GAIP participates in budding of membrane carriers at the trans-Golgi network.
Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans-Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.
Wylie
Fiona G
FG
Institute for Molecular Bioscience and Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland, Australia.
Lock
John G
JG
Jamriska
Lubomira
L
Khromykh
Tatiana
T
Brown
Darren L
DL
Stow
Jennifer L
JL
D000818:Animals; D002458:Cell Fractionation; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018715:Microscopy, Video; D016296:Mutagenesis; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D021601:trans-Golgi Network / Q000502:physiology* / Q000648:ultrastructure*
D020690:GTPase-Activating Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins