peroxisome-staining-mammalian-cells-using-peroxisome-specific

Peroxisome Staining in Mammalian Cells Using Peroxisome-Specific Probes

Peroxisomes are ubiquitous organelles with essential roles in human health, and their dysfunction leads to a spectrum of genetic disorders. Visualization ...

Bar-Ilan University

Crucial metabolic functions of peroxisomes rely on a variety of peroxisomal membrane proteins (PMPs). While mRNA transcripts of PMPs were shown to be colocalized with peroxisomes, the process by which PMPs efficiently couple translation with targeting to the peroxisomal membrane remained elusive. Here, we combine quantitative electron microscopy with proximity-specific ribosome profiling and reveal that translation of specific PMPs occurs on the surface of peroxisomes in the yeast . This places peroxisomes alongside chloroplasts, mitochondria, and the endoplasmic reticulum as organelles that use localized translation for ensuring correct insertion of hydrophobic proteins into their membranes. Moreover, the correct targeting of these transcripts to peroxisomes is crucial for peroxisomal and cellular function, emphasizing the importance of localized translation for cellular physiology.

Peroxisome function relies on organelle-associated mRNA translation.

Most cellular proteins require targeting to a distinct cellular compartment to function properly. A subset of proteins is distributed to two or more destinations in the cell and little is known about the mechanisms controlling the process of dual/multiple targeting. Here, we provide insight into the mechanism of dual targeting of proteins between mitochondria and peroxisomes. We perform a high throughput microscopy screen in which we visualize the location of the model tail-anchored proteins Fis1 and Gem1 in the background of mutants in virtually all yeast genes. This screen identifies three proteins, whose absence results in a higher portion of the tail-anchored proteins in peroxisomes: the two paralogues Tom70, Tom71, and the uncharacterized gene YNL144C that we rename mitochondria and peroxisomes factor 1 (Mpf1). We characterize Mpf1 to be an unstable protein that associates with the cytosolic face of the mitochondrial outer membrane. Furthermore, our study uncovers a unique contribution of Tom71 to the regulation of dual targeting. Collectively, our study reveals, for the first time, factors that influence the dual targeting of proteins between mitochondria and peroxisomes.

Mpf1 affects the dual distribution of tail-anchored proteins between mitochondria and peroxisomes.

Chanarin-Dorfman syndrome (CDS) is a multisystemic autosomal recessive rare disorder. CDS is caused by variants in the abhydrolase domain containing 5 (ABHD5) encoding gene (CGI-58), which ultimately leads to excessive lipid storage, and therefore a high abundance of cellular lipid droplets (LDs). Although the molecular etiology of the disease was described many years ago, no treatment for CDS is currently available.

Using chanarin-dorfman syndrome patient fibroblasts to explore disease mechanisms and new treatment avenues.

Proper protein targeting to organelles is crucial for maintaining eukaryotic cellular function and homeostasis. This necessity has driven the evolution of specific targeting signals on proteins and the targeting factors that recognize them. A prominent example is peroxisomal matrix proteins, most of which depend on the targeting factor Pex5 to localize and function correctly. Although most Pex5 cargoes contain a peroxisomal targeting signal type 1 (PTS1), they are not all targeted similarly. Some undergo priority targeting, facilitated either by stronger binding to specific subsets of PTS1 signals or by additional interaction interfaces. These observations highlight the extensive complexity of Pex5-mediated targeting. In this study, we reveal that the Saccharomyces cerevisiae (yeast) matrix protein Eci1 can reach peroxisomes and bind Pex5 in the absence of PTS1. By solving the structure of the yeast Pex5-Eci1 complex using cryo-electron microscopy, we identified additional binding interfaces. Our findings provide new insights into the versatile interactions between Pex5 and its cargo, Eci1. More broadly, this work highlights the intricate, dynamic nature of the interactions between cargo factors and their cargoes to meet the complex environment within eukaryotic cells.

A cryo-electron microscopy structure of yeast Pex5 in complex with a cargo uncovers a novel binding interface.

Peroxisomes are metabolic organelles essential for human health. Defects in peroxisomal biogenesis proteins (also known as peroxins (PEXs)) cause devastating disease. PEX7 binds proteins containing a type 2 peroxisomal targeting signal (PTS2) to enable their import from the cytosol into peroxisomes, although many aspects of this process remain enigmatic. Utilizing in vitro assays, yeast and human cells, we show that PEX39, a previously uncharacterized protein, is a cytosolic peroxin that facilitates the import of PTS2-containing proteins by binding PEX7 and stabilizing its interaction with cargo proteins containing a PTS2. PEX39 and PEX13, a peroxisomal membrane translocon protein, both possess an (R/K)PWE motif necessary for PEX7 binding. Handover of PEX7 from PEX39 to PEX13 via these motifs provides a new paradigm for peroxisomal protein import and biogenesis. Collectively, this work reveals how PEX39 and (R/K)PWE motifs facilitate the import of PTS2-containing proteins and advances our understanding of peroxisomal disease.

Article	Year
Peroxisome function relies on organelle-associated mRNA translation. Science advances\| PubMed ID: 35020435	2022
Mpf1 affects the dual distribution of tail-anchored proteins between mitochondria and peroxisomes. EMBO reports\| PubMed ID: 40175596	2025
Using chanarin-dorfman syndrome patient fibroblasts to explore disease mechanisms and new treatment avenues. Orphanet journal of rare diseases\| PubMed ID: 40275410	2025
A cryo-electron microscopy structure of yeast Pex5 in complex with a cargo uncovers a novel binding interface. Journal of cell science\| PubMed ID: 40376748	2025
PEX39 facilitates the peroxisomal import of PTS2-containing proteins. Nature cell biology\| PubMed ID: 40739340	2025

Einat Zalckvar

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