The Targeting Mechanisms and Membrane Topology of Tail-Anchored Proteins

Abstract

<p>Tail-anchored proteins are integral membrane proteins that are characterized by a transmembrane domain at the carboxyl-terminus of protein. The correct targeting and localization of tail-anchored proteins is essential for cell function and viability. In this thesis, the targeting mechanism and membrane topology of tail-anchored proteins were examined using a cell-free assay system. Initially, the targeting of tail-anchored proteins was thought to occur via spontaneous integration into the membrane bilayer. However evidence is presented in this thesis to suggest that more than one targeting mechanism exists. Unlike the spontaneous insertion of tail-anchored proteins such as cytochrome b5, one family of proteins (that includes proteins called Vamps) is shown to require both ATP and a membrane-bound receptor to mediate integration into the bilayer of the endoplasmic reticulum. The minimal region on Vamps that is necessary and sufficient to confer ATP-dependent and receptor mediated targeting was identified. Within this region are four lysine residues spaced along an amphiphatic helix that was found to be necessary for binding of Vamps to endoplasmic reticulum. While spontaneous insertion is sufficient to account for membrane integration for cyctochrome b5 and Bcl-2 data was obtained suggesting that specific localization is achieved by regulating targeting. The membrane topology of one of this class of tail-anchored protein wa examined using chemical modifying techniques. The membrane topology of Bcl-2 is shown to be dynamic, as it changes in cells treated with chemotherapy drugs. This represents the first evidence of a tail-anchored protein that changes membrane topology in response to a cellular signal</p>