Biology subjects

Sonal,

Publications and source records attributed to Sonal,.

3 recordsLinked to original sources

bioRxiv2023

When is an auxotroph not an auxotroph: how budding yeast lacking MET17 collectively overcome their metabolic defect

Assimilation of sulfur is vital to all organisms. In S. cerevisiae, inorganic sulfate is first reduced to sulfide, which is then reacted to an organic carbon backbone by the Met17 enzyme. The resulting homocysteine can then be converted to all other essential organosulfurs such as methionine, cysteine, and glutathione. This pathway has been known for nearly half a century, and met17 mutants have long been classified as organosulfur auxotrophs which are unable to grow on sulfate as the sole sulfur source. Surprisingly, we found that met17{Delta} could grow on sulfate, albeit only at sufficiently high cell densities. We show that the accumulation of hydrogen sulfide gas underpins this density-dependent growth of met17{Delta} on sulfate, and that the locus YLL058W (HSU1) enables met17{Delta} cells to assimilate hydrogen sulfide. Hsu1 protein is induced during sulfur starvation and under exposure to high sulfide in wildtype cells, suggesting multiple functions of this gene. In a mathematical model, the low efficiency of sulfide assimilation in met17{Delta} can explain the observed density-dependent growth of met17{Delta} on sulfate. Thus, having uncovered and explained the paradoxical growth of a commonly used "auxotroph", our findings may impact the design of future studies in yeast genetics, metabolism, and volatile-mediated microbial interactions.

microbiology↗

bioRxiv2022

One-pot synthesis of prenylated proteins utilizing E. coli cell-free expression

Bottom-up synthetic biology is a powerful tool for uncovering the mechanisms underlying vital biological processes, such as signaling and cell polarization. The core principle of reconstituting cellular functions in their minimal forms can be achieved through modular protein design. However, assembling multiple purified proteins into a functional and synchronized system remains a technical challenge. The fact that many regulatory proteins show direct or indirect membrane interactions further exacerbates the complications. Here, we introduce the Cell-Free prenylated Protein Synthesis (CFpPS) system which enables the production of prenylated proteins in a single reaction mix, through reconstituted prenylation machinery. Not only does the CFpPS system offer a fast and reliable method for producing solubilized prenylated proteins, but it can also produce the protein of interest directly in the vicinity of biomimetic membranes, thus enabling microscopy-based functional assessment. As proof of principle, we demonstrate synthesis and solubilization of various important signaling proteins from the Ras superfamily, as well as membrane binding and extraction of the key polarity regulator Cdc42. Furthermore, our method can be used to confer membrane affinity to any protein, simply by adding a 4-peptide motif to the C-terminus of the protein. In sum, the CFpPS system offers a versatile and effective platform for designing peripheral membrane proteins for synthetic biology applications.

synthetic biology↗

bioRxiv2018

Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortex

Dynamic reorganization of the actomyosin cytoskeleton allows a fine-tuning of cell shape that is vital to many cellular functions. It is well established that myosin-II motors generate the forces required for remodeling the cell surface by imparting contractility to actin networks. An additional, less understood, role of myosin-II in cytoskeletal dynamics is believed to be in the regulation of actin turnover; it has been proposed that myosin activity increases actin turnover in various cellular contexts, presumably by contributing to disassembly. In vitro reconstitution of actomyosin networks has confirmed the role of myosin in actin network disassembly, but factors such as diffusional constraints and the use of stabilized filaments have thus far limited the observation of myosin-assisted actin turnover in these networks. Here, we present the reconstitution of a minimal dynamic actin cortex where actin polymerization is catalyzed on the membrane in the presence of myosin-II activity. We demonstrate that myosin activity leads to disassembly and redistribution in this simplified cortex. Consequently, a new dynamic steady state emerges in which actin filaments undergo constant turnover. Our findings suggest a multi-faceted role of myosin-II in fast remodeling of the eukaryotic actin cortex.

biophysics↗