Transport of a fluorescent macromolecule via endosomes to the vacuole in Saccharomyces cerevisiae.
Open Access
- 1 January 1987
- journal article
- research article
- Published by Rockefeller University Press in The Journal of cell biology
- Vol. 104 (1), 67-75
- https://doi.org/10.1083/jcb.104.1.67
Abstract
Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) is internalized by endocytosis into the lysosome-like vacuoles of Saccharomyces cerevisiae (Makarow, M., 1985, EMBO (Eur. Mol. Biol. Organ.) J. 4:1861-1866). Here we show that under energy depletion conditions FITC-dextran accumulated in a cytoplasmic compartment, from which it could be chased to the vacuole when the energy block was removed. The internal pH of the intermediate compartment under energy depletion was determined by fluorometry to be 5.8. The pH could be raised by the lysosomotropic agent ammonium chloride, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (CCCP) and the ATPase inhibitors dicyclohexylcarbodiimide (DCCD) and sodium vanadate. The pH of the vascuole was found to be 6.5. It was raised by ammonium chloride, CCCP, and DCCD, but not with sodium vanadate. Efrapeptin had no effect on the internal pH of either compartment. By dissecting the endocytic pathway, two portions of the route leading to the vascuole could be studied separately. The internalization of FITC-dextran from the extracellular fluid to the intermediate compartment followed linear kinetics, was independent of energy, and occurred at temperatures of between 15.degree. and 37.degree. C. Transfer of the marker from the intermediate compartment to the vacuole required energy, took place at temperatures between 19.degree. and 37.degree. C, and had a half-time of 7 min at 37.degree. C. Transport of the marker from the exterior of the cell to the vacuole did not require acidic pH values in the intermediate compartment of the vacuole. We suggest that the cytoplasmic compartment revealed by FITC-dextran, under energy depletion, represents the equivalent of the endosomes of mammalian cells.This publication has 49 references indexed in Scilit:
- Reconstitution of an endocytic fusion event in a cell-free systemCell, 1985
- A Test of Clathrin Function in Protein Secretion and Cell GrowthScience, 1985
- Transcytosis of the G protein of vesicular stomatitis virus after implantation into the apical plasma membrane of Madin-Darby canine kidney cells. I. Involvement of endosomes and lysosomes.The Journal of cell biology, 1984
- Identification of coated vesicles in Saccharomyces cerevisiae.The Journal of cell biology, 1984
- Semliki Forest virus penetration from endosomes: a morphological studyBiology of the Cell, 1984
- Penetration of semliki forest virus from acidic prelysosomal vacuolesCell, 1983
- Serial-section analysis of coated pits and vesicles involved in adsorptive pinocytosis in cultured fibroblasts.The Journal of cell biology, 1983
- Endocytosis and the recycling of plasma membrane.The Journal of cell biology, 1983
- Fusion of Semliki forest virus with the plasma membrane can be induced by low pH.The Journal of cell biology, 1980
- Adsorptive endocytosis of Semliki Forest virusJournal of Molecular Biology, 1980