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© The Rockefeller University Press, 0021-9525/1997//803 $5.00
The Journal of Cell Biology, Volume 136, Number 4, , 1997 803-810

Article

Identification of Novel Vesicles in the Cytosol to Vacuole Protein Degradation Pathway



Pei-Hsin Huang and Hui-Ling Chiang

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

The key gluconeogenic enzyme, fructose1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are starved of glucose. FBPase is targeted from the cytosol to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. Several vid mutants defective in the glucose-induced degradation of FBPase in the vacuole have been isolated. In some vid mutants, FBPase is found in punctate structures in the cytoplasm. When extracts from these cells are fractionated, a substantial amount of FBPase is sedimentable in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these vid mutants. In this paper we investigated whether FBPase association with intracellular structures also existed in wild-type cells. We report the purification of novel FBPase-associated vesicles from wild-type cells to near homogeneity. Kinetic studies indicate that FBPase association with these vesicles is stimulated by glucose and occurs only transiently, suggesting that these vesicles are intermediate in the FBPase degradation pathway. Fractionation analysis demonstrates that these vesicles are distinct from known organelles such as the vacuole, ER, Golgi, mitochondria, peroxisomes, endosomes, COPI, or COPII vesicles. Under EM, these vesicles are 30–40 nm in diam. Proteinase K experiments indicate that the majority of FBPase is sequestered inside the vesicles. We propose that FBPase is imported into these vesicles before entering the vacuole.

Abbreviations used in this paper: API, aminopeptidase I; CPY, carboxypeptidase Y; ECL, enhanced chemiluminescence; FBPase, fructose-1,6bisphosphatase.

Address all correspondence to Hui-Ling Chiang, Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. Tel. and Fax: (617) 432-0140. e-mail: chiangne{at}warren.med.harvard.edu


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