Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris

The pas2 mutant of the methylotrophic yeast Pichia pastoris is characterized by a deficiency in peroxisome biogenesis. We have cloned the PpPAS2 gene by functional complementation and show that it encodes a protein of 455 amino acids with a molecular mass of 52 kDa. In a Pppas2 null mutant, import of both peroxisomal targeting signal 1 (PTS1)- and PTS2-containing proteins is impaired as shown by biochemical fractionation and fluorescence microscopy. No morphologically distinguishable peroxisomal structures could be detected by electron microscopy in Pppas2 null cells induced on methanol and oleate, suggesting that PpPas2p is involved in the early stages of peroxisome biogenesis. PpPas2p is a peroxisomal membrane protein (PMP) and is resistant to extraction by 1 M NaCl or alkaline sodium carbonate, suggesting that it is a peroxisomal integral membrane protein. Two hydrophobic domains can be distinguished which may be involved in anchoring PpPas2p to the peroxisomal membrane. PpPas2p is homologous to the Saccharomyces cerevisiae Pas3p. The first 40 amino acids of PpPas2p, devoid of the hydrophobic domains, are sufficient to target a soluble fluorescent reporter protein to the peroxisomal membrane, with which it associates tightly, A comparison with the membrane peroxisomal targeting signal of PMP47 of Candida boidinii revealed a stretch of positively charged amino acids common to both sequences. The role of peroxisomal membrane targeting signals and transmembrane domains in anchoring PMPs to the peroxisomal membrane is discussed.</p

The FLUXNET2015 dataset and the ONEFlux processing pipeline for eddy covariance data

The FLUXNET2015 dataset provides ecosystem-scale data on CO2, water, and energy exchange between the biosphere and the atmosphere, and other meteorological and biological measurements, from 212 sites around the globe (over 1500 site-years, up to and including year 2014). These sites, independently managed and operated, voluntarily contributed their data to create global datasets. Data were quality controlled and processed using uniform methods, to improve consistency and intercomparability across sites. The dataset is already being used in a number of applications, including ecophysiology studies, remote sensing studies, and development of ecosystem and Earth system models. FLUXNET2015 includes derived-data products, such as gap-filled time series, ecosystem respiration and photosynthetic uptake estimates, estimation of uncertainties, and metadata about the measurements, presented for the first time in this paper. In addition, 206 of these sites are for the first time distributed under a Creative Commons (CC-BY 4.0) license. This paper details this enhanced dataset and the processing methods, now made available as open-source codes, making the dataset more accessible, transparent, and reproducible.Peer reviewe

Structural characterisation of peroxisomal import receptor complexes

Peroxisomes are dynamic eukaryotic organelles that require import of their membrane and matrix proteins by soluble receptors in the cytosol. Their biogenesis, protein import, and proliferation are regulated by a distinct set of proteins, collectively called peroxins.Two peroxins, Pex3 and Pex19, are involved in the insertion of various Peroxisomal Membrane Proteins (PMPs) in the peroxisomal membrane and are thus crucial for its formation. Pex3 is a PMP that acts as a docking receptor for Pex19. The role of Pex19, in turn, is to bind, stabilise, and guide PMPs to the membrane-docked Pex3, where they are inserted into the peroxisomal membrane by an unknown mechanism. Pex14 is a PMP protein that is a key component of the peroxisomal import pore. Its interaction with Pex19 is established, yet little structural information is available about their full-length complex. In this thesis we investigated the conformation of the full-length human PEX19 in complex with the cytosolic domain of PEX3. A hybrid structural and biochemical approach was employed in order to characterize this interaction. Furthermore, the role of PEX19-PEX14 binding was addressed and the full-length complex was structurally characterised, providing insight into the stoichiometry, binding and shape of the never-before-described full-length assembly, using a variety of biochemical, biophysical and structural methods.The second part of this thesis aims to shed light on the peroxisomal protein import mechanism process. Matrix proteins (cargoes) can be imported into the peroxisomal lumen using peroxisomal targeting signal 1 (PTS1), or peroxisomal targeting signal 2 (PTS2) import pathways. The PTS1 pathway is the most common, utilising peroxin Pex5p. Pex5p is a soluble receptor, cycling between a free cytoplasmic state -where it recognises and binds peroxisomal matrix proteins- and a membrane bound state, as part of the transient PTS1 pore. The interaction between Pex5p and cargo proteins occurs via the TPR domains (tetratricopeptide repeats) of Pex5p and the PTS1 sequence at the C-terminus of the cargo proteins. Two peroxisomal cargoes, Pcs60 and MIF1, were studied in this part of the PhD project. Pcs60 is a yeast peroxisomal oxalyl-CoA synthetase, while MIF1 is a plant peroxisomal cargo associated with stress response, both of which contain a PTS1 recognition signal peptide. These proteins were crystallised and structurally characterised by means of X-ray crystallography. Further characterisation of Pcs60 and its complex with Pex5p was performed by biophysical methods, small angle X-ray scattering (SAXS), and negative stain electron microscopy. Elucidation of the structure of the Pex5p-Pcs60 complex will lead to enhancing our understanding of peroxisomal protein import