Computational Evaluation of Peroxisomal Targeting Signals in Metazoa.

Most soluble proteins enclosed in peroxisomes encode either type-1 or type-2 peroxisomal targeting signals (PTS1 or PTS2), which act as postal codes and define the proteins' intracellular destination. Thus, various computational programs have been developed to evaluate the probability of specific peptide sequences for being a functional PTS or to scan the primary sequence of proteins for such signals. Among these prediction algorithms the PTS1-predictor ( https://mendel.imp.ac.at/pts1/ ) has been amply used, but the research logic of this and other PTS1 prediction tools is occasionally misjudged giving rise to characteristic pitfalls. Here, a proper utilization of the PTS1-predictor is introduced together with a framework of additional tests to increase the validity of the interpretation of results. Moreover, a list of possible causes for a mismatch between results of such predictions and experimental outcomes is provided. However, the foundational arguments apply to other prediction tools for PTS1 motifs as well.

The deubiquitination of the PTS1-import receptor Pex5p is required for peroxisomal matrix protein import.

Peroxisomal biogenesis depends on the correct import of matrix proteins into the lumen of the organelle. Most peroxisomal matrix proteins harbor the peroxisomal targeting-type 1 (PTS1), which is recognized by the soluble PTS1-receptor Pex5p in the cytosol. Pex5p ferries the PTS1-proteins to the peroxisomal membrane and releases them into the lumen. Finally, the PTS1-receptor is monoubiquitinated on the conserved cysteine 6 in Saccharomyces cerevisiae. The monoubiquitinated Pex5p is recognized by the peroxisomal export machinery and is retrotranslocated into the cytosol for further rounds of protein import. However, the functional relevance of deubiquitination has not yet been addressed. In this study, we have analyzed a Pex5p-truncation lacking Cys6 [(Δ6)Pex5p], a construct with a ubiquitin-moiety genetically fused to the truncation [Ub-(Δ6)Pex5p], as well as a construct with a reduced susceptibility to deubiquitination [Ub(G75/76A)-(Δ6)Pex5p]. While the (Δ6)Pex5p-truncation is not functional, the Ub-(Δ6)Pex5p chimeric protein can facilitate matrix protein import. In contrast, the Ub(G75/76A)-(Δ6)Pex5p chimera exhibits a complete PTS1-import defect. The data show for the first time that not only ubiquitination but also deubiquitination rates are tightly regulated and that efficient deubiquitination of Pex5p is essential for peroxisomal biogenesis.

Predicting Peroxisomal Targeting Signals to Elucidate the Peroxisomal Proteome of Mammals.

Peroxisomes harbor a plethora of proteins, but the peroxisomal proteome as the entirety of all peroxisomal proteins is still unknown for mammalian species. Computational algorithms can be used to predict the subcellular localization of proteins based on their amino acid sequence and this method has been amply used to forecast the intracellular fate of individual proteins. However, when applying such algorithms systematically to all proteins of an organism the prediction of its peroxisomal proteome in silico should be possible. Therefore, a reliable detection of peroxisomal targeting signals (PTS ) acting as postal codes for the intracellular distribution of the encoding protein is crucial. Peroxisomal proteins can utilize different routes to reach their destination depending on the type of PTS. Accordingly, independent prediction algorithms have been developed for each type of PTS, but only those for type-1 motifs (PTS1) have so far reached a satisfying predictive performance. This is partially due to the low number of peroxisomal proteins limiting the power of statistical analyses and partially due to specific properties of peroxisomal protein import, which render functional PTS motifs inactive in specific contexts. Moreover, the prediction of the peroxisomal proteome is limited by the high number of proteins encoded in mammalian genomes, which causes numerous false positive predictions even when using reliable algorithms and buries the few yet unidentified peroxisomal proteins. Thus, the application of prediction algorithms to identify all peroxisomal proteins is currently ineffective as stand-alone method, but can display its full potential when combined with other methods.

Unraveling of the Structure and Function of Peroxisomal Protein Import Machineries.

Peroxisomes are dynamic organelles of eukaryotic cells performing a wide range of functions including fatty acid oxidation, peroxide detoxification and ether-lipid synthesis in mammals. Peroxisomes lack their own DNA and therefore have to import proteins post-translationally. Peroxisomes can import folded, co-factor bound and even oligomeric proteins. The involvement of cycling receptors is a special feature of peroxisomal protein import. Complex machineries of peroxin (PEX) proteins mediate peroxisomal matrix and membrane protein import. Identification of PEX genes was dominated by forward genetic techniques in the early 90s. However, recent developments in proteomic techniques has revolutionized the detailed characterization of peroxisomal protein import. Here, we summarize the current knowledge on peroxisomal protein import with emphasis on the contribution of proteomic approaches to our understanding of the composition and function of the peroxisomal protein import machineries.

Artificial import substrates reveal an omnivorous peroxisomal importomer.

The peroxisome matrix protein importomer has the remarkable ability to transport oligomeric protein substrates across the bilayer. However, the selectivity and relation between import and overall peroxisome homeostasis remain unclear. Here, we microinject artificial import substrates and employ quantitative microscopy to probe limits and capabilities of the importomer. DNA and polysaccharides are "piggyback" imported when noncovalently bound by a peroxisome targeting signal (PTS)-bearing protein. A dimerization domain that can be tuned to systematically vary the binding dissociation constant (Kd ) shows that a Kd in the millimolar range is sufficient to promote piggyback import. Microinjection of import substrate at high levels results in peroxisome growth and a proportional accumulation of peroxisome membrane proteins (PMPs). However, corresponding PMP mRNAs do not accumulate, suggesting that this response is posttranscriptionally regulated. Together, our data show that the importomer can tolerate diverse macromolecular species. Coupling between matrix import and membrane biogenesis suggests that matrix protein expression levels can be sufficient to regulate peroxisome size.