title: A Novel Assay to Identify the Trafficking Proteins that Bind to Specific Vesicle Populations.
abstract: Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin binding domain (FRB)-tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled.
authors.affiliation: Jungers Center for Neurosciences Research, Oregon Health & Science University, Portland, Oregon.
authors.forename: Marvin
authors.initials: M
authors.lastname: Bentley
authors.affiliation: Jungers Center for Neurosciences Research, Oregon Health & Science University, Portland, Oregon.
authors.forename: Gary
authors.initials: G
authors.lastname: Banker
mesh_terms: D000818:Animals; D002460:Cell Line; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D016547:Kinesins / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism*
chemical_list: D020409:Molecular Motor Proteins; D049452:Green Fluorescent Proteins; D058570:TOR Serine-Threonine Kinases; D016547:Kinesins; D022061:Tacrolimus Binding Protein 1A
keywords: FKBP; FRB; Rab; kinesin; membrane trafficking; vesicle