The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is subjected to catabolite inactivation and degradation when glucose-starved cells are replenished with fresh glucose. In various studies, the proteasome and the vacuole have each been reported to be the major site of FBPase degradation. Because different growth conditions were used in these studies, we examined whether variations in growth conditions could alter the site of FBPase degradation. Here, we demonstrated that FBPase was degraded outside the vacuole (most likely in the proteasome), when glucose was added to cells that were grown in low glucose media for a short period of time. By contrast, cells that were grown in the same low glucose media for longer periods of time degraded FBPase in the vacuole in response to glucose. Another gluconeogenic enzyme malate dehydrogenase (MDH2) showed the same degradation characteristics as FBPase in that the short term starvation of cells led to a non-vacuolar degradation, whereas long term starvation resulted in the vacuolar degradation of this protein.The N-terminal proline is required for the degradation of FBPase and MDH2 for both the vacuolar and nonvacuolar proteolytic pathways. The cAMP signaling pathway and the phosphorylation of glucose were needed for the vacuolar-dependent degradation of FBPase and MDH2. By contrast, the cAMP-dependent signaling pathway was not involved in the non-vacuolar degradation of these proteins, although the phosphorylation of glucose was required.

The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded in the vacuole when glucose is added to glucose-starved cells. Before it is delivered to the vacuole, however, FBPase is imported into intermediate carriers called Vid (vacuole import and degradation) vesicles. Here, using biochemical and genetic approaches, we identified a requirement for SEC28 in FBPase degradation. SEC28 encodes the ⑀-COP subunit of COPI (coat protein complex I) coatomer proteins. When SEC28 and other coatomer genes were mutated, FBPase degradation was defective and FBPase association with Vid vesicles was impaired. Coatomer proteins were identified as components of Vid vesicles, and they formed a protein complex with a Vid vesicle-specific protein, Vid24p. Furthermore, Vid24p association with Vid vesicles was impaired when coatomer genes were mutated. Kinetic studies indicated that Sec28p traffics to multiple locations. Sec28p was in Vid vesicles, endocytic compartments, and the vacuolar membrane in various mutants that block the FBPase degradation pathway. Sec28p was also found in vesicles adjacent to the vacuolar membrane in the ret2-1 coatomer mutant. We propose that Sec28p resides in Vid vesicles, and these vesicles converge with the endocytic pathway. After fusion, Sec28p is distributed on the vacuolar membrane, where it concentrates on vesicles that pinch off from this organelle. FBPase also utilizes the endocytic pathway for transport to the vacuole, as demonstrated by its presence in endocytic compartments in the ⌬vph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway.Transport of proteins and lipids between organelles is an important function of all eukaryotic cells. In many cases, vesicles facilitate the transport of cargo proteins or lipids from donor membranes to acceptor membranes (1-10). The most thoroughly studied transporters are coat protein complex I (COPI), 2 COPII, and clathrin-coated vesicles. COPI vesicles mediate the retrograde transport from the Golgi to the ER as well as intra-Golgi transport, whereas COPII-coated vesicles conduct anterograde transport from the ER to Golgi. The clathrin-coated vesicles regulate trafficking from the plasma membrane to early endosomes and from the Golgi to endosomes (1-10).Characterization of COPI vesicles has revealed the presence of a protein complex called "coatomer." Coatomer is a cytosolic protein complex comprised of seven subunits, ␣-COP (160 kDa), ␤-COP (110 kDa), ␤ Ј -COP (102 kDa), ␥-COP (98 kDa), ␦-COP (61 kDa), ⑀-COP (35 kDa), and -COP (20 kDa) (11-13). The COPI coats have been detected on the Golgi complex and on the ER (14, 15). Coatomer subunits have also been found in endocytic compartments in mammalian cells and yeast (16 -21). Furthermore, they play an important role in endocytic trafficking in both mammalian cells (16 -20), and in yeast (21). In addition to coatomer, the GTPase ARF (ADP-ribosylation factor) associates with COPI vesicles in its GTP bound form. This prot...