Biology subjects

Schliebs, W.

Publications and source records attributed to Schliebs, W..

8 recordsLinked to original sources

bioRxiv2023

High confidence glycosomal membrane protein inventory unveils trypanosomal Peroxin PEX15

Infections by trypanosomatid parasites cause Chagas disease, Human African Trypanosomiasis, and Leishmaniasis, affecting over 12 million people worldwide. Glycosomes, the unique peroxisome-related organelles of trypanosomes are essential for their survival, and hence their metabolic functions and biogenesis mediated by peroxins (PEX) are suitable as drug targets. Here we report on a comprehensive protein inventory of glycosomal membranes through advanced subcellular membrane protein profiling employing quantitative mass spectrometry. Our quantitative analysis resulted in the identification of 28 novel high confidence glycosomal membrane proteins. Our in-depth protein inventory of glycosomal membranes serves as an important resource for characterizing glycosome biology and drug development. We validated four so far unknown glycosomal membrane proteins, including two tail-anchored (TA) proteins, a homolog of human peroxisomal PXMP4, and a Macrodomain-containing protein. Using a structure-based approach, we identified one of the TA proteins as the long-sought Trypanosoma PEX15. Despite its low sequence similarity, Trypanosoma PEX15 exhibits structural and topological similarities with its yeast (Pex15) and human counterparts (PEX26). We show that PEX15 is an essential integral glycosomal membrane protein that interacts with PEX6. Accordingly, RNAi knockdown of PEX15 in bloodstream form trypanosomes demonstrates that it is essential for glycosome biogenesis and parasite survival. Considering the low degree of conservation with its human counterpart, PEX15 is a promising molecular target for drug development.

cell biology↗

bioRxiv2023

A novel PEX14/PEX5 interface links peroxisomal protein import and receptor recycling

Newly synthesized peroxisomal proteins are recognized in the cytosol by the cycling receptor PEX5 and directed to a docking complex comprising PEX14 and PEX13 at the peroxisomal membrane. After cargo translocation, the unloaded PEX5 is recycled in an ATP-dependent manner. Receptor docking involves the WxxxF-motifs in the N-terminal domain (NTD) of PEX5 that are recognized by the N-terminal domain of PEX14. Here, we combine biochemical methods and NMR spectroscopy to identify a novel binding interface between human PEX5 and PEX14. The interaction involves the PEX5 C-terminal cargo-binding TPR domain and a conserved IPSWQI peptide motif in the C-terminal intrinsically disordered region of PEX14. The three-dimensional structure of the PEX14 IPSWQI peptide bound the PEX5 TPR domain, shows the PEX14 interaction is non-overlapping with PTS1 binding to the TPR domain. Notably, PEX14 IPSWQI motif binding to a hinge region in the TPR domain shows a more open supercoil of the TPR fold that resembles the apo conformation in the absence of PTS1 peptide. Mutation of binding site residues in PEX5 or PEX14 leads to a partial protein import defect and decrease of the steady-state-concentration of PEX5. This resembles the mutant phenotype of cells affected in receptor recycling, suggesting a role in this process.

biochemistry↗

bioRxiv2023

PEX1 is essential for the glycosome biogenesis and trypanosomatid parasite survival.

Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we identified the Trypanosoma PEX1 orthologue using sequence and structural similarities. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggests that these parasites possess a highly efficient quality control mechanisms that export the import receptors from glycosomes to the cytosol, in the absence of a functional TbPEX1-TbPEX6 complex.

cell biology↗

bioRxiv2022

Intramolecular autoinhibition of human PEX13 modulates peroxisomal import

Targeting and import of peroxisomal proteins depends on PEX5, PEX14 and PEX13. We present a biochemical and structural characterization of the PEX13 C-terminal region. By combining NMR spectroscopy, X-ray crystallography and biochemical methods, we show that the PEX13 SH3 domain mediates intramolecular interactions with a newly identified proximal FxxxF motif and also binds to WxxxF peptide motifs from the PEX5 NTD, demonstrating evolutionary conservation of this interaction from yeast to human. Strikingly, the C-terminal FxxxF motif autoinhibits the WxxxF/Y binding surface on the PEX13 SH3 domain. This is supported by high-resolution crystal structures, which show FxxxF or WxxxF/Y binding to the same, non-canonical surface on the SH3 domain. The FxxxF motif also binds the PEX14 NTD with micromolar affinity. Surprisingly, the canonical binding surface for PxxP motifs on the human PEX13 SH3 fold does not recognize PxxP motifs in PEX14, distinct from the yeast ortholog. The dynamic network of PEX13, PEX14 and PEX5 interactions mediated by diaromatic peptide motifs fine-tunes and modulates peroxisomal matrix import in cells.

biochemistry↗

bioRxiv2022

Peroxisomal ATPase ATAD1 acts in quality control of the protein import machinery

ATAD1 is an AAA-ATPase which shows a dual localization at mitochondria and peroxisomes. While its peroxisomal function is not known, in mitochondria ATAD1 is part of a quality control mechanism extracting mislocalised tail-anchored and accumulated precursor proteins from the outer membrane. Here, we studied the peroxisomal interactome of ATAD1 and could show that human ATAD1 interacts with PEX5, a cytosolic receptor for peroxisomal matrix proteins which transiently inserts into peroxisomal membranes. Upon cargo-release, mono-ubiquitinated PEX5 is recycled into the cytosol by the AAA-peroxins PEX1 and PEX6. The accumulation of ubiquitinated PEX5 is known to trigger degradation of whole organelles called pexophagy. Here, we used ATAD1-, PEX1- and ATAD1/PEX1-CRISPR-Knockout cell lines to investigate the physiological role of an ATAD1-PEX5 interaction. We could show an influence of ATAD1 on the stability of accumulated PEX5 and hypothesize a role in a peroxisomal quality control mechanism enabling clearance of ubiquitinated receptor from the membrane.

biochemistry↗

bioRxiv2022

Determining the targeting specificity of the selective peroxisomal targeting factor Pex9

Targeting proteins to their correct cellular location is a fundamental process that allows them to carry out their cellular functions. Peroxisomes utilize two paralog targeting factors, Pex5 and Pex9, for proteins with a Peroxisomal Targeting Signal 1 (PTS1). However, in spite of their similarity, Pex9 targets only a subset of Pex5 cargo proteins. Here, we studied the properties that facilitate the targeting specificity of Pex9, both by unbiased screens and by site-directed mutagenesis of the PTS1 motifs of either binders or non-binders. We find that the binding specificity of Pex9 is largely determined by the hydrophobic nature of the amino acid preceding the PTS1 tripeptide of its cargos. This is in line with structural modeling of the PTS1-binding cavity of the two factors, showing that while Pex5 has large negative electrostatic patches at the area surrounding the PTS1 binding cavity, Pex9 is mostly hydrophobic. Our work outlines the mechanism by which targeting specificity is achieved, enabling dynamic rewiring of the peroxisomal proteome in changing metabolic needs.

cell biology↗

bioRxiv2021

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics. SummaryThe peroxisomal import receptor PEX5 transports newly synthesized proteins from the cytosol to the peroxisomal matrix. Here the cytosolic diffusion and interaction dynamics of PEX5 are characterized by advanced microscopic spectroscopy methods, revealing a so far unknown interaction partner.

biophysics↗

bioRxiv2020

Competitive Microtubule Binding of PEX14 Coordinates Peroxisomal Protein Import and Motility

PEX14 functions as peroxisomal docking protein for the import receptor PEX5. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human {beta}-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the {beta}-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.

biochemistry↗