Selective Uptake of Cytosolic, Peroxisomal, and Plasma Membrane Proteins into the Yeast Lysosome for Degradation

Abstract

When glucose-starved cells are replenished with glucose, the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is selectively targeted from the cytosol to the yeast lysosome (vacuole) for degradation. The glucose-induced targeting of FBPase to the vacuole for degradation occurs in cells grown under a variety of metabolic conditions. Immunoelectron microscopic studies demonstrate that the uptake of FBPase by the vacuole is mediated in part by an autophagic process. FBPase can be found on the vacuolar membrane and also at the sites of membrane invaginations. Furthermore, FBPase is associated with different forms of vesicles, which are induced to accumulate inside the vacuole. We have identified peroxisomes as the organelles that are delivered to the vacuole for degradation when cells are replenished with glucose. Ultrastructural studies indicate that peroxisomes are engulfed by the vacuole by an autophagic process, leading to the destruction of whole organelles in the vacuole. Furthermore, the galactose transporter (Gal2p) is also delivered from the plasma membrane to the vacuole for degradation in response to glucose. Gal2p is delivered to the vacuole through the endocytic pathway, as mutants defective in receptor-mediated endocytosis fail to degrade Gal2p in response to glucose.

Cyclophilin A Mediates Vid22p Function in the Import of Fructose-1,6-bisphosphatase into Vid Vesicles

“…Although the site of FBPase degradation has been a matter of debate (21,22), a PEP4-dependent degradation of FBPase has been shown by our laboratory and confirmed by an independent research group (23). Using immunofluorescence and immunoelectron microscopy, we showed that FBPase is localized to the vacuole in the pep4 strain (18,19). In addition, FBPase trafficking to the vacuole has been reconstituted using a semi-intact pep4 strain (20).…”

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“…Presumably, glucose stimulates a receptor protein in the membrane of the vacuole. Whether the mechanism of vacuolar uptake involves microautophagy or a chaperonin-mediated direct translocation of fructose 1,6-diphosphatase into the vacuole, or a combination of both, is not entirely clear (Chiang et al, 1996). An alternative view is that fructose 1,6-phosphatase is not targeted to the vacuole but, instead, is degraded via the ubiquitinproteasome pathway (Schork et al, 1995).…”

“…However, when glucose replaces methanol, peroxisomes are taken up directly by the vacuoles by a process resembling microautophagy, which is sensitive to inhibition by cycloheximide (Tuttle & Dunn, 1995). Similarly, when glucose-starved S. cerevisiae cells are replenished with glucose, peroxisomes are degraded by microautophagy (Chiang et al, 1996).…”

Degradation of the Gluconeogenic Enzymes Fructose-1,6-bisphosphatase and Malate Dehydrogenase Is Mediated by Distinct Proteolytic Pathways and Signaling Events

“…For these experiments, aliquots of cells (1 A 600 ) were used for each time point. Rabbit anti-FBPase antibodies were produced as described previously (30,31). The ⌬pep4⌬prb1⌬prc1 strain (45) was obtained from Dr. A. Kornberg (Stanford University).…”