The Vid vesicle to vacuole trafficking event requires components of the SNARE membrane fusion machinery.
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-delta60Pho8p 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-delta60Pho8p. 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.
Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania 17033, USA. crb13@psu.edu
C Randell
CR
Brown
Jingjing
J
Liu
Guo-Chiuan
GC
Hung
Donald
D
Carter
Dongying
D
Cui
Hui-Ling
HL
Chiang
D000469:Alkaline Phosphatase / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013997:Time Factors; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C107706:VAM3 protein, S cerevisiae; C111452:VID24 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D000469:Alkaline Phosphatase; D006597:Fructose-Bisphosphatase; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases; D005947:Glucose