The vacuolar import and degradation pathway merges with the endocytic pathway to deliver fructose-1,6-bisphosphatase to the vacuole for degradation.
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 epsilon-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 Deltavph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway.
Brown
C Randell
CR
Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Wolfe
Allison B
AB
Cui
Dongying
D
Chiang
Hui-Ling
HL
D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D020755:Coat Protein Complex I / Q000737:chemistry; D020756:Coatomer Protein / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004705:Endocytosis; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
D020755:Coat Protein Complex I; D020756:Coatomer Protein; D003432:Cross-Linking Reagents; D029701:Saccharomyces cerevisiae Proteins; C532448:Sec28 protein, S cerevisiae; D049452:Green Fluorescent Proteins; D006597:Fructose-Bisphosphatase