The Pseudomonas syringae cyclic lipodepsipeptide syringomycin inhibits the growth of Saccharomyces cerevisiae. A novel yeast gene, SYR2, was found to complement two syringomycin-resistant S. cerevisiae mutants. SYR2 was cloned, sequenced, and shown to encode a 349 amino acid protein located in the endoplasmic reticulum. SYR2 was identical to SUR2, which is involved in survival during nutritional starvation. Gene disruption or overexpression of SYR2 did not affect cell viability or ergosterol levels, but did influence cellular phospholipid levels. The findings suggest that phospholipids are important for the growth inhibitory action of syringomycin.
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http://dx.doi.org/10.1099/13500872-142-3-477 | DOI Listing |
Methods Mol Biol
January 2025
Department of Immunobiology, University of Lausanne, Epalinges, Switzerland.
Fluorescence recovery after photobleaching (FRAP) can be employed to investigate membrane lipid mixing of vacuoles in live budding yeast cells and distinguish the fused, hemi-fused or non-fused states of these organelles under physiological conditions. Here, we describe a protocol for labeling the outer and inner leaflets of vacuoles in live cells that allow to detect hemifusion intermediates and, thus, identify components necessary for fusion pore opening.
View Article and Find Full Text PDFMethods Mol Biol
January 2025
School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, South Korea.
Cell-free in vitro assays offer several advantages for elucidating molecular mechanisms underlying various biological processes. Here, we describe a simple and quantitative in vitro assay using isolated yeast microsomes to measure homotypic ER membrane fusion. In this assay, membrane fusion between ER microsomes is monitored by reconstitution of luciferase activity from split luciferase fragments.
View Article and Find Full Text PDFMethods Mol Biol
January 2025
Estrella Mountain Community College, Phoenix, AZ, USA.
Vacuole fusion is driven by SNARE proteins that require activation-or priming-by the AAA+ protein Sec18 (NSF) before they can bring membranes together and trigger the merger of two bilayers into a continuous membrane. Sec18 resides on vacuoles prior to engaging inactive cis-SNARE complexes through its interaction with the regulatory lipid phosphatidic acid (PA). Binding PA causes Sec18 to undergo large conformational changes that keeps it bound to the membrane, thus precluding its interactions with SNAREs.
View Article and Find Full Text PDFMethods Mol Biol
January 2025
Dept of Biochemistry & Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA.
Bio-Layer Interferometry (BLI) is a technique that uses optical biosensing to analyze interactions between molecules. The analysis of molecular interactions is measured in real-time and does not require fluorescent tags. BLI uses disposable biosensors that come in a variety of formats to bind different ligands including biotin, hexahistidine, GST, and the Fc portion of antibodies.
View Article and Find Full Text PDFNat Struct Mol Biol
January 2025
Laboratory of Regulation of Gene Expression, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic.
Transfer RNAs (tRNAs) serve as a dictionary for the ribosome translating the genetic message from mRNA into a polypeptide chain. In addition to this canonical role, tRNAs are involved in other processes such as programmed stop codon readthrough (SC-RT). There, tRNAs with near-cognate anticodons to stop codons must outcompete release factors and incorporate into the ribosomal decoding center to prevent termination and allow translation to continue.
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