The outer membrane form of the mitochondrial protein Mcr1 follows a TOM-independent membrane insertion pathway

The yeast gene MCR1 encodes two isoforms of the mitochondrial NADH-cytochrome b5 reductase. One form is embedded in the outer membrane whereas the other is located in the intermembrane space (IMS). In the present work we investigated the biogenesis of the outer membrane form. We demonstrate that while the IMS form crosses the outer membrane via the translocase of the outer mitochondrial membrane (TOM) complex, the other form is integrated into the outer membrane by a process that does not require any of the known import components at the outer membrane. Thus, the import pathways of the two forms diverge in a stage before the encounter with the TOM complex and their mechanism of biogenesis represents a unique example how to achieve dual localization within one organelle.     

Mim1 functions in an oligomeric form to facilitate the integration of Tom20 into the mitochondrial outer membrane

The translocase of the outer mitochondrial membrane (TOM) complex is the general entry site into the organelle for newly synthesized proteins. Despite its central role in the biogenesis of mitochondria, the assembly process of this complex is not completely understood. Mim1 (mitochondrial import protein 1) is a mitochondrial outer membrane protein with an undefined role in the assembly of the TOM complex. The protein is composed of an N-terminal cytosolic domain, a central putative transmembrane segment (TMS) and a C-terminal domain facing the intermembrane space. Here we show that Mim1 is required for the integration of the import receptor Tom20 into the outer membrane. We further investigated what the structural characteristics allowing Mim1 to fulfil its function are. The N- and C-terminal domains of Mim1 are crucial neither for the function of the protein nor for its biogenesis. Thus, the TMS of Mim1 is the minimal functional domain of the protein. We show that Mim1 forms homo-oligomeric structures via its TMS, which contains two helix-dimerization GXXXG motifs. Mim1 with mutated GXXXG motifs did not form oligomeric structures and was inactive. With all these data taken together, we propose that the homo-oligomerization of Mim1 allows it to fulfil its function in promoting the integration of Tom20 into the mitochondrial outer membrane.     

Compaction of a Prokaryotic Signal-Anchor Transmembrane Domain Begins within the Ribosome Tunnel and Is Stabilized by SRP during Targeting

Cotranslational targeting of membrane proteins is mediated by the universally conserved signal recognition particle (SRP). In eukaryotes, SRP attenuates translation during targeting; however, in prokaryotes, a simplified SRP is believed to carry out targeting during continuing translation. Here, we show a detailed stepwise analysis of the targeting of subunit c of the F₀ component of the bacterial ATP synthase (F₀c) to the inner membrane. We show that the first transmembrane (TM) signal-anchor domain of F₀c forms a compacted structure within the distal portion of the ribosome tunnel. This structure is formed just prior to the interaction with SRP. In the absence of SRP this structure is lost as the TM domain exits the tunnel; however in the presence of SRP it is stabilized. Our results suggest differences in early protein folding of substrates for prokaryotic SRP‐dependent membrane protein targeting pathways, from that of eukaryotic SRP targeting. These results imply that early TM domain recognition by targeting factors acts to ensure that the efficiency of membrane targeting is maintained.     

Probing conformational changes in lipoxygenases upon membrane binding: Fine-tuning by the active site inhibitor ETYA

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme–membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors.     

Structural elucidation of transmembrane transporter protein bilitranslocase: Conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy

Membrane proteins represent about a third of the gene products in most organisms, as revealed by the genome sequencing projects. They account for up to two thirds of known drugable targets, which emphasizes their critical pharmaceutical importance. Here we present a study on bilitranslocase (BTL) (TCDB 2.A.65), a membrane protein primarily involved in the transport of bilirubin from blood to liver cells. Bilitranslocase has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its 3D structure. However, at present, only a limited knowledge is available beyond the primary structure of BTL. It has been recently confirmed experimentally that one of the four computationally predicted transmembrane segments of bilitranslocase, TM3, has a helical structure with hydrophilic amino acid residues oriented towards one side, which is typical for transmembrane domains of membrane proteins. In this study we confirmed by the use of multidimensional NMR spectroscopy that the second transmembrane segment, TM2, also appears in a form of α-helix. The stability of this polypeptide chain was verified by molecular dynamics (MD) simulation in dipalmitoyl phosphatidyl choline (DPPC) and in sodium dodecyl sulfate (SDS) micelles. The two α-helices, TM2 corroborated in this study, and TM3 confirmed in our previous investigation, provide reasonable building blocks of a potential transmembrane channel for transport of bilirubin and small hydrophilic molecules, including pharmaceutically active compounds.     

Specific heteromeric association of four transmembrane peptides derived from platelet glycoprotein Ib-IX complex

As the receptor on the platelet surface for von Willebrand factor, glycoprotein (GP) Ib-IX complex is critically involved in hemostasis and thrombosis. How the complex is assembled from GP Ibα, GP Ibβ and GP IX subunits, all of which are type I transmembrane proteins, is not entirely clear. Genetic and mutational analyses have identified the transmembrane (TM) domains of these subunits as active participants in assembly of the complex. In this study, peptides containing the transmembrane domain of each subunit have been produced and their interaction with one another characterized. Only the Ibβ TM sequence, but not the Ibα and IX counterparts, can form homo-oligomers in SDS-PAGE and TOXCAT assays. Following up on our earlier observation that a Ibβ-Ibα-Ibβ peptide complex (αβ₂) linked through native juxtamembrane disulfide bonds could be produced from isolated Ibα and Ibβ TM peptides in detergent micelles, we show here that addition of the IX TM peptide facilitates formation of the native αβ₂ complex, reproducing the same effect by the IX subunit in cells expressing the GP Ib-IX complex. Specific fluorescence resonance energy transfer was observed between donor-labeled αβ₂ peptide complex and acceptor-conjugated IX TM peptide in micelles. Finally, the mutation D135K in the IX TM peptide could hamper both the formation of the αβ₂ complex and the energy transfer, consistent with its reported effect in the full-length complex. Overall, our results have demonstrated directly the native-like heteromeric interaction among the isolated Ibα, Ibβ and IX TM peptides, which provides support for the four-helix bundle model of the TM domains in the GP Ib-IX complex and paves the way for further structural analysis. The methods developed in this study may be applicable to other studies of heteromeric interaction among multiple TM helices.     

Tandem duplications of a degenerated GTP-binding domain at the origin of GTPase receptors Toc159 and thylakoidal SRP

The evolutionary origin of some nuclear encoded proteins that translocate proteins across the chloroplast envelope remains unknown. Therefore, sequences of GTPase proteins constituting the Arabidopsis thaliana translocon at the outer membrane of chloroplast (atToc) complexes were analyzed by means of HCA. In particular, atToc159 and related proteins (atToc132, atToc120, and atToc90) do not have proven homologues of prokaryotic or eukaryotic ancestry. We established that the three domains commonly referred to as A, G, and M originate from the GTPase G domain, tandemly repeated, and probably evolving toward an unstructured conformation in the case of the A domain. It resulted from this study a putative common ancestor for these proteins and a new domain definition, in particular the splitting of A into three domains (A1, A2, and A3), has been proposed. The family of Toc159, previously containing A. thaliana and Pisum sativum, has been extended to Medicago truncatula and Populