Biology subjects

Zalckvar, E.

Publications and source records attributed to Zalckvar, E..

10 recordsLinked to original sources

bioRxiv2025

Structure of Pex8 in complex with peroxisomal receptor Pex5 reveals its essential role in peroxisomal cargo translocation

Peroxisomes are essential cellular organelles that enable the sequestered execution of a broad range of metabolic processes. Due to the lack of an internal protein synthesis machinery, they entirely depend on the import of target proteins to carry out their functions within peroxisomes. While the process of cargo/receptor recognition is well understood, knowledge about the molecular mechanisms of the subsequent translocation steps, including cargo release and receptor recycling, is lacking behind. Here, we provide structural and functional evidence on the role of Pex8 in these processes. First, we show that Pex8 in yeast is essential for peroxisomal cargo translocation, irrespective of the mechanism of receptor/cargo recognition. Next, we reveal that Pex8 binds through an irregular twelvefold HEAT repeat array to a short three-helical bundle within the otherwise unfolded N-terminal domain of the Pex5 receptor. Impairing this interaction abolishes peroxisomal protein translocation. It is complemented by a secondary autonomous Pex8 cargo-like interaction site with the C-terminal domain of Pex5, thus generating a bipartite interaction between the two proteins. Our data support a model in which Pex5/Pex8 complex formation allows assembly with the peroxisomal Pex2/Pex10/Pex12 E3-ubiquitin ligase complex to initiate recycling of the receptor. In summary, our findings provide in-depth insight into the transition from cargo release into peroxisomes to receptor recycling, which is essential to uncover the overall process of the peroxisomal cargo translocation.

biophysics↗

bioRxiv2024

An unconventional interaction interface between the peroxisomal targeting factor Pex5 and Eci1 enables PTS1 independent import

Accurate and regulated protein targeting to organelles is crucial for eukaryotic cellular function and homeostasis. This has driven the evolution of targeting signals on proteins and the targeting factors that recognize them. One example for this is peroxisomal matrix proteins, the majority of which rely on the targeting factor Pex5 to correctly localize and function. While most Pex5 cargos contain a Peroxisomal Targeting Signal type 1 (PTS1), in recent years it has become clear that more binding interfaces exist, and that targeting by Pex5 is more complex than previously thought. Here, we uncover that the matrix protein Eci1 can reach peroxisomes in the absence of its PTS1. By solving the structure of a complex between full length yeast Pex5 and Eci1 using Cryo-Electron Microscopy, we could identify their binding interfaces. This allowed us to map an additional binding interface that is independent of the canonical PTS1-mediated binding site. Our work brings forward a solution to a long-standing mystery regarding Eci1 targeting to peroxisomes. More globally, it demonstrates the intricate and complex nature of organelle targeting and how it has evolved to serve the complex eukaryotic environment.

cell biology↗

bioRxiv2024

PEX39 facilitates the peroxisomal import of PTS2 proteins

Peroxisomes are metabolic organelles essential for human health. Defects in peroxisomal biogenesis proteins (peroxins/PEXs) cause devastating disease. PEX7 binds newly synthesized proteins containing a type 2 peroxisomal targeting signal (PTS2) to enable their import from the cytosol into peroxisomes, although many aspects of this import pathway remain enigmatic. Utilizing in vitro assays, yeast, and human cells, we show that PEX39, a previously uncharacterized protein, is a cytosolic peroxin that facilitates PTS2-protein import by binding PEX7 and stabilizing its interaction with PTS2 cargo. PEX39 and PEX13, a peroxisomal membrane translocon protein, both possess a KPWE motif necessary for PEX7 binding. Sequential binding of PEX7 to this motif in PEX39 and PEX13 provides a novel paradigm for how PTS2 cargo engage the translocation machinery. Collectively, our work uncovers an ancient and functionally important relationship among PEX39, PEX7, and PEX13, offering insights that will advance our understanding of peroxisomal biogenesis and disease.

cell biology↗

bioRxiv2024

Mpf1 is a novel factor that affects the dual distribution of tail-anchored proteins between mitochondria and peroxisomes

Over half of cellular proteins must target upon their translation to distinct cellular compartments to function. Whereas considerable progress has been made in our understanding of targeting to individual organelles, we know truly little about how proteins distribute their targeting between two, or more, destinations - a process called dual/multiple targeting. In this study, we shine mechanistic insight into the process of dual targeting of proteins between the yeast mitochondria and peroxisomes. We performed a high throughput systematic microscopy screen in which we visualized the location of the model tail-anchored (TA) proteins Fis1 and Gem1 on the background of mutants in all yeast genes. This screen identified three proteins, whose absence resulted in a higher portion of the TA proteins in peroxisomes: the two paralogues Tom70, Tom71, as well as the uncharacterized gene YNL144C that we renamed mitochondria peroxisomes factor 1 (Mpf1). We characterized Mpf1 to be an unstable protein that associated with the cytosolic face of the mitochondrial outer membrane. Furthermore, our study uncovers a unique contribution of Tom71 for the regulation of the dual targeting and reveals a link between TOM70/71 and the quantity of transcripts of MPF1. Collectively, our study revealed, for the first-time, factors that influence the dual targeting of proteins between mitochondria and peroxisomes.

