Analysis of general secretion in 41X<i>HAclc1</i> cells.

<p>A. Micrographs of wild-type (WT) and 41X<i>HAclc1</i> cells bearing Apm1-GFP and Chc1-mCherry fusion proteins; cells were incubated in the absence (−T) and the presence (+T) of thiamine for 6 hours. The micrographs are projections of z-stack images taken with a DeltaVision deconvolution microscope. B. Upper panels, micrographs of wild-type (WT) and 41X<i>HAclc1</i> cells bearing Syb1-GFP incubated in minimal medium in the absence (−T) and the presence (+T) of thiamine for 6 hours. The micrographs are medial sections. Lower panels, the same as before but the cells were incubated in the presence of thiamine and sorbitol for 48 hours. C. Micrographs of the indicated strains carrying a Sec8-GFP fusion protein; the photographs were taken with a conventional fluorescence microscope. Bars, 10 μm. D. Acid phosphatase activity was measured in the culture medium and cell extracts as indicated in Materials and methods; all samples were collected from logarithmic cultures at a density of 2×10⁷ cells per ml. Left panel, activity as arbitrary units in the culture medium and cell extracts from the indicated strains grown in the presence of thiamine for 6 hours. The activity in the cell extracts was estimated per number of cells in the same volume of culture that was used to estimate activity in the medium, so that both data can be compared directly. Right panel, the secretion of acid phosphatase was calculated as the percentage of activity in the culture medium with respect to the total activity detected (activity in the medium plus activity in cell extracts). The experiment was performed a minimum of five times with duplicates; statistically significant defects are indicated (*; p<0.05; Student's t-test).