The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins.

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.

Hydrophobic and basic domains target proteins to lipid droplets.

In recent years, progress in the study of the lateral organization of the plasma membrane has led to the proposal that mammalian cells use two different organelles to store lipids: intracellular lipid droplets (LDs) and plasma membrane caveolae. Experimental evidence suggests that caveolin (CAV) may act as a sensitive lipid-organizing molecule that physically connects these two lipid-storing organelles. Here, we determine the sequences necessary for efficient sorting of CAV to LDs. We show that targeting is a process cooperatively mediated by two motifs. CAV's central hydrophobic domain (Hyd) anchors CAV to the endoplasmic reticulum (ER). Next, positively charged sequences (Pos-Seqs) mediate sorting of CAVs into LDs. Our findings were confirmed by identifying an equivalent, non-conserved but functionally interchangeable Pos-Seq in ALDI, a bona fide LD-resident protein. Using this information, we were able to retarget a cytosolic protein and convert it to an LD-resident protein. Further studies suggest three requirements for targeting via this mechanism: the positive charge of the Pos-Seq, physical proximity between Pos-Seq and Hyd and a precise spatial orientation between both motifs. The study uncovers remarkable similarities with the signals that target proteins to the membrane of mitochondria and peroxisomes.

Evolutionary analysis and molecular dissection of caveola biogenesis.

Caveolae are an abundant feature of mammalian cells. Integral membrane proteins called caveolins drive the formation of caveolae but the precise mechanisms underlying caveola formation, and the origin of caveolae and caveolins during evolution, are unknown. Systematic evolutionary analysis shows conservation of genes encoding caveolins in metazoans. We provide evidence for extensive and ancient, local and genomic gene duplication, and classify distinct caveolin gene families. Vertebrate caveolin-1 and caveolin-3 isoforms, as well as an invertebrate (Apis mellifera, honeybee) caveolin, all form morphologically identical caveolae in caveolin-1-null mouse cells, demonstrating that caveola formation is a conserved feature of evolutionarily distant caveolins. However, coexpression of flotillin-1 and flotillin-2 did not cause caveola biogenesis in this system. In contrast to the other tested caveolins, C. elegans caveolin is efficiently transported to the plasma membrane but does not generate caveolae, providing evidence of diversity of function in the caveolin gene family. Using C. elegans caveolin as a template to generate hybrid caveolin constructs we now define domains of caveolin required for caveolae biogenesis. These studies lead to a model for caveola formation and novel insights into the evolution of caveolin function.

A novel switch region regulates H-ras membrane orientation and signal output.

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 alpha4 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 beta2-beta3 loop and helix alpha5 are involved in a novel conformational switch that operates through helix alpha4 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.

Lipid rafts and membrane traffic.

Membrane rafts are regions of increased lipid acyl chain order that differ in their lipid and protein composition from the surrounding membrane. By providing an additional level of compartmentalization they have been proposed to serve many functions in cellular signal transduction and trafficking. We will review their potential involvement in different forms of membrane traffic, explicitly excluding signalling, and discuss select aspects of the raft hypothesis in its current form.

Structure and dynamics of the full-length lipid-modified H-Ras protein in a 1,2-dimyristoylglycero-3-phosphocholine bilayer.

Ras proteins regulate signal transduction processes that control cell growth and proliferation. Their disregulation is a common cause of human tumors. Atomic level structural and dynamical information in a membrane environment is crucial for understanding signaling specificity among Ras isoforms and for the design of selective anti-cancer agents. Here, the structure of the full-length H-Ras protein in complex with a 1,2-dimyristoylglycero-3-phosphocholine (DMPC) bilayer obtained from modeling and all-atom explicit solvent molecular dynamics simulations, as well as experimental validation of the main results, are presented. We find that, in addition to the lipid anchor, H-Ras membrane binding involves direct interaction of residues in the catalytic domain with DMPC phosphates. Two modes of binding (possibly modulated by GTP/GDP exchange) differing in the orientation and bilayer contact of the soluble domain as well as in the participation of the flexible linker in membrane binding are proposed. These results are supported by our initial in vivo experiments. The overall structures of the protein and the bilayer remain similar to those of the isolated components, with few localized structural and dynamical changes. The implications of the results to membrane lateral segregation and other aspects of Ras signaling are discussed.

Biogenesis of caveolae: a structural model for caveolin-induced domain formation.

Caveolae are striking morphological features of the plasma membrane of mammalian cells. Caveolins, the major proteins of caveolae, play a crucial role in the formation of these invaginations of the plasma membrane; however, the precise mechanisms involved are only just starting to be unravelled. Recent studies suggest that caveolae are stable structures first generated in the Golgi complex. Their formation and exit from the Golgi complex is associated with caveolin oligomerisation, acquisition of detergent insolubility, and association with cholesterol. Modelling of caveolin-membrane interactions together with in vitro studies of caveolin peptides are providing new insights into how caveolin-lipid interactions could generate the unique architecture of the caveolar domain.

Properties of the interaction of Arf-like protein 2 with PDEdelta.

Arf-like proteins (Arl) share certain characteristic features with the Arf subfamily of Ras superfamily proteins, but their function is unknown. Here, we show by a variety of spectroscopic techniques that Arl2, unlike most other Ras-related proteins, has micromolar rather than picomolar affinity for nucleotides. As a consequence of low affinity, nucleotide dissociation rates are rather fast, arguing that it is not regulated by guanine nucleotide exchange factors. Arl2 is isolated as prey in a yeast double hybrid screen using phosphodiesterase 6delta (PDEdelta) as bait. This interaction is dependent on GTP, and the binding of PDEdelta substantially stabilizes GTP binding, increasing affinity and decreasing dissociation rates by a similar factor. Among all Arl proteins tested, PDEdelta only interacted with the closely related proteins Arl2 and Arl3, strongly suggesting that Arl2/3 are specific regulators of PDEdelta.

The complex of Arl2-GTP and PDE delta: from structure to function.

Arf-like (Arl) proteins are close relatives of the Arf regulators of vesicular transport, but their function is unknown. Here, we present the crystal structure of full-length Arl2-GTP in complex with its effector PDE delta solved in two crystal forms (Protein Data Bank codes 1KSG, 1KSH and 1KSJ). Arl2 shows a dramatic conformational change from the GDP-bound form, which suggests that it is reversibly membrane associated. PDE delta is structurally closely related to RhoGDI and contains a deep empty hydrophobic pocket. Further experiments show that H-Ras, Rheb, Rho6 and G alpha(i1) interact with PDE delta and that, at least for H-Ras, the intact C-terminus is required. We suggest PDE delta to be a specific soluble transport factor for certain prenylated proteins and Arl2-GTP a regulator of PDE delta-mediated transport.