C-terminal Membrane Research Articles

Membrane mediated regulation in free peptides of HIV-1 gp41: minimal modulation of the hemifusion phase

Membrane-mediated structural modulation in two short fragments of the human HIV-1 envelope protein gp41 is demonstrated. Derived from the C-terminal membrane proximal external (MPE) and N-terminal fusion peptide proximal (FPP) regions, these peptides are widely separated in the primary sequence but form tertiary contacts during the intermediate (hemifusion) phase of HIV infection. The structural perturbations observed at the membrane interface offer evidence of rudimentary regulatory mechanisms operating in the free peptides which may be relevant in the biological system. No such regulatory phenomena were observed for the individual peptides in a membrane environment or between the peptides in aqueous solutions. Structure determination is made using a combination of circular and linear dichroism spectroscopy (supported by calorimetric measurements) and molecular dynamics simulations. Specifically, we show that these peptides interact locally without the conformational support of helical heptad repeat regions in native gp41 and that the modulation is not mutual with the FPP peptide operating as a primary regulator of the MPE-FPP interactions in the hemifusion phase.

Subcellular targeting domains of sphingomyelin synthase 1 and 2.

Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. Furthermore, its product SM has been implicated in atherogenic processes such as retention of lipoproteins in the blood vessel intima. There are two mammalian sphingomyelin synthases: SMS1 and SMS2. SMS1 is found exclusively in the Golgi at steady state, whereas SMS2 exists in the Golgi and plasma membrane. Conventional motifs responsible for protein targeting to the plasma membrane or Golgi are either not present in, or unique to, SMS1 and SMS2. In this study, we examined how SMS1 and SMS2 achieve their respective subcellular localization patterns. Brefeldin A treatment prevented SMS1 and SMS2 from exiting the ER, demonstrating that they transit through the classical secretory pathway. We created truncations and chimeras of SMS1 and SMS2 to define their targeting signals. We found that SMS1 contains a C-terminal Golgi targeting signal and that SMS2 contains a C-terminal plasma membrane targeting signal.

Biochemistry and Biophysics of HIV-1 gp41 - Membrane Interactions and Implications for HIV-1 Envelope Protein Mediated Viral-Cell Fusion and Fusion Inhibitor Design

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

Functional anatomy of the Arabidopsis cytokinesis‐specific syntaxin KNOLLE

In plant cytokinesis, Golgi/trans-Golgi network-derived vesicles are targeted to the plane of cell division where they fuse with one another to form the partitioning membrane (cell plate). This membrane fusion requires a specialised syntaxin (Qa-SNARE), named KNOLLE in Arabidopsis. KNOLLE is only made during the M-phase of the cell cycle, targeted to the plane of cell division and degraded in the vacuole at the end of cytokinesis. To identify the parts of KNOLLE required for proper targeting and function in membrane fusion, we generated chimeric syntaxins comprising complementary fragments from KNOLLE and MVB-localized PEP12 (SYP21). Surprisingly, targeting of the chimeric protein was not specified by the C-terminal membrane anchor. Rather the N-terminal region including helix Ha and the adjacent linker to helix Hb appeared to played a critical role. However, deletion of this N-terminal fragment from KNOLLE (KN(Δ1-82) ) had the same effect as its presence in the chimeric protein (KN(1-82) -PEP12(64-279) ), suggesting that targeting to the plane of cell division occurs by default, i.e. when no sorting signal would target the syntaxin to a specific endomembrane compartment. Once the full-length syntaxin accumulated at the plane of division, phenotypic rescue of the knolle mutant only required the SNARE domain plus the adjacent linker connecting helix Hc to the SNARE domain from KNOLLE. Our results suggest that targeting of syntaxin to the plane of cell division occurs without active sorting, whereas syntaxin-mediated membrane fusion requires sequence-specific features.

