A Designed Probe for Acidic Phospholipids Reveals the Unique Enriched Anionic Character of the Cytosolic Face of the Mammalian Plasma Membrane

Nicole M Okeley,Michael H Gelb

doi:10.1074/jbc.m313469200

Abstract

It is generally accepted that the cytosolic face of the plasma membrane of mammalian cells is enriched in acidic phospholipids due to an asymmetric distribution of neutral and anionic phospholipids in the two bilayer leaflets. However, the phospholipid asymmetry across intracellular membranes is not known. Two models have been proposed for the selective targeting of K-Ras4B, which contains a C-terminal farnesyl cysteine methyl ester adjacent to a polybasic peptide segment, to the cytosolic face of the plasma membrane. One involves electrostatic interaction of the lipidated polybasic domain with anionic phospholipids in the plasma membrane, and the other involves binding of K-Ras4B to a specific protein receptor. To address this issue, we prepared by semi-synthesis a green fluorescent protein variant that is linked to a farnesylated, polybasic peptide corresponding to the K-Ras4B C terminus as well as a variant that contains an all-d amino acid version of the K-Ras4B peptide. As expected based on electrostatics, both constructs showed preferential in vitro binding to anionic phospholipid vesicles versus those composed only of zwitterionic phospholipid. Both constructs fully targeted to the plasma membrane when microinjected into live Chinese hamster ovary and Madin-Darby canine kidney cells. Because the all-d amino acid peptide should be devoid of binding affinity to a putative highly specific K-Ras membrane receptor, these results support an electrostatic basis for the targeting of K-Ras4B to the plasma membrane, and they support an intracellular landscape of phospholipids in which the cytosolic face of the plasma membrane is the most enriched in acidic phospholipids.

Highlights

It is well known that the lipids that constitute the plasma membrane of eukaryotic cells are asymmetrically distributed across the two bilayer leaflets [1, 2]
It is generally accepted that the cytosolic face of the plasma membrane of mammalian cells is enriched in acidic phospholipids due to an asymmetric distribution of neutral and anionic phospholipids in the two bilayer leaflets
It has been suggested that the electrostatic interaction of a polybasic domain with acidic phospholipids is the basis of K-Ras4B and SRC binding to the plasma membrane

Summary

Introduction

It is well known that the lipids that constitute the plasma membrane of eukaryotic cells are asymmetrically distributed across the two bilayer leaflets [1, 2]. Other proteins display preferential in vitro binding to PS-rich membranes compared with those rich in PC and are known to localize to the cytosolic face of the plasma membrane, for example K-Ras4B [12, 13] and Src kinase (14 –16), which contain lipid anchors adjacent to a string of lysine and arginine residues. Similar results have been observed with the SRC N-terminal domain [19, 20], which contains an N-terminal myristoyl group followed by a peptide segment enriched in basic residues Despite this support for a major electrostatic role in K-Ras4B and SRC targeting, the mechanism by which these proteins are targeted to the cytosolic face of the plasma membrane is still debated. We have developed such a probe based on the C terminus of K-Ras4B and have studied its targeting in mammalian cells

Methods

Results

Conclusion