title: Identification of motor protein cargo by yeast 2-hybrid and affinity approaches.
abstract: Identification of the molecular composition of the cargo transported by individual kinesin motors is critical to an understanding of both motor function and regulation of the proper intracellular placement of numerous cellular components including proteins, RNA, and organelles. In this chapter, we describe methods to identify the motor tail sequences responsible for cargo binding by expression of green fluorescent protein (GFP)-motor tail fusion proteins in mammalian cells. In addition, we detail two complementary approaches to identify specific proteins associated with these targeting sequences: a yeast 2-hybrid screen and affinity chromatography.
authors.affiliation: Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA, USA.
authors.forename: Yuguo
authors.initials: Y
authors.lastname: Zhang
authors.forename: Rong
authors.initials: R
authors.lastname: Wang
authors.forename: Holly
authors.initials: H
authors.lastname: Jefferson
authors.forename: Ann O
authors.initials: AO
authors.lastname: Sperry
mesh_terms: D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D016547:Kinesins / Q000737:chemistry*; D051379:Mice; D008870:Microtubules / Q000737:chemistry*; D041681:NIH 3T3 Cells; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques
chemical_list: D005656:Fungal Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D016547:Kinesins