Biology subjects

Onischenko, E.

Publications and source records attributed to Onischenko, E..

3 recordsLinked to original sources

bioRxiv2025

Vsb1, Ypq1 and Ypq2 control dynamic cationic amino acid storage in the yeast vacuole

Although the yeast vacuole plays a crucial role in storing and mobilizing cationic amino acids (CAA), CAA transport at the vacuolar membrane remains poorly understood. Here, by combining analysis of CAA pools, uptake and permeabilization assays, we establish Vsb1 as the principal vacuolar lysine transporter, enabling its strong accumulation in the vacuole while mitigating its toxicity. We further show that, although Ypq1 can mediate proton-independent vacuolar lysine import, it mainly functions as a lysine exporter necessary for lysine mobilization under conditions of lysine scarcity and downregulated as lysine stores are exhausted. Using quantitative models based on dynamic metabolic labeling, we further show that, surprisingly, in growing cells, CAA rapidly exchange between vacuolar and cytosolic compartments, a process involving the export activity of Ypq1 and its paralogue Ypq2, specific for lysine and arginine, respectively. Together, our findings reveal the unexpectedly complex function of Vsb1 and Ypq1/2 as the key transporters mediating dynamic vacuolar CAA storage. 40-word summaryZaremba et al. characterize Vsb1 as the main yeast vacuolar lysine importer and Ypq1/2 as bi- directional vacuolar transporters of lysine and arginine. Their study highlights the role of vacuolar transporters in regulating cationic amino acid homeostasis under fluctuating nutrient availability.

cell biology↗

bioRxiv2022

An amphipathic helix in Brl1 is required for membrane fusion during nuclear pore complex biogenesis in S. cerevisiae

The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-ET analyses reveal multi-layered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.

cell biology↗

bioRxiv2017

Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

The nuclear pore complex (NPC) is an 8-fold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ~30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure, however, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed Nuclear Rim Intensity Measurement or NuRIM, to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188 that their altered expression leads to significant changes in Nup stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.\n\nSignificanceThe nuclear pore complex (NPC) is one of the largest protein complexes in eukaryotes comprising over 500 nucleoporin subunits. The NPC is essential for transport of biomolecules across the nuclear envelope, however, due to its enormous size, it has been a challenge to characterize its molecular architecture. Herein, we have developed a novel, widely applicable imaging pipeline to determine the absolute nucleoporin abundances in native yeast NPCs. This reveals that the NPC composition dramatically differs between yeast and human despite an overall conservation of individual subunits. We also applied our imaging pipeline to examine yeast mutants revealing a remarkable compositional plasticity of NPCs. Our stoichiometry analyses provide an important resource for the generation of high-resolution structure models of the NPC.

cell biology↗