Figure 1.

Localization of intracellular fractions of Nce102 in exponentially growing and stationary cell cultures. Subcellular distribution of Nce102-GFP fluorescence in strains Y1039 (A, upper row) and Y240 (A, mid and lower row; B) was monitored in a liquid cell culture cultivated for different time periods, indicated by abbreviations (log: 6 h, stationary: 48 h, A) or by numbers (in hours, B). In A, the GFP fluorescence signal (left; cyan in right column) was co-localized with the red fluorescence of organelle-specific markers (middle; magenta in right column). These were ER luminal marker ss-DsRed-HDEL (upper row, [25]) and vacuolar membrane-concentrated lipophilic dye FM 4-64 (mid and lower row; see Methods for details). Transversal optical sections are presented. In single channel images, inverted fluorescence intensities in false-coloured lookup table are shown to maximize contrast. Cell nuclei (arrowheads) and vacuoles (asterisks) are denoted in A. Bars: 5 μm. (C) The amount of whole-cell and vacuolar Nce102 was monitored by western blot. Vacuoles were isolated as described in Methods. Samples were taken after spheroplast homogenization (homogenate), ultracentrifugation on 4% Ficoll 400 gradient (intact vacuoles) and after the final ultracentrifugation following vacuole lysis (vacuolar membranes). Proteins used as organelle markers: vacuolar carboxypeptidase Y (Prc1; vacuolar membranes), 1-acyldihydroxyacetone-phosphate reductase (Ayr1; lipid droplets; vacuolar membrane fraction purity control). Glyceraldehyde 3-phosphate dehydrogenase GAPDH was used as a loading control. Note the markedly increased intensity of Nce102 in each fraction of the 72 h old culture (relatively to 24 h; ~2.5 times in homogenates [normalized to GAPDH]; ~1.7 times in vacuolar membranes [normalized to Prc1]).