Biology subjects

Asraf, Y.

Publications and source records attributed to Asraf, Y..

6 recordsLinked to original sources

bioRxiv2026

Pex9 regulates TORC2 during the cellular response to oxidative stress

Peroxisomes are known for their detoxification role in cellular reactive oxygen species (ROS) homeostasis, yet their regulatory roles in cellular recovery from oxidative stress remain unclear. Here, using yeast, we systematically screened a panel of fluorescently tagged peroxisome-related proteins and identified widespread stress-induced changes in their localization. Notably, we discovered that the peroxisomal import receptor, Pex9, rapidly enters the nucleus upon H2O2 exposure, independently of the canonical oxidative stress regulators-Yap1 and Skn7, suggesting a non-canonical signaling function. We further show that Pex9 nuclear entry is modulated by Avo1, an essential Tor complex 2 (TORC2) subunit. In turn, Pex9 downregulates the essential TOR complexes kinase, Tor2, at both the transcriptional and protein levels. Finally, we show that this Pex9-TORC2 regulatory loop during oxidative stress is facilitated by the cellular envelope stress response. Our findings uncover an unexpected peroxisome-TORC2 signaling axis and highlight the importance of organelles in shaping global cellular responses to stress.

cell biology↗

bioRxiv2025

A pipeline for screening condition-specific enzymes uncovers a function for the alcohol dehydrogenase Bdh2

Cells possess intricate metabolic networks comprised of hundreds of enzymes. Despite extensive research, many of these enzymes remain uncharacterized. Identifying such enzymes is crucial for advancing our understanding of metabolism. However, multiple enzymes are not expressed in standard conditions, making them challenging to study. To overcome this challenge, we created a pipeline for characterizing the expression of condition-specific enzymes in yeast. We assembled a collection of 110 yeast strains, each containing an uncharacterized putative enzyme fused to a fluorophore under the regulation of their own promotor. By subjecting them to 43 diverse growth or stress environments, we identified the biologically relevant conditions for the expression of 19 proteins. We focused on one such putative alcohol dehydrogenase, Bdh2, and functionally characterized it. More broadly, our discovery pipeline lays the foundation for uncovering new condition-specific enzymes. This has implications in cell biology and biotechnology and should expand our understanding of metabolism.

cell biology↗

bioRxiv2025

Creation and Validation of a Proteome-Wide Yeast Library for Protein Detection and Analysis

A significant challenge in cell biology is to uncover the function of uncharacterized proteins. Surprisingly a quarter of the proteome is still poorly understood even in the most well studied model organisms. Systematic methodologies, including the use of tagged protein collections, have emerged as a powerful approach to address this gap. Despite the availability of proteome- wide collections featuring various fused proteins, the impact of tag size on protein function highlighted the need for using minimally disruptive tags for functional genomic studies. To rise to this challenge, we have created a proteome-wide collection of yeast strains in which proteins are N-terminally tagged with the Hemagglutinin (HA) epitope. The library leverages the compact size of the HA tag to minimize drawbacks associated with larger tags while enabling efficient functional analysis. We showcase the potential uses of our library for systematically evaluating protein size, abundance and localization using an in vivo labeling approach. Our characterization underscores the potential utility of a proteome-wide HA-tagged library in revealing novel aspects of cell biology, providing an additional powerful tool for functional genomics.

molecular biology↗

bioRxiv2024

A proteome-wide yeast degron collection for the dynamic study of protein function.

Genome-wide collections of yeast strains, known as libraries, revolutionized the way systematic studies are carried out. Specifically, libraries that involve a cellular perturbation, such as the deletion collection, have facilitated key biological discoveries. However, short-term rewiring and long-term accumulation of suppressor mutations often obscure the functional consequences of such perturbations. We present the AID library which supplies "on demand" protein depletion to overcome these limitations. Here, each protein is tagged with a Green Fluorescent Protein (GFP) and an Auxin inducible degron (AID), enabling rapid protein depletion that can be quantified systematically using the GFP element. We characterized the degradation response of all strains and demonstrated its utility by revisiting seminal yeast screens for genes involved in cell cycle progression as well as mitochondrial distribution and morphology. In addition to recapitulating known phenotypes, we also uncovered proteins with previously unrecognized roles in these central processes. Hence, our tool expands our knowledge of cellular biology and physiology by enabling access to phenotypes that are central to cellular physiology and therefore rapidly equilibrated.

cell biology↗

bioRxiv2024

A systematic bi-genomic split-GFP assay illuminates the mitochondrial matrix proteome and protein targeting routes

The majority of mitochondrial proteins are encoded in the nuclear genome and often lack clear targeting signals. Therefore, what constitutes the entire mitochondrial proteome is still unclear. We here build on our previously developed bi-genomic (BiG) split-GFP assay (Bader et al. 2020) to solidify the list of matrix and inner membrane mitochondrial proteins. The assay relies on one fragment (GFP1-10) encoded in the mitochondrial DNA enabling specific visualization of only the proteins tagged with a smaller fragment, GFP11, and localized to the mitochondrial matrix or the inner membrane. We used the SWAp-Tag (SWAT) strategy to tag every protein with GFP11 and mated them with the BiG GFP strain. Imaging the collection in six different conditions allowed us to visualize almost 400 mitochondrial proteins, 50 of which were never visualized in mitochondria before, and many are poorly studied dually localized proteins. We use structure-function analysis to characterize the dually localized protein Gpp1, revealing an upstream start codon that generates a mitochondrial targeting signal and explore its unique function. We also show how this data can be applied to study mitochondrial inner membrane protein topology and sorting. This work brings us closer to finalizing the mitochondrial proteome and the freely distributed library of GFP11-tagged strains will be a useful resource to study protein localization, biogenesis and interactions.

cell biology↗

bioRxiv2024

Profiling the LAM family of contact site tethers provides insights into their regulation and function

Membrane contact sites are molecular bridges between organelles that are sustained by tethering proteins and enable organelle communication. The endoplasmic reticulum (ER) membrane harbors many distinct families of tether proteins that enable the formation of contacts with all other organelles. One such example is the LAM (Lipid transfer protein At Membrane contact sites) family, composed of six members, each containing a lipid binding and transfer domain and an ER-embedded transmembrane segment. The family is divided into three homologous pairs each unique in their molecular architecture and localization to different ER subdomains. However, what determines the distinct localization of the different LAMs and which specific roles they carry out in each contact are still open questions. To address these, we utilized a labeling approach to profile the proximal protein landscape of the entire family. Focusing on unique interactors we could support that Lam5 resides at the ER-mitochondria contact site and demonstrate a role for it in sustaining mitochondrial activity. Capturing shared interactors of multiple LAMs, we show how the Lam1/3 and Lam2/4 paralogous pairs could be associated specifically with the plasma membrane. Overall, our work provides new insights into the regulation and function of the LAM family members. More globally it demonstrates how proximity labeling can help identify the shared or unique functions of paralogous proteins.

cell biology↗