Biochemical Analysis of Fructose-1,6-bisphosphatase Import into Vacuole Import and Degradation Vesicles Reveals a Role for UBC1 in Vesicle Biogenesis

Abstract

When Saccharomyces cerevisiae are shifted from medium containing poor carbon sources to medium containing fresh glucose, the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is imported into Vid (vacuole import and degradation) vesicles and then to the vacuole for degradation. Here, we show that FBPase import is independent of vacuole functions and proteasome degradation. However, FBPase import required the ubiquitin-conjugating enzyme Ubc1p. A strain containing a deletion of the UBC1 gene exhibited defective FBPase import. Furthermore, FBPase import was inhibited when cells overexpressed the K48R/K63R ubiquitin mutant that fails to form multiubiquitin chains. The defects in FBPase import seen for the ⌬ubc1 and the K48R/K63R mutants were attributed to the Vid vesicle fraction. In the ⌬ubc1 mutant, the level of the Vid vesicle-specific marker Vid24p was reduced in the vesicle fraction, suggesting that UBC1 is required for either Vid vesicle production or Vid24p binding to Vid vesicles. However, the K48R/K63R mutant did not prevent Vid24p binding to Vid vesicles, indicating that ubiquitin chain formation is dispensable for Vid24p binding to these structures. Our results support the findings that ubiquitin conjugation and ubiquitin chain formation play important roles in a number of cellular processes including organelle biogenesis.

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

“…A reduction in free ubiquitin levels can potentially impair the functions of conjugating enzymes to ubiquitinate substrate proteins. In our previous studies, we showed that at least one of the ubiquitin-conjugating enzymes, Ubc1p, is required for Vid vesicle formation and function in the vacuole pathway (38). Therefore, when the free ubiquitin pool is reduced, Ubc1p activity may be affected.…”

“…For the proteasomal degradation pathway, the N-terminal proline is necessary for the ubiquitination of FBPase (25). However, the vacuolar degradation pathway is unlikely to utilize this system, because FBPase ubiquitination was not observed under the vacuolar degradation conditions (38). FBPase is known to interact with MDH2 under glucose starvation conditions (44).…”

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

“…Interestingly, we have identified a role for ubiquitination in the formation of Vid vesicles. The ⌬ubc1 ubiquitin mutant exhibits a decrease in the amount of Vid vesicles and a concurrent defect in FBPase import and degradation (26). The levels of Vid vesicles were also reduced when cells overexpressed the R48R63 ubiquitin mutant, a mutation that inhibits the formation of multiubiquitin chains (26).…”

The Type 1 Phosphatase Reg1p-Glc7p Is Required for the Glucose-induced Degradation of Fructose-1,6-bisphosphatase in the Vacuole

“…Although Vid vesicle formation is compromised in the absence of the UBC1 gene (17), this is the only gene that is known to be essential for this process. To determine whether Reg1p plays a role in Vid vesicle formation, we performed a differential centrifugation analysis using Vid24p as a specific marker of Vid vesicles.…”

“…These vacuole import and degradation (Vid) vesicles have been purified to near homogeneity and partially characterized (14). The formation of Vid vesicles requires the ubiquitin-conjugating enzyme Ubc1p (17). FBPase import into Vid vesicles is dependent on the heat shock protein Ssa2p (18), cyclophilin A (19), and Vid22p (20).…”