Rab8-optineurin-myosin VI: Analysis of interactions and functions in the secretory pathway

Abstract The small GTPase Rab8 has been shown to regulate polarized membrane trafficking pathways from the TGN to the cell surface. Optineurin is an effector protein of Rab8 and a binding partner of the actin‐based motor protein myosin VI. We used various approaches to study the interactions between myosin VI and its binding partners and to analyze their role(s) in intracellular membrane trafficking pathways. In this chapter, we describe the use of the mammalian two‐hybrid assay to demonstrate protein–protein interactions and to identify binding sites. We describe a secretion assay that was used in combination with RNA interference technology to analyze the function of myosin VI, optineurin, and Rab8 in exocytic membrane trafficking pathways.