Release of ActA into a medium via membrane anchor cleavage is required for Listeria monocytogenes host cell invasion

The ActA protein, which is involved in intra- and intercellular Listeria monocytogenes movement, is anchored in the bacterial membrane by a hydrophobic C-tail, and it is a membrane-bound form of ActA that is indispensable for intracellular movement. However, soluble ActA is detected in the culture supernatant of L. monocytogenes as a major protein. In this study, the mechanisms of ActA release into the culture supernatant and the role that soluble ActA plays in intracellular virulence were investigated. Our results indicated that the cleavage of the C-terminal membrane anchor is required for ActA release. The MALDI-TOF MS analysis of trypsin-cut soluble ActA suggested that the cleavage occurred between His582 and Thr583. The proposed cleavage site was confirmed by introduction of the His582Pro substitution preventing ActA release, although this did not disturb its surface presentation. The virulence-associated metalloprotease Mpl, which is responsible for pH-dependent ActA degradation, is not involved in membrane anchor cleavage, which requires another still unidentified protease. The mutant strain had a lower invasive effectiveness into HeLa epithelial cells, whereas it did not differ from the wild strain in intracellular proliferation and the ability to form plaques of lysis in the HeLa confluent monolayer. The obtained results suggested that ActA release is important for the invasion of L. monocytogenes into epithelial cells but is not required for intracellular motility and cell-to-cell spread.

ChemInform Abstract: Autotransporters with GDSL Passenger Domains: Molecular Physiology and Biotechnological Applications

AbstractReview: 77 refs.

Autotransporters with GDSL Passenger Domains: Molecular Physiology and Biotechnological Applications

Autotransporters are large proteins produced and secreted by Gram-negative bacteria. They consist of an N-terminal passenger domain, which typically harbours enzymatic activity and exerts a virulence function, and a C-terminal membrane anchor domain. Somehow, the membrane domain facilitates the transport of the passenger domain into the extracellular space. Several autotransporters possess hydrolase passenger domains that belong to the GDSL family of lipolytic enzymes. GDSL autotransporters represent a functionally distinct family and are characterized by several features of their passenger domains; these include 1) the absence of a conserved right-handed parallel β-helix, 2) lipolytic activity, and thus the capability to hydrolyse membranes, and 3) covalent attachment to the respective C-terminal β-domain, with the hydrolase domain exposed to the exterior. The esterase EstA of Pseudomonas aeruginosa is a typical enzyme of this type. Its physiological role was studied, its potential biotechnological application has been demonstrated, and its crystal structure was solved recently. Furthermore, it is capable of displaying different classes of enzymes in a range of Gram-negative bacteria including Escherichia coli, and FACS-based high-throughput screening for enantioselective esterases could be achieved using EstA.

Ras Signaling in Yeast

Since the study of yeast RAS and adenylate cyclase in the early 1980s, yeasts including budding and fission yeasts contributed significantly to the study of Ras signaling. First, yeast studies provided insights into how Ras activates downstream signaling pathways. Second, yeast studies contributed to the identification and characterization of GAP and GEF proteins, key regulators of Ras. Finally, the study of yeast provided many important insights into the understanding of C-terminal processing and membrane association of Ras proteins.

An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability

Membrane proteins account for about 30% of the genomes sequenced to date and play important roles in a variety of cellular functions. However, determining the three-dimensional structures of membrane proteins continues to pose a major challenge for structural biologists due to difficulties in recombinant expression and purification. We describe here a high throughput pipeline for Escherichia coli based membrane protein expression and purification. A ligation-independent cloning (LIC)-based vector encoding a C-terminal green fluorescence protein (GFP) tag was used for cloning in a high throughput mode. The GFP tag facilitated expression screening in E. coli through both cell culture fluorescence measurements and in-gel fluorescence imaging. Positive candidates from the GFP screening were subsequently sub-cloned into a LIC-based, GFP free vector for further expression and purification. The expressed, C-terminal His-tagged membrane proteins were purified via membrane enrichment and Ni-affinity chromatography. Thermofluor technique was applied to screen optimal buffers and detergents for the purified membrane proteins. This pipeline has been successfully tested for membrane proteins from E. coli and can be potentially expanded to other prokaryotes.

