Fig 4. Bidirectional transport of ergosterol from the ER to the PM is unaffected in Δ-s-tether cells.
A. Outline of the transport assay. B. Characterization of subcellular fractions. Top, immunoblots using antibodies against Pma1 (PM), Dpm1 (ER), and Vph1 (vacuole). Fraction 2 is designated ER* to indicate that it contains membranes in addition to ER membranes. Data correspond to fractionation of a homogenate of WT cells prepared at the end of the labeling pulse. Middle, quantification of Dpm1 and Pma1 in fractions prepared from homogenates of WT and Δ-s-tether cells taken after a 30 min chase period. The blots were quantified by ImageJ. Bottom, quantification of ergosterol in the different fractions from the middle panel. C. WT and Δ-s-tether cells were processed as in panel A. The SR of ergosterol in each fraction ([3H]ergosterol [cpm] ÷ ergosterol mass) was normalized to the SR of the total homogenate at each time point to obtain an RSR. The figure shows RSR versus time (t = 0 min is the start of the labeling pulse). The lower portion of the graph (solid symbols) is based on 3–4 independent experiments; the upper portion (open symbols) is based on 2–5 independent experiments. The lines are mono-exponential fits of the data that plateau at RSR = 1. D. Transport of ergosterol in Δ-s-tether cells with block in vesicular transport. Mid-log cultures of WT, sec18-1ts (CBY2859), Δ-s-tether (CBY5838), and Δ-s-tether sec18-1ts (CBY5851) cells were grown at 24 °C, shifted to the restrictive temperature of 37 °C for 20 min, pulse-labeled with [3H]methyl-methionine for 4 min and chased for 15 min at the same temperature. The bar chart shows the RSR of the PM fraction from samples taken at the end of the pulse and chase periods. Data are mean ± SEM (n = 3). Numerical data presented in this figure may be found in S1 Data. Δ-s-tether, Δ-super-tether; cpm, counts per minute; ER, endoplasmic reticulum; PM, plasma membrane; RSR, relative specific radioactivity; SR, specific radioactivity; WT, wild type.