Interactions of Lipidated Ras Proteins With Raft Membranes Studied By Time-Lapse Atomic Force Microscopy

Abstract

The existence of membrane subdomains with different lipid composition and the relationship between lipid-domain formation and the conformation and functional properties of membrane-associated proteins is one of the central questions in the fields of membrane biochemistry and biophysics. It has been suggested that raft domains play a role in signal transduction processes by acting as “signaling platforms”. The three Ras isoforms are posttranslationally modified via lipidation on their C-termini, which is essential for correct functioning and localization at the inner leaflet of the plasma membrane.By using semisynthetic fully functional lipidated N- and K-Ras proteins, the partitioning of Ras in liquid-disordered (ld) and liquid-ordered (lo, i.e., raft-like) subdomains of different artificial and natural membranes was studied by time-lapse atomic force microscopy. The results provide direct evidence that partitioning of Ras occurs preferentially into ld domains, independent of the lipid anchor system and GDP/GTP-loading [1,2]. Whereas N-Ras proteins bearing at least one farnesyl showed a time dependent diffusion and subsequent clustering in the lo/ld phase boundary region, formation of new domains with accumulated protein inside a fluid-like environment was observed for the farnesylated, inactive K-Ras protein. The inserted farnesylated N-Ras proteins are expelled to the interfacial region, probably due to the lack of a particular phase preference, while for the K-Ras protein the strong electrostatic interaction between its positively charged lysines and negatively charged lipids of the membrane seems to control the partitioning behavior. Minimizing the line energy is likely to be one of the key parameters controlling not only the size and dynamic properties of rafts but also of signaling platforms.