Outer Membrane Secretion Efficiency of Autotransporter Virulence Proteins Correlates with Passenger Domain Folding Properties

Autotransporters (ATs) are the largest class of extracellular virulence proteins secreted from Gram-negative bacteria. Each AT is synthesized as a tripartite pre-protein containing an N-terminal signal sequence that directs secretion across the inner membrane, a central “passenger” domain that becomes the mature extracellular virulence protein, and a C-terminal outer membrane (OM) porin domain that is essential for OM transport. AT passenger domains have highly diverse sequences, lengths, and functions, but almost all are predicted to contain β-helical structure. It was originally proposed that an AT protein autonomously catalyzes transport of its own passenger domain across the OM, but recent results have cast doubt on this model. Moreover, in the absence of a significant quantity of ATP or proton gradient across the OM, the driving force for efficient OM secretion remains unclear. Here we demonstrate a direct correlation between localized regions of AT passenger domain stability (ΔGunfolding) and OM secretion efficiency. Destabilizing the C-terminus of a passenger domain β-helix significantly reduced OM secretion efficiency. In contrast, destabilizing more N-terminal portions of the passenger domain produced a linearly correlated increase in OM secretion efficiency. Thus, C-terminal passenger domain stability facilitates OM secretion, whereas N-terminal stability hinders it. The contributions of regional passenger stability to OM secretion efficiency demonstrate a crucial role for the passenger domain itself in directing its secretion across the OM. These results indicate that the folding properties of distinct regions of the passenger domain provide a directed molecular driving force, for use as a generalized transporter device. The regionalized distribution of AT passenger domain stability provides a unique solution for the directed transport of macromolecules across biological membranes, defining a new category of ATP-independent Brownian motor.

Tracking Glideosome-Associated Protein 50 Reveals the Development and Organization of the Inner Membrane Complex of Plasmodium falciparum

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion, and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Ras-mediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth-transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar, and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively activated RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Ras-mediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.

Adhesive Activity of the Haemophilus Cryptic Genospecies Cha Autotransporter Is Modulated by Variation in Tandem Peptide Repeats

The Haemophilus cryptic genospecies is an important cause of maternal genital tract and neonatal systemic infections and initiates infection by colonizing the genital or respiratory epithelium. In recent work, we identified a unique Haemophilus cryptic genospecies protein called Cha, which mediates efficient adherence to genital and respiratory epithelia. The Cha adhesin belongs to the trimeric autotransporter family and contains an N-terminal signal peptide, an internal passenger domain that harbors adhesive activity, and a C-terminal membrane anchor domain. The passenger domain in Cha contains clusters of YadA-like head domains and neck motifs as well as a series of tandem 28-amino-acid peptide repeats. In the current study, we report that variation in peptide repeat number gradually modulates Cha adhesive activity, associated with a direct effect on the length of Cha fibers on the bacterial cell surface. The N-terminal 404 residues of the Cha passenger domain mediate binding to host cells and also facilitate bacterial aggregation through intermolecular Cha-Cha binding. As the tandem peptide repeats expand, the Cha fiber becomes longer and Cha adherence activity decreases. The expansion and contraction of peptide repeats represent a novel mechanism for modulating adhesive capacity, potentially balancing the need of the organism to colonize the genital and respiratory tracts with the ability to attach to alternative substrates, disperse within the host, or evade the host immune system.

Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

BackgroundPathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region.Methodology/Principal FindingsPathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 Å resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs.Conclusions/SignificanceThe modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics.

Nix Is Critical to Two Distinct Phases of Mitophagy, Reactive Oxygen Species-mediated Autophagy Induction and Parkin-Ubiquitin-p62-mediated Mitochondrial Priming

Damaged mitochondria can be eliminated by autophagy, i.e. mitophagy, which is important for cellular homeostasis and cell survival. Despite the fact that a number of factors have been found to be important for mitophagy in mammalian cells, their individual roles in the process had not been clearly defined. Parkin is a ubiquitin-protein isopeptide ligase able to translocate to the mitochondria that are to be removed. We showed here in a chemical hypoxia model of mitophagy induced by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP) that Parkin translocation resulted in mitochondrial ubiquitination and p62 recruitment to the mitochondria. Small inhibitory RNA-mediated knockdown of p62 significantly diminished mitochondrial recognition by the autophagy machinery and the subsequent elimination. Thus Parkin, ubiquitin, and p62 function in preparing mitochondria for mitophagy, here referred to as mitochondrial priming. However, these molecules were not required for the induction of autophagy machinery. Neither Parkin nor p62 seemed to affect autophagy induction by CCCP. Instead, we found that Nix was required for the autophagy induction. Nix promoted CCCP-induced mitochondrial depolarization and reactive oxygen species generation, which inhibited mTOR signaling and activated autophagy. Nix also contributed to mitochondrial priming by controlling the mitochondrial translocation of Parkin, although reactive oxygen species generation was not involved in this step. Deletion of the C-terminal membrane targeting sequence but not mutations in the BH3 domain disabled Nix for these functions. Our work thus distinguished the molecular events responsible for the different phases of mitophagy and placed Nix upstream of the events.

Regulation of the Rho Family Small GTPase Wrch-1/RhoU by C-Terminal Tyrosine Phosphorylation Requires Src

Wrch-1 is an atypical Rho family small GTPase with roles in migration, epithelial cell morphogenesis, osteoclastogenesis, and oncogenic transformation. Here, we observed rapid relocalization of Wrch-1 from the plasma membrane upon serum stimulation. Studies revealed a requirement for serum-stimulated tyrosine phosphorylation of Wrch-1 at residue Y254 within its C-terminal membrane targeting domain, mediated by the nonreceptor tyrosine kinase Src. Genetic or pharmacological loss of Src kinase activity blocked both phosphorylation and relocalization of Wrch-1. Functionally, Y254 was required for proper Wrch-1 modulation of cystogenesis in three-dimensional culture, and the phospho-deficient mutant, Y254F, was enhanced in Wrch-1-mediated anchorage-independent growth. Mechanistically, C-terminal tyrosine phosphorylation and subsequent relocalization of Wrch-1 downregulated its ability to interact with and activate its effectors by decreasing active Wrch-1-GTP, perhaps by altering proximity to a GEF or GAP. Phospho-deficient Wrch-1(Y254F) remained at the plasma membrane and GTP bound and continued to recruit and activate its effector PAK, even upon serum stimulation. In contrast, a phospho-mimetic mutant, Y254E, was constitutively endosomally localized and GDP bound and failed to recruit PAK unless mutated to be constitutively active/GAP insensitive. C-terminal tyrosine phosphorylation thus represents a new paradigm in posttranslational control of small GTPase localization, activation, and biological function.

Comparative analysis of Drosophila and mammalian complexins as fusion clamps and facilitators of neurotransmitter release

The SNARE-binding protein complexin (Cpx) has been demonstrated to regulate synaptic vesicle fusion. Previous studies are consistent with Cpx functioning either as a synaptic vesicle fusion clamp to prevent premature exocytosis, or as a facilitator to directly stimulate release. Here we examined conserved roles of invertebrate and mammalian Cpx isoforms in the regulation of neurotransmitter release using the Drosophila neuromuscular junction as a model synapse. We find that SNARE binding by Cpx is required for its role as a fusion clamp. All four mammalian Cpx proteins (mCpx), which have been demonstrated to facilitate release, also function as fusion clamps when expressed in Drosophila cpx null mutants, though their clamping abilities vary between isoforms. Moreover, expression of mCpx I, II or III isoforms dramatically enhance evoked release compared to mCpx IV or Drosophila Cpx. Differences in the clamping and facilitating properties of complexin isoforms can be partially attributed to differences in the C-terminal membrane tethering domain. Our findings indicate that the function of complexins as fusion clamps and facilitators of fusion are conserved across evolution, and that these roles are genetically separable within an isoform and across different isoforms.

The Drosophila Anion Exchanger (DAE) lacks a detectable interaction with the spectrin cytoskeleton

BackgroundCurrent models suggest that the spectrin cytoskeleton stabilizes interacting ion transport proteins at the plasma membrane. The human erythrocyte anion exchanger (AE1) was the first membrane transport protein found to be associated with the spectrin cytoskeleton. Here we evaluated a conserved anion exchanger from Drosophila (DAE) as a marker for studies of the downstream effects of spectrin cytoskeleton mutations.ResultsSequence comparisons established that DAE belongs to the SLC4A1-3 subfamily of anion exchangers that includes human AE1. Striking sequence conservation was observed in the C-terminal membrane transport domain and parts of the N-terminal cytoplasmic domain, but not in the proposed ankyrin-binding site. Using an antibody raised against DAE and a recombinant transgene expressed in Drosophila S2 cells DAE was shown to be a 136 kd plasma membrane protein. A major site of expression was found in the stomach acid-secreting region of the larval midgut. DAE codistributed with an infolded subcompartment of the basal plasma membrane of interstitial cells. However, spectrin did not codistribute with DAE at this site or in anterior midgut cells that abundantly expressed both spectrin and DAE. Ubiquitous knockdown of DAE with dsRNA eliminated antibody staining and was lethal, indicating that DAE is an essential gene product in Drosophila.ConclusionsBased on the lack of colocalization and the lack of sequence conservation at the ankyrin-binding site, it appears that the well-characterized interaction between AE1 and the spectrin cytoskeleton in erythrocytes is not conserved in Drosophila. The results establish a pattern in which most of the known interactions between the spectrin cytoskeleton and the plasma membrane in mammals do not appear to be conserved in Drosophila.

NMR structure note: oxidized microsomal human cytochrome b5

Cytochrome b5 (cytb5) is a small membrane-bound hemoprotein present in all eukaryotic organisms. In most eukaryotic cells, cytb5 is attached to the cytosolic face of the endoplasmic reticulum and is described as the microsomal form (Mc). In vertebrates, two supplementary cytb5 can be found: soluble in the erythrocytes and membrane-bound attached to the internal face of the outer membrane in the mitochondria (OM) (Wang et al. 2007). Cytb5 can be divided into three domains: an N-terminal hydrosoluble globular domain that contains approximately 90 residues, a C-terminal hydrophobic domain about 25 residues long and a linker between the two above-mentioned domains. The hydrophilic domain contains the redox center, an iron protoporphyrin IX, ligated to the apoprotein by two histidyl residues. Mc cytb5 transfers electrons to various acceptors and is consequently involved in many different metabolic pathways. In the mixed function oxidase system, Mc cytb5 is a facultative electron donor to cytochromes P450 (Vergeres and Waskell 1995). Several mechanisms for the recognition between cytb5 and its physiological or artificial acceptors have been hypothesized. The implication of electrostatic interactions has been mostly studied with the cytochromes P450 as acceptors and is now widely accepted as a dominant factor (Schenkman et al. 1994). Nonetheless, several other factors may contribute positively to the recognition mechanism. First, the soluble domain of cytb5 lacking its C-terminal membrane domain cannot transfer electrons to membrane electron acceptors like cytochrome P450 (Dailey and Strittmatter 1978) while the full-length cytb5 can transfer electrons to both soluble and membranebound electron acceptor proteins (Vergeres and Waskell 1995). The linker is also recognized as an important factor controlling cytb5 to P450 interactions (Clarke et al. 2004). The three dimensional structures of the oxidized or reduced soluble domain of wild-type and mutant cytb5 from different species have been determined, either by X-ray or NMR. So far, no three dimensional structure of the full length protein has been reported, although some solid-state NMR data of the full-length rabbit cytb5 inserted in bicelles was Electronic supplementary material The online version of this article (doi:10.1007/s10858-010-9428-6) contains supplementary material, which is available to authorized users.

A Myosin IK-Abp1-PakB Circuit Acts as a Switch to Regulate Phagocytosis Efficiency

Actin dynamics and myosin (Myo) contractile forces are necessary for formation and closure of the phagocytic cup. In Dictyostelium, the actin-binding protein Abp1 and myosin IK are enriched in the closing cup and especially at an actin-dense constriction furrow formed around the neck of engulfed budded yeasts. This phagocytic furrow consists of concentric overlapping rings of MyoK, Abp1, Arp3, coronin, and myosin II, following an order strikingly reminiscent of the overall organization of the lamellipodium of migrating cells. Mutation analyses of MyoK revealed that both a C-terminal farnesylation membrane anchor and a Gly-Pro-Arg domain that interacts with profilin and Abp1 were necessary for proper localization in the furrow and efficient phagocytosis. Consequently, we measured the binding affinities of these interactions and unraveled further interactions with profilins, dynamin A, and PakB. Due to the redundancy of the interaction network, we hypothesize that MyoK and Abp1 are restricted to regulatory roles and might affect the dynamic of cup progression. Indeed, phagocytic uptake was regulated antagonistically by MyoK and Abp1. MyoK is phosphorylated by PakB and positively regulates phagocytosis, whereas binding of Abp1 negatively regulates PakB and MyoK. We conclude that a MyoK-Abp1-PakB circuit acts as a switch regulating phagocytosis efficiency of large particles.