Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran

Concurrent with Riezman's report (Riezman, H. 1985, Cell. 40:1001-1009) that fluid-phase endocytosis of the small molecule Lucifer yellow occurs in the yeast Saccharomyces cerevisiae, Makarow (Makarow, M. 1985. EMBO [Eur. Mol. Biol. Organ.] J. 4:1861-1866) reported the endocytotic uptake of 70-kD FITC-dextran (FD) and its subsequent compartmentation into the yeast vacuole. Samples of FD synthesized and purified here failed to label yeast vacuoles under conditions that allowed labeling using commercial FD. Chromatography revealed that the commercial FD was heavily contaminated with at least three low molecular weight fluorescent compounds. Dialysis was ineffective for removing the contaminants. After purification (Sephadex G25, ethanol extraction), commercial FD was incapable of labeling vacuoles. Extracts of cells labeled with partially purified FD contained FITC, not FD, based on Sephadex and thin layer chromatography. In either the presence or absence of unlabeled 70-kD dextran, authentic FITC (10 micrograms/ml) was an effective labeling agent for vacuoles. The rapid kinetics (0.28 pmol/min per 10(6) cells at pH 5.5) and the pH dependence of FITC uptake suggest that the mechanism of FITC uptake involves diffusion rather than endocytosis. In view of these results, labeling experiments that use unpurified commercial FD should be interpreted with caution.