A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5'-3' exonuclease. S. cerevisiae EXO1 interacted with both S. cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments. exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway. exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants.
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http://dx.doi.org/10.1073/pnas.94.14.7487 | DOI Listing |
Bioresour Technol
January 2025
School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR China; Frontiers Science Center for Synthetic Biology, Tianjin University, Tianjin, PR China; Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, PR China. Electronic address:
Miltiradiene serves as a pivotal precursor for the synthesis of numerous abietane-type diterpenes with important pharmacological activities. The endogenous mevalonate (MVA) pathway is tightly regulated in Saccharomyces cerevisiae, which limits the availability of precursors for the heterologous production of miltiradiene. In this study, the orthogonal isopentenol utilization pathway (IUP) was constructed and investigated for its adaptability with mitochondria and peroxisomes in S.
View Article and Find Full Text PDFACS Synth Biol
January 2025
State Key Laboratory of Fine Chemicals, Frontiers Science Centre for Smart Materials Oriented Chemical Engineering, School of Bioengineering, Dalian University of Technology, Dalian 116024, China.
Cannabichromene (CBC), a valuable but extremely low-abundance component of cannabinoids in L., is known for its ability to promote neurogenesis. The scarcity of CBC in natural is primarily attributed to the inefficiency of the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4 phosphate (DOXP/MEP) and fatty acid metabolism pathways, along with the limited competitive advantage of cannabichromenic acid synthetase (CBCAS) compared to other cannabinoid synthases.
View Article and Find Full Text PDFMethods 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.
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