Lipid Phase Specific Organisation of the Transmembrane Anchor of Influenza Hemagglutinin

Abstract

Correct transport and localization of membrane proteins to distinct cellular organelles are crucial for cell growth but also for the infection cycle of enveloped viruses. Viral membrane proteins like hemagglutinin of influenza virus, anchored in the membrane via a single transmembrane domain (TMD), are transported to the plasma membrane and - together with other virus components - recruited to the budding site. Cholesterol and glycosphingolipid-enriched membrane microdomains are considered as assembly and budding sites for enveloped viruses such as influenza virus. Besides a most likely location of a sorting signal within the TMD sequence also the width of the bilayer is important, which is, amongst other factors, controlled by the cholesterol content of the membrane.Using different fluorescent-based approaches we study the impact of membrane hydrophobic thickness and membrane packing properties on the sorting behavior of virus derived TMD peptides containing a Trp residue in its center. The emission λmax depending on the hydrophobicity of the surrounding of the Trp residue and parallax analysis of fluorescence quenching are used to determine the Trp location in the lipid bilayer revealing a transmembrane orientation. Membrane thickness is altered by the use of lipids having different acyl chain length and also by the addition of cholesterol or decane shifting λmax. Incorporation of Rhodamine labeled TMD into giant unilamellar vesicles prepared from lipids with varying length and also from ternary lipid mixtures forming distinct liquid phases allows us to study phase dependent TMD localization by fluorescence microscopy. Typically, in the model system the TMD sorts into the liquid disordered phase in contrary to the raft association of hemagglutinin in cells. To address the latter by a more appropriate system, we used viral lipids for vesicle preparation mimicking the natural environment of the TMD.