Experimental Validation of Plant Peroxisomal Targeting Prediction Algorithms by Systematic Comparison of In Vivo Import Efficiency and In Vitro PTS1 Binding Affinity

Most peroxisomal matrix proteins possess a C-terminal targeting signal type 1 (PTS1). Accurate prediction of functional PTS1 sequences and their relative strength by computational methods is essential for determination of peroxisomal proteomes in silico but has proved challenging due to high levels of sequence variability of non-canonical targeting signals, particularly in higher plants, and low levels of availability of experimentally validated non-canonical examples. In this study, in silico predictions were compared with in vivo targeting analyses and in vitro thermodynamic binding of mutated variants within the context of one model targeting sequence. There was broad agreement between the methods for entire PTS1 domains and position-specific single amino acid residues, including residues upstream of the PTS1 tripeptide. The hierarchy Leu>Met>Ile>Val at the C-terminal position was determined for all methods but both experimental approaches suggest that Tyr is underweighted in the prediction algorithm due to the absence of this residue in the positive training dataset. A combination of methods better defines the score range that discriminates a functional PTS1. In vitro binding to the PEX5 receptor could discriminate among strong targeting signals while in vivo targeting assays were more sensitive, allowing detection of weak functional import signals that were below the limit of detection in the binding assay. Together, the data provide a comprehensive assessment of the factors driving PTS1 efficacy and provide a framework for the more quantitative assessment of the protein import pathway in higher plants

Peroxisome biogenesis, protein targeting mechanisms and PEX gene functions in plants

Peroxisomes play diverse and important roles in plants. The functions of peroxisomes are dependent upon their steady state protein composition which in turn reflects the balance of formation and turnover of the organelle. Protein import and turnover of constituent peroxisomal proteins is controlled by the state of cell growth and environment. The evolutionary origin of the peroxisome and the role of the endoplasmic reticulum in peroxisome biogenesis is discussed, as informed by studies of the trafficking of peroxisome membrane proteins. The process of matrix protein import in plants and its similarities and differences with peroxisomes in other organisms is presented and discussed in the context of peroxin distribution across the green plants

Covalent Label Transfer Between Peroxisomal Importomer Components Reveals Export-Driven Import Interactions

Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal Malate Dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes, owing to high degrees of protease susceptibility or resistance, respectively. Here, we present a means for analysis of in vitro import through a covalent biotin label transfer, and employ this method to the import of PEX5C. Label transfer demonstrates that PEX5C construct is monomeric in the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labelling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest, and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import

Peroxisome protein import: a complex journey.

The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognised by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor-cargo import with release of unloaded receptor from the peroxisome. Here we provide an update on recent literature concerning mechanisms of protein import into peroxisomes

Translation, rewriting and the marginal city in Geraldine Monk's Escafeld Hangings

Geraldine Monk's 2005 poetry collection Escafeld Hangings presents the city of Sheffield via a reworking of letters and textual fragments by Mary Queen of Scots and other sources. This article explores a set of connections between Monk's approach to language and her representation of city space. Looking at the city as a site of encounter between different languages, I argue that Monk's work uses forms of translation to reconfigure relationships between place and language, addressing both constructions of nationhood and the homogenizing effects of English as a global language by focusing on locality, heterogeneity and diachronic variation. I show firstly how her interrogation of proper nouns becomes a means of questioning the structures that define the city and its relationships of class and gender, and secondly how the transpositions between texts by Mary Queen of Scots and Monk's versions of them simultaneously evoke and displace the poetic subject, presenting the city as haunted by its margins