Phosphatidylserine (PS) plays a central role in cell signaling and in the biosynthesis of other lipids. To date, however, the subcellular distribution and transmembrane topology of this crucial phospholipid remain ill-defined. We transfected cells with a GFP-tagged C2 domain of lactadherin to detect by light and electron microscopy PS exposed on the cytosolic leaflet of the plasmalemma and organellar membranes. Cytoplasmically exposed PS was found to be clustered on the plasma membrane, and to be associated with caveolae, the trans-Golgi network, and endocytic organelles including intraluminal vesicles of multivesicular endosomes. This labeling pattern was compared with the total cellular distribution of PS as visualized using a novel on-section technique. These complementary methods revealed PS in the interior of the ER, Golgi complex, and mitochondria. These results indicate that PS in the lumenal monolayer of the ER and Golgi complex becomes exposed cytosolically at the trans-Golgi network. Transmembrane flipping of PS may contribute to the exit of cargo from the Golgi complex.

Caveolae are an abundant feature of the plasma membrane in many cells. Until recently, they were generally considered to be membrane invaginations whose formation primarily driven by integral membrane proteins called caveolins. However, the past decade has seen the emergence of the cavin family of peripheral membrane proteins as essential coat components and regulators of caveola biogenesis. In this Commentary, we summarise recent data on the role of cavins in caveola formation, highlighting structural studies that provide new insights into cavin coat assembly. In mammals, there are four cavin family members that associate through homo- and hetero-oligomerisation to form distinct subcomplexes on caveolae, which can be released into the cell in response to stimuli. Studies from several labs have provided a better understanding of cavin stoichiometry and the molecular basis for their oligomerisation, as well as identifying interactions with membrane phospholipids that may be important for caveola function. We propose a model in which coincident, low-affinity electrostatically controlled protein–protein and protein–lipid interactions allow the formation of caveolae, generating a meta-stable structure that can respond to plasma membrane stress by release of cavins.

The plasma membrane nanoscale distribution of H‐ras is regulated by guanine nucleotide binding. To explore the structural basis of H‐ras membrane organization, we combined molecular dynamic simulations and medium‐throughput FRET measurements on live cells. We extracted a set of FRET values, termed a FRET vector, to describe the lateral segregation and orientation of H‐ras with respect to a large set of nanodomain markers. We show that mutation of basic residues in helix α4 or the hypervariable region (HVR) selectively alter the FRET vectors of GTP‐ or GDP‐loaded H‐ras, demonstrating a critical role for these residues in stabilizing GTP‐ or GDP‐H‐ras interactions with the plasma membrane. By a similar analysis, we find that the β2–β3 loop and helix α5 are involved in a novel conformational switch that operates through helix α4 and the HVR to reorient the H‐ras G‐domain with respect to the plasma membrane. Perturbation of these switch elements enhances MAPK activation by stabilizing GTP‐H‐ras in a more productive signalling conformation. The results illustrate how the plasma membrane spatially constrains signalling conformations by acting as a semi‐neutral interaction partner.