Molecular and cellular characterization of GCC185: A tethering protein of the trans-Golgi network

Abstract

Transport vesicle tethers are proteins that link partner membranes together to permit subsequent SNARE protein pairing and fusion. Despite the identification of a relatively large number of tethering proteins, little is known about the precise mechanisms by which they act. Biochemical isolation of tethers permits direct analysis of their physical characteristics and molecular interactions. Here, we describe the expression and purification of GCC185, a trans -Golgi-localized, 190-kDa coiled-coil tethering protein. In addition, we present a gene rescue approach to analyze the function of this tether after its depletion from cells using siRNA.