Biochemical analysis of fructose-1,6-bisphosphatase import into vacuole import and degradation vesicles reveals a role for UBC1 in vesicle biogenesis.
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 Deltaubc1 and the K48R/K63R mutants were attributed to the Vid vesicle fraction. In the Deltaubc1 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.
Shieh
H L
HL
Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, Hershey 17033, USA.
Chen
Y
Y
Brown
C R
CR
Chiang
H L
HL
D001692:Biological Transport; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000378:metabolism* / Q000502:physiology*; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C066752:UBC1 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D006597:Fructose-Bisphosphatase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; D005947:Glucose