biochemistry↗

bioRxiv2022

Dually targeted proteins regulate proximity between peroxisomes and partner organelles

Peroxisomes play a central role in fatty acid metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. One mystery in the field is sorting of proteins that carry both a targeting signal for peroxisomes as well as for other organelles such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these dually localized proteins in Saccharomyces cerevisiae, we observed that they can act as dynamic tethers bridging organelles together through an affinity for organelle-destined targeting factors. We show that this mode of tethering involves the peroxisome import machinery, the ER- mitochondria encounter structure (ERMES) in the case of mitochondria and the GET complex in the case of the ER. Depletion of each of the targeting factors resulted in the accumulation of smaller peroxisomes. We propose that dual targeting of proteins occurs at contact sites and that protein import per se contributes to the maintenance of these membrane proximities. This introduces a previously unexplored concept of how targeting of dual affinity proteins can support organelle attachment, growth and communication.

cell biology↗

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

Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions

Seventy years following the discovery of peroxisomes, their proteome remains undefined. Uncovering the complete peroxisomal proteome, the peroxi-ome, is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover the peroxi-ome of the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known organellar proteome by [~]40% and paved the way for performing systematic, whole-organellar proteome assays. Coupled with targeted experiments this allowed us to discover new peroxisomal functions. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly-identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

cell biology↗

bioRxiv2021

Identification of Pex34p as a component of the peroxisomal de novo biogenesis machinery in yeast

Cells can regulate the abundance and composition of peroxisomes to adapt to environmental changes. In the bakers yeast, S. cerevisiae, peroxisomes represent the only site for degradation of fatty acids. Hence, it is not surprising that growth of yeast cells on oleic acid results in a massive proliferation of peroxisomes. New peroxisomes can form either by division of pre-existing peroxisomes or de novo in a Pex25p-dependent process with the involvement of the Endoplasmic Reticulum (ER). In search for further factors involved in de novo formation of peroxisomes, we screened ~6,000 yeast mutants that were depleted of peroxisomes by conditional inhibition of PEX19 expression. Screening the mutants for the reappearance of peroxisomes upon expression of PEX19 identified Pex34p, in addition to the well-known component Pex25p, as crucial determinants for de novo biogenesis. Pex34p interacts with Pex19p and with different Peroxisomal Membrane Proteins (PMPs) in a PEX19-dependent manner. Depletion of Pex34p results in reduced numbers of import-competent peroxisomes formed de novo and Pex3p is partly retained and distributed in ER-like structures. We suggest that Pex25p and Pex34p are both required to maintain peroxisome number in a cell and that they perform non-redundant roles in the de novo formation of peroxisomes.

cell biology↗

bioRxiv2020

Pex14p phosphorylation modulates import of citrate synthase 2 into peroxisomes in Saccharomyces cerevisiae

The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and nonphosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes on oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Our data point to the control of the peroxisomal import of Cit2p via the state of Pex14p phosphorylation at S266, which may help S. cerevisiae cells to rapidly adjust their carbohydrate metabolism according to the nutritional conditions.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsPTSperoxisomal targeting signal;LC-MSliquid chromatography-mass spectrometry;TEVtobacco etch virus;TPATEV protease cleavage site-Protein AView Full Text

cell biology↗

bioRxiv2018

An alternative membrane topology permits lipid droplet localization of peroxisomal fatty acyl-CoA reductase 1

Fatty acyl-CoA reductase 1 (Far1) is an ubiquitously expressed peroxisomal membrane protein generating fatty alcohols required for the biosynthesis of ether lipids.\n\nLipid droplet localization of human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization and N-glycosylation tagging suggest that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively. Two closely spaced hydrophobic domains are contained within the C-terminal region. When analyzed separately, the second domain was sufficient for the localization of a fluorescent reporter to lipid droplets. Targeting of Far1 to lipid droplets was not impaired in either pex19 or TRC40/ASNA1 CRISPR/Cas9 knockout cells.\n\nIn conclusion, our data suggest that Far1 is a novel member of the rather exclusive group of dual topology membrane proteins. At the same time, Far1 shows lipid metabolism-dependent differential subcellular localizations to peroxisomes and lipid droplets.

cell biology↗