Volume 398, Issue 5-6 -

GTPases are central switches in cells. Their dysfunctions are involved in severe diseases. The small GTPase Ras regulates cell growth, differentiation and apoptosis by transmitting external signals to the nucleus. In one group of oncogenic mutations, the ‘switch-off’ reaction is inhibited, leading to persistent activation of the signaling pathway. The switch reaction is regulated by GTPase-activating proteins (GAPs), which catalyze GTP hydrolysis in Ras, and by guanine nucleotide exchange factors, which catalyze the exchange of GDP for GTP. Heterotrimeric G-proteins are activated by G-protein coupled receptors and are inactivated by GTP hydrolysis in the Gα subunit. Their GAPs are called regulators of G-protein signaling. In the same way that Ras serves as a prototype for small GTPases, Gα i1 is the most well-studied Gα subunit. By utilizing X-ray structural models, time-resolved infrared-difference spectroscopy, and biomolecular simulations, we elucidated the detailed molecular reaction mechanism of the GTP hydrolysis in Ras and Gα i1 . In both proteins, the charge distribution of GTP is driven towards the transition state, and an arginine is precisely positioned to facilitate nucleophilic attack of water. In addition to these mechanistic details of GTP hydrolysis, Ras dimerization as an emerging factor in signal transduction is discussed in this review.

The prenyl binding protein PDEδ enhances the diffusion of farnesylated Ras proteins in the cytosol, ultimately affecting their correct localization and signaling. This has turned PDEδ into a promising target to prevent oncogenic KRas signaling. In this review we summarize and describe the structure-guided-development of the three different PDEδ inhibitor chemotypes that have been documented so far. We also compare both their potency for binding to the PDEδ pocket and their in vivo efficiency in suppressing oncogenic KRas signaling, as a result of the inhibition of the PDEδ/KRas interaction.

The partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into various membrane systems, ranging from single-component fluid bilayers, binary fluid mixtures, heterogeneous raft model membranes up to complex native-like lipid mixtures (GPMVs) in the absence and presence of integral membrane proteins have been explored in the last decade in a combined chemical-biological and biophysical approach. These studies have revealed pronounced isoform-specific differences regarding the lateral distribution in membranes and formation of protein-rich membrane domains. In this context, we will also discuss the effects of lipid head group structure and charge density on the partitioning behavior of the lipoproteins. Moreover, the dynamic properties of N-Ras and K-Ras4B have been studied in different model membrane systems and native-like crowded milieus. Addition of crowding agents such as Ficoll and its monomeric unit, sucrose, gradually favors clustering of Ras proteins in forming small oligomers in the bulk; only at very high crowder concentrations association is disfavored.

Our understanding of the manner in which Rab proteins regulate intracellular vesicular transport has progressed remarkably in the last one or two decades by application of a wide spectrum of biochemical, biophysical and cell biological methods, augmented by the methods of chemical biology. Important additional insights have arisen from examination of the manner in which certain bacteria can manipulate vesicular transport mechanisms. The progress in these areas is summarized here.

Ras GTPases are key players in cellular signalling because they act as binary switches. These states manifest through toggling between an active (GTP-loaded) and an inactive (GDP-loaded) form. The hydrolysis and replenishing of GTP is controlled by two additional protein classes: GAP (GTPase-activating)- and GEF (Guanine nucleotide exchange factors)-proteins. The complex interplay of the proteins is known as the GTPase-cycle. Several point mutations of the Ras protein deregulate this cycle. Mutations in Ras are associated with up to one-third of human cancers. The three isoforms of Ras (H, N, K) exhibit high sequence similarity and mainly differ in a region called HVR (hypervariable region). The HVR governs the differential action and cellular distribution of the three isoforms. Rheb is a Ras-like GTPase that is conserved from yeast to mammals. Rheb is mainly involved in activation of cell growth through stimulation of mTORC1 activity. In this review, we summarise multidimensional NMR studies on Rheb and Ras carried out to characterise their structure-function relationship and explain how the activity of these small GTPases can be modulated by low molecular weight compounds. These might help to design GTPase-selective antagonists for treatment of cancer and brain disease.

The small GTPase Rheb was originally detected as an immediate early response protein whose expression was induced by NMDA-dependent synaptic activity in the brain. Rheb’s activity is highly regulated by its GTPase activating protein (GAP), the tuberous sclerosis complex protein, which stimulates the conversion from the active, GTP-loaded into the inactive, GDP-loaded conformation. Rheb has been established as an evolutionarily conserved molecular switch protein regulating cellular growth, cell volume, cell cycle, autophagy, and amino acid uptake. The subcellular localization of Rheb and its interacting proteins critically regulate its activity and function. In stem cells, constitutive activation of Rheb enhances differentiation at the expense of self-renewal partially explaining the adverse effects of deregulated Rheb in the mammalian brain. In the context of various cellular stress conditions such as oxidative stress, ER-stress, death factor signaling, and cellular aging, Rheb activation surprisingly enhances rather than prevents cellular degeneration. This review addresses cell type- and cell state-specific function(s) of Rheb and mainly focuses on neurons and their surrounding glial cells. Mechanisms will be discussed in the context of therapy that interferes with Rheb’s activity using the antibiotic rapamycin or low molecular weight compounds.

In peroxisomal matrix protein import two processes directly depend on the binding and hydrolysis of ATP, both taking place at the late steps of the peroxisomal import cycle. First, ATP hydrolysis is required to initiate a ubiquitin-transfer cascade to modify the import (co-)receptors. These receptors display a dual localization in the cytosol and at the peroxisomal membrane, whereas only the membrane bound fraction receives the ubiquitin modification. The second ATP-dependent process of the import cycle is carried out by the two AAA + -proteins Pex1p and Pex6p. These ATPases form a heterohexameric complex, which is recruited to the peroxisomal import machinery by the membrane anchor protein Pex15p. The Pex1p/Pex6p complex recognizes the ubiquitinated import receptors, pulls them out of the membrane and releases them into the cytosol. There the deubiquitinated receptors are provided for further rounds of import. ATP binding and hydrolysis are required for Pex1p/Pex6p complex formation and receptor export. In this review, we summarize the current knowledge on the peroxisomal import cascade. In particular, we will focus on the ATP-dependent processes, which are so far best understood in the model organism Saccharomyces cerevisiae .

Cellular proteomes are dynamic and adjusted to permanently changing conditions by ATP-fueled proteolytic machineries. Among the five AAA + proteases in Escherichia coli FtsH is the only essential and membrane-anchored metalloprotease. FtsH is a homohexamer that uses its ATPase domain to unfold and translocate substrates that are subsequently degraded without the need of ATP in the proteolytic chamber of the protease domain. FtsH eliminates misfolded proteins in the context of general quality control and properly folded proteins for regulatory reasons. Recent trapping approaches have revealed a number of novel FtsH substrates. This review summarizes the substrate diversity of FtsH and presents details on the surprisingly diverse recognition principles of three well-characterized substrates: LpxC, the key enzyme of lipopolysaccharide biosynthesis; RpoH, the alternative heat-shock sigma factor and YfgM, a bifunctional membrane protein implicated in periplasmic chaperone functions and cytoplasmic stress adaptation.

Crystal structures of small GTP binding protein complexes with their effectors and regulators reveal that one particularly flat side of the G domain that contains helix α4 and the C-terminal helix α5 is practically devoid of contacts. Although this observation seems trivial as the main binding targets are the switch I and II regions opposite of this side, the fact that all interacting proteins, even the largest ones, seem to avoid occupying this area (except for Ran, that does not localize to membranes) is very striking. An orientation with this ‘flat’ side parallel to the membrane was proposed before and would allow simultaneous interaction of the lipidated C-terminus and positive charges in the α4 helix with the membrane while being bound to effector or regulator molecules. Furthermore, this ‘flat’ side might be involved in regulatory mechanisms: a Ras dimer that is found in different crystal forms interacts exactly at this side. Additional interface analysis of GTPase complexes nicely confirms the effect of different flexibilities of the GTP and GDP forms. Besides Ran proteins, guanine nucleotide exchange factors (GEFs) bury the largest surface areas to provide the binding energy to open up the switch regions for nucleotide exchange.

Chloroplasts derive from a prokaryotic symbiont that lost most of its genes during evolution. As a result, the great majority of chloroplast proteins are encoded in the nucleus and are posttranslationally imported into the organelle. The chloroplast genome encodes only a few proteins. These include several multispan thylakoid membrane proteins which are synthesized on thylakoid-bound ribosomes and cotranslationally inserted into the membrane. During evolution, ancient prokaryotic targeting machineries were adapted and combined with novel targeting mechanisms to facilitate post- and cotranslational protein transport in chloroplasts. This review focusses on the chloroplast signal recognition particle (cpSRP) protein transport system, which has been intensively studied in higher plants. The cpSRP system derived from the prokaryotic SRP pathway, which mediates the cotranslational protein transport to the bacterial plasma membrane. Chloroplasts contain homologs of several components of the bacterial SRP system. The function of these conserved components in post- and/or cotranslational protein transport and chloroplast-specific modifications of these transport mechanisms are described. Furthermore, recent studies of cpSRP systems in algae and lower plants are summarized and their impact on understanding the evolution of the cpSRP system are discussed.

Small GTP-hydrolyzing enzymes (GTPases) of the RhoA family play manifold roles in cell biology and are regulated by upstream guanine nucleotide exchange factors (GEFs). Herein, we focus on the GEFs of the Vav subfamily. Vav1 was originally described as a proto-oncogene of the hematopoietic lineage. The GEFs Vav2 and Vav3 are more broadly expressed in various tissues. In particular, the GEF Vav3 may play important roles in the developing nervous system during the differentiation of neural stem cells into the major lineages, namely neurons, oligodendrocytes and astrocytes. We discuss its putative regulatory roles for progenitor differentiation in the developing retina, polarization of neurons and formation of synapses, migration of oligodendrocyte progenitors and establishment of myelin sheaths. We propose that Vav3 mediates the response of various neural cell types to environmental cues.

The class III phosphatidylinositol 3-kinase Vps34 (vacuolar protein sorting 34) catalyzes for the formation of the signaling lipid phosphatidylinositol-3-phopsphate, which is a central factor in the regulation of autophagy, endocytic trafficking and vesicular transport. In this article, we discuss the functional role of the lipid kinase Vps34 in Saccharomyces cerevisiae .

Quantitative mass spectrometry approaches are used for absolute and relative quantification in global proteome studies. To date, relative and absolute quantification techniques are available that differ in quantification accuracy, proteome coverage, complexity and robustness. This review focuses on most common relative or absolute quantification strategies exemplified by three experimental studies. A label-free relative quantification approach was performed for the investigation of the membrane proteome of sensory cilia to the depth of olfactory receptors in Mus musculus . A SILAC-based relative quantification approach was successfully applied for the identification of core components and transient interactors of the peroxisomal importomer in Saccharomyces cerevisiae . Furthermore, AQUA using stable isotopes was exemplified to unraveling the prenylome influenced by novel prenyltransferase inhibitors. Characteristic enrichment and fragmentation strategies for a robust quantification of the prenylome are also summarized.

Ras-mediated apoptotic signaling is expected to be mediated via Rassf-MST complexes, but the system has been poorly characterized in vitro until now. Here we demonstrate that active H-Ras, Nore1A and MST1 form a stable ternary complex in vitro without other external factors, Nore1A interacting simultaneously with H-Ras and MST1 via its RBD and SARAH domain, respectively. Moreover, our data show for the first time that the SARAH domain of Nore1A plays a role in the Nore1A binding to H-Ras. Finally, we analyze the relation between the electrostatic and hydrophobic forces and kinetic constants of the Nore1A – H-Ras complex.