Abstract
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.
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Selected References
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- Cain C. C., Murphy R. F. Growth inhibition of 3T3 fibroblasts by lysosomotropic amines: correlation with effects on intravesicular pH but not vacuolation. J Cell Physiol. 1986 Oct;129(1):65–70. doi: 10.1002/jcp.1041290110. [DOI] [PubMed] [Google Scholar]
- Chvatchko Y., Howald I., Riezman H. Two yeast mutants defective in endocytosis are defective in pheromone response. Cell. 1986 Aug 1;46(3):355–364. doi: 10.1016/0092-8674(86)90656-2. [DOI] [PubMed] [Google Scholar]
- DULBECCO R., VOGT M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 1954 Feb;99(2):167–182. doi: 10.1084/jem.99.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jenness D. D., Spatrick P. Down regulation of the alpha-factor pheromone receptor in S. cerevisiae. Cell. 1986 Aug 1;46(3):345–353. doi: 10.1016/0092-8674(86)90655-0. [DOI] [PubMed] [Google Scholar]
- Makarow M. Endocytosis in Saccharomyces cerevisiae: internalization of alpha-amylase and fluorescent dextran into cells. EMBO J. 1985 Jul;4(7):1861–1866. doi: 10.1002/j.1460-2075.1985.tb03861.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Makarow M., Nevalainen L. T. Transport of a fluorescent macromolecule via endosomes to the vacuole in Saccharomyces cerevisiae. J Cell Biol. 1987 Jan;104(1):67–75. doi: 10.1083/jcb.104.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ohkuma S., Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327–3331. doi: 10.1073/pnas.75.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pick U., Bassilian S. Modification of the ATP binding site of the Ca2+ -ATPase from sarcoplasmic reticulum by fluorescein isothiocyanate. FEBS Lett. 1981 Jan 12;123(1):127–130. doi: 10.1016/0014-5793(81)80035-x. [DOI] [PubMed] [Google Scholar]
- Riezman H. Endocytosis in yeast: several of the yeast secretory mutants are defective in endocytosis. Cell. 1985 Apr;40(4):1001–1009. doi: 10.1016/0092-8674(85)90360-5. [DOI] [PubMed] [Google Scholar]