The Vid Vesicle to Vacuole Trafficking Event Requires Components of the SNARE Membrane Fusion Machinery

• Published in: The Journal of biological chemistry Vol. 278; no. 28; pp. 25688 - 25699
• Main Authors: Brown, C.Randell, Liu, Jingjing, Hung, Guo-Chiuan, Carter, Donald, Cui, Dongying, Chiang, Hui-Ling
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11-07-2003; American Society for Biochemistry and Molecular Biology
• Subjects: Alkaline Phosphatase - metabolism; Aspartic Acid Endopeptidases - metabolism; Cytosol - metabolism; Fructose-Bisphosphatase - metabolism; Glucose - pharmacology; Membrane Fusion; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Plasmids - metabolism; Qa-SNARE Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - metabolism; SNARE Proteins; Time Factors; Vacuoles - metabolism; Vesicular Transport Proteins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M210549200

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is targeted to Vid vesicles when glucose-starved cells are replenished with glucose. Vid vesicles then deliver FBPase to the vacuole for degradation. A modified alkaline phosphatase assay was developed to study the trafficking of Vid vesicles to the vacuole. For this assay, FBPase was fused with a truncated form of alkaline phosphatase. Under in vivo conditions, FBPase-Δ60Pho8p was targeted to the vacuole via Vid vesicles, and it exhibited Pep4p- and Vid24p-dependent alkaline phosphatase activation. Vid vesicle-vacuole targeting was reconstituted using Vid vesicles that contained FBPase-Δ60Pho8p. These vesicles were incubated with vacuoles in the presence of cytosol and an ATP-regenerating system. Under in vitro conditions, alkaline phosphatase was also activated in a Pep4p- and Vid24p-dependent manner. The GTPase Ypt7p was identified as an essential component in Vid vesicle-vacuole trafficking. Likewise, a number of v-SNAREs (Ykt6p, Nyv1p, Vti1p) and homotypic fusion vacuole protein sorting complex family members (Vps39p and Vps41p) were required for the proper function of Vid vesicles. In contrast, the t-SNARE Vam3p was a necessary vacuolar component. Vid vesicle-vacuole trafficking exhibits characteristics similar to heterotypic membrane fusion events.