Steady-state localization of transmembrane proteins of the trans Golgi network (TGN) is achieved by dynamic recycling between TGN and endosomal compartments. These transport pathways localize processing enzymes (e.g. Kex2 protease and furin) to their sites of action, effect lysosomal/vacuolar biogenesis and regulate levels of plasma membrane transporters and receptors. The fundamental importance of these pathways is underscored by their evolutionary conservation. The need to define TGN-endosomal transport pathways biochemically led us in the last grant period to begin to develop rapid, quantitative assays for cell-free reconstitution of these transport pathways. In this grant period, we will specifically focus on vesicular transport from the TGN to the late endosome, a key pathway both in lysosomal biogenesis and in processing protein localization that has not been reconstituted in any other system. Our overall goals will be to define mechanisms of vesicle budding, cargo sorting and regulation and vesicle scission in the formation of the clathrin coated vesicles that function in this step. Strong preliminary data, both published and unpublished, form the basis for the proposed work.
The Specific Aims of this proposal are: 1. To determine rigorously whether there is a clathrin-coated vesicle intermediate in the reaction, to purify and characterize this intermediate and determine its molecular composition and to devise a vesicle budding assay that provides a direct assay for cargo recruitment. 2. To elucidate the roles of Gga1/2p versus AP1 adaptors in vesicle formation and cargo selection and determine how they are recruited to membranes. 3. To determine how cargo proteins are recruited to TGN-PVC transport vesicles and how they interact with adaptors or other components of the vesicle coat.
|De, Mithu; Oleskie, Austin N; Ayyash, Mariam et al. (2017) The Vps13p-Cdc31p complex is directly required for TGN late endosome transport and TGN homotypic fusion. J Cell Biol 216:425-439|
|De, Mithu; Abazeed, Mohamed E; Fuller, Robert S (2013) Direct binding of the Kex2p cytosolic tail to the VHS domain of yeast Gga2p facilitates TGN to prevacuolar compartment transport and is regulated by phosphorylation. Mol Biol Cell 24:495-509|
|Komiyama, Tomoko; Coppola, Julia M; Larsen, Martha J et al. (2009) Inhibition of furin/proprotein convertase-catalyzed surface and intracellular processing by small molecules. J Biol Chem 284:15729-38|
|Abazeed, Mohamed E; Fuller, Robert S (2008) Yeast Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins are but adaptor protein-1 is not required for cell-free transport of membrane proteins from the trans-Golgi network to the prevacuolar compartment. Mol Biol Cell 19:4826-36|
|Brace, E J; Parkinson, Leah P; Fuller, Robert S (2006) Skp1p regulates Soi3p/Rav1p association with endosomal membranes but is not required for vacuolar ATPase assembly. Eukaryot Cell 5:2104-13|
|Abazeed, Mohamed E; Blanchette, Jennifer M; Fuller, Robert S (2005) Cell-free transport from the trans-golgi network to late endosome requires factors involved in formation and consumption of clathrin-coated vesicles. J Biol Chem 280:4442-50|
|Blanchette, Jennifer M; Abazeed, Mohamed E; Fuller, Robert S (2004) Cell-free reconstitution of transport from the trans-golgi network to the late endosome/prevacuolar compartment. J Biol Chem 279:48767-73|
|Sipos, Gyorgy; Brickner, Jason H; Brace, E J et al. (2004) Soi3p/Rav1p functions at the early endosome to regulate endocytic trafficking to the vacuole and localization of trans-Golgi network transmembrane proteins. Mol Biol Cell 15:3196-209|
|Sipos, Gyorgy; Fuller, Robert S (2002) Separation of Golgi and endosomal compartments. Methods Enzymol 351:351-65|
|MacLeod, K J; Fuller, R S; Scholten, J D et al. (2001) Conserved cysteine and tryptophan residues of the endothelin-converting enzyme-1 CXAW motif are critical for protein maturation and enzyme activity. J Biol Chem 276:30608-14|
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