Identification of motor protein cargo by yeast 2-hybrid and affinity approaches.
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.
Zhang
Yuguo
Y
Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA, USA.
Wang
Rong
R
Jefferson
Holly
H
Sperry
Ann O
AO
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
D005656:Fungal Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D016547:Kinesins