Research Paper Volume 5, Issue 7 pp 551—574

Figure 1. In yeast cultured with exogenously added LCA in the presence or absence of DMSO, this bile acid enters cells and accumulates in a subcellular fraction consisting of mitochondria, ER, Golgi, vacuoles, PM and nuclei. (A and B) Cells were cultured in the nutrient-rich YP medium initially containing 0.2% glucose with 50 μM LCA or without it, in the presence of 1% DMSO (A) or in its absence (B). Survival curves of chronologically aging yeast are shown; data are presented as means ± SEM (n = 11-14). (C and D) The age-related dynamics of changes in the levels of LCA associated with cells or remaining in the cultural medium in yeast cultures that were incubated with exogenously added 50 μM LCA in the presence of 1% DMSO (C) or in its absence (D); data are presented as means ± SEM (n = 5-6). (E and F) The age-related dynamics of changes in the levels of LCA recovered in various subcellular fractions that were separated by differential centrifugation of yeast cell homogenates; data for subcellular fractions recovered from yeast cultures that were incubated with exogenously added 50 μM LCA in the presence of DMSO (E) or in its absence (F) are presented as means ± SEM (n = 4). The efficacy of spheroplast formation is also shown as means ± SEM (n = 4). (G) Outline of a procedure for subcellular fractionation of yeast cell homogenates through sequential centrifugation steps of increasing force and duration. Abbreviations: Diauxic (D), logarithmic (L), post-diauxic (PD) or stationary (ST) growth phase.