Lipoprotein insertion into membranes of various complexity: lipid sorting, interfacial adsorption and protein clustering

Abstract

In a combined chemical-biological and biophysical approach we explored the membrane partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into membrane systems of different complexity, ranging from one-component lipid bilayers and anionic binary and ternary heterogeneous membrane systems even up to partitioning studies on protein-free and protein-containing giant plasma membrane vesicles (GPMVs). To yield a pictorial view of the localization process, imaging using confocal laser scanning and atomic force microscopy was performed. The results reveal pronounced isoform-specific differences regarding the lateral distribution and formation of protein-rich membrane domains. Line tension is one of the key parameters controlling not only the size and dynamic properties of segregated lipid domains but also the partitioning process of N-Ras that acts as a lineactant. The formation of N-Ras protein clusters is even recorded for almost vanishing hydrophobic mismatch. Conversely, for K-Ras4B, selective localization and clustering are electrostatically mediated by its polybasic farnesylated C-terminus. The formation of K-Ras4B clusters is also observed for the multi-component GPMV membrane, i.e., it seems to be a general phenomenon, largely independent of the details of the membrane composition, including the anionic charge density of lipid headgroups. Our data indicate that unspecific and entropy-driven membrane-mediated interactions play a major role in the partitioning behavior, thus relaxing the need for a multitude of fine-tuned interactions. Such a scenario seems also to be reasonable recalling the high dynamic nature of cellular membranes. Finally, we note that even relatively simple models of heterogeneous membranes are able to reproduce many of the properties of much more complex biological membranes.

Unveiling the Dynamics of KRAS4b on Lipid Model Membranes

“…Thicker arrows correspond to faster rates confirming experimental findings (Chakrabarti et al 2016;Li et al 2018b;Mazhab-Jafari et al 2015;McCarthy et al 2016;Gorfe 2013, 2014;Prakash et al 2016;Travers et al 2018). More specifically, molecular dynamic (MD) simulations suggest that the association of the G-domain to the membrane is lipid-dependent (Cao et al 2019;Gregory et al 2017;Prakash et al 2016), highlighting the role of the C-terminal polybasic hypervariable region (HVR, residues 167-185) (Banerjee et al 2016;Janosi and Gorfe 2010;Neale and Garcia 2018;Zhou et al 2017) and its farnesyl group, both of which are involved in the recruitment of KRAS4b within the anionic-rich lipid domains of membranes (Brunsveld et al 2009;Erwin et al 2016;Jang et al 2015;Plowman et al 2008;Rowat et al 2004;Vogel et al 2009). A recent massive MD simulation (Neale and Garcia 2020) confirmed the lipid-modulatory effect on the dynamics of KRAS4b.…”

The microdomain-organizing protein MPP1 is required for insulin-stimulated activation of H-Ras

“…Both GEFs and GAPs which turn Ras on and off need to be recruited to the PM. Also Raf kinase, effector molecule activated by Ras, needs to be recruited to the PM sites displaying distinct lipid specificity [ 45 ] and it is a critical step for further signaling.…”

RAS Proteins and Their Regulators in Human Disease

“…KRAS4b clusters in “spatially distinct” nucleotide-dependent l d nanoclusters and the polybasic KRAS4b HVR orders lipids as part of a unique signaling complex. These distinct RAS micro-foci may contribute to isoform-specific signaling output as many of the effectors that interact with GTP-bound RAS have their own membrane interaction domains that display lipid specificity (Erwin et al, 2016). …”