In vitro reconstitution of glucose-induced targeting of fructose-1,6- bisphosphatase into the vacuole in semi-intact yeast cells

Hui Ling Shieh, Hui Ling Chiang

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

Fructose-1,6-bisphosphatase (FBPase), the key enzyme in gluconeogenesis in the yeast Saccharomyces cerevisiae, is induced when cells are grown in medium containing poor carbon sources. FBPase is targeted from the cytosol to the vacuole for degradation when glucose-starved yeast cells are replenished with fresh glucose. In this study, we report the reconstitution of the glucose-induced import of FBPase into the vacuole in semi-intact yeast cells using radiolabeled FBPase, an ATP regenerating system and cytosol. The import of FBPase was defined as the fraction of the FBPase that was sequestered inside a membrane-sealed compartment. FBPase import requires ATP hydrolysis and is stimulated by cytosolic proteins. Furthermore, the import of FBPase is a saturable process. FBPase import is low in the glucose-starved cells and is stimulated in the glucose-replenished cells. FBPase accumulates to a higher level in the pep4 cell, suggesting that FBPase is targeted to the vacuole for degradation. Indirect immunofluorescence microscopy studies demonstrate that the imported FBPase is localized to the vacuole in the permeabilized cells. Thus, the glucose-induced targeting of FBPase into the vacuole can be reproduced in our in vitro system.

Original languageEnglish (US)
Pages (from-to)3381-3387
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number6
DOIs
StatePublished - Feb 6 1998

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'In vitro reconstitution of glucose-induced targeting of fructose-1,6- bisphosphatase into the vacuole in semi-intact yeast cells'. Together they form a unique fingerprint.

Cite this