pex3p is the key component for both pathways and its targeting to the ER is thought to initiate the de novo formation of peroxisomes. Here, we addressed the question whether Pex3p also can induce peroxisome formation from mitochondrial membranes. Pex3p was targeted to mitochondria by fusion with the mitochondrial targeting signal of Tom20p. The Tom20p-Pex3p-fusion protein was expressed in Pex3p-deficient cells, which are characterized by the lack of peroxisomal membranes. De novo formation of import-competent peroxisomes was observed upon expression of the mitochondrial Pex3p in the mutant cells. This de novo synthesis is independent of the GTPases Vps1p and Dnm1p, two proteins required for peroxisome fission. We conclude that natural or artificial targeting of Pex3p to any endomembrane may initiate peroxisome formation and that also Pex3p-containing mitochondria can serve as source for the de novo synthesis of peroxisomes.

Cotranslational assembly of the yeast SET1C histone methyltransferase complex.

While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

Farnesylation of pex19p is required for its structural integrity and function in peroxisome biogenesis.

The conserved CaaX box peroxin Pex19p is known to be modified by farnesylation. The possible involvement of this lipid modification in peroxisome biogenesis, the degree to which Pex19p is farnesylated, and its molecular function are unknown or controversial. We resolve these issues by first showing that the complete pool of Pex19p is processed by farnesyltransferase in vivo and that this modification is independent of peroxisome induction or the Pex19p membrane anchor Pex3p. Furthermore, genomic mutations of PEX19 prove that farnesylation is essential for proper matrix protein import into peroxisomes, which is supposed to be caused indirectly by a defect in peroxisomal membrane protein (PMP) targeting or stability. This assumption is corroborated by the observation that mutants defective in Pex19p farnesylation are characterized by a significantly reduced steady-state concentration of prominent PMPs (Pex11p, Ant1p) but also of essential components of the peroxisomal import machinery, especially the RING peroxins, which were almost depleted from the importomer. In vivo and in vitro, PMP recognition is only efficient when Pex19p is farnesylated with affinities differing by a factor of 10 between the non-modified and wild-type forms of Pex19p. Farnesylation is likely to induce a conformational change in Pex19p. Thus, isoprenylation of Pex19p contributes to substrate membrane protein recognition for the topogenesis of PMPs, and our results highlight the importance of lipid modifications in protein-protein interactions.

The N-domain of Pex22p can functionally replace the Pex3p N-domain in targeting and peroxisome formation.

Pex3p is a central component of the import machinery for peroxisomal membrane proteins (PMPs) that can reach peroxisomes via the endoplasmic reticulum (ER) and even trigger de novo peroxisome formation from the ER. Pex19p is the import receptor for type I PMPs, whereas targeting of type II PMPs, of which Pex3p so far represents the only species, does not require Pex19p. Pex3p possesses two domains with distinct function: a short N-terminal domain, which harbors the information for peroxisomal (and ER) targeting, and a C-terminal domain, which faces the cytosol and serves as a docking site for Pex19p, thereby delivering newly synthesized PMPs to the peroxisome. Here we show that the N-terminal domain of Pex3p can be functionally replaced by the N-terminal peroxisomal membrane targeting signal (mPTS) of Pex22p, a supposedly unrelated component of the import machinery for peroxisomal matrix proteins. An exchange of the mPTS of Pex22p by that of Pex3p likewise fully preserved the function of Pex22p. Neither of the two mPTS interacted with Pex19p, and in the absence of Pex19p, colocalization of Pex3p and Pex22p was observed, indicating that also Pex22p is targeted to peroxisomes by a type II mPTS. When a type I mPTS was hooked to the C-terminal domains of Pex22p and Pex3p, function was retained in the case of Pex22p and in part even for Pex3p. The C-terminal domain of Pex3p thus contains the relevant information required for de novo peroxisome formation, thereby challenging the concept of the N terminus of Pex3p being key in that process.

Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes.

Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (Saccharomyces cerevisiae and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane. In this report, we show that the specificity of peroxisomal membrane protein binding for Trypanosoma brucei PEX19 is very similar to those previously identified for human and yeast PEX19. Our studies show that trafficking is conserved across these distant phyla and that both a PEX19 binding site and a transmembrane domain are required for the insertion of two test proteins into the glycosomal membrane. However, in contrast to T. brucei PEX10 and PEX12, T. brucei PEX14 does not traffic to human peroxisomes, indicating that it is not recognized by the human PEX14 import mechanism.

Targeting of the tail-anchored peroxisomal membrane proteins PEX26 and PEX15 occurs through C-terminal PEX19-binding sites.

Tail-anchored proteins contain a single transmembrane domain (TMD) followed by a short C-terminal domain extending into the organellar lumen. Tail-anchored proteins are thought to target to the correct subcellular compartment by virtue of general physicochemical properties of their C-termini; however, the machineries that enable correct sorting remain largely elusive. Here we analyzed targeting of the human peroxisomal tail-anchored protein PEX26. Its C-terminal-targeting signal contains two binding sites for PEX19, the import receptor for several peroxisomal membrane proteins. One PEX19-binding site overlapped with the TMD, the other was contained within the luminal domain. Although the PEX19-binding site containing the TMD targeted to peroxisomes to some extent, the luminal site proved essential for correct targeting of the full-length protein, as it prevented PEX26 from mislocalization to mitochondria. Its function as a targeting motif was proved by its ability to insert a heterologous TMD-containing fragment into the peroxisomal membrane. Finally we show that PEX19 is essential for PEX26 import. Analysis of the yeast tail-anchored protein Pex15p revealed that it also harbors a luminal PEX19-binding site that acts as a peroxisomal-targeting motif. We conclude that C-terminal PEX19-binding sites mark tail-anchored proteins for delivery to peroxisomes.

Function of the PEX19-binding site of human adrenoleukodystrophy protein as targeting motif in man and yeast. PMP targeting is evolutionarily conserved.

We predicted in human peroxisomal membrane proteins (PMPs) the binding sites for PEX19, a key player in the topogenesis of PMPs, by virtue of an algorithm developed for yeast PMPs. The best scoring PEX19-binding site was found in the adrenoleukodystrophy protein (ALDP). The identified site was indeed bound by human PEX19 and was also recognized by the orthologous yeast PEX19 protein. Likewise, both human and yeast PEX19 bound with comparable affinities to the PEX19-binding site of the yeast PMP Pex13p. Interestingly, the identified PEX19-binding site of ALDP coincided with its previously determined targeting motif. We corroborated the requirement of the ALDP PEX19-binding site for peroxisomal targeting in human fibroblasts and showed that the minimal ALDP fragment targets correctly also in yeast, again in a PEX19-binding site-dependent manner. Furthermore, the human PEX19-binding site of ALDP proved interchangeable with that of yeast Pex13p in an in vivo targeting assay. Finally, we showed in vitro that most of the predicted binding sequences of human PMPs represent true binding sites for human PEX19, indicating that human PMPs harbor common PEX19-binding sites that do resemble those of yeast. Our data clearly revealed a role for PEX19-binding sites as PMP-targeting motifs across species, thereby demonstrating the evolutionary conservation of PMP signal sequences from yeast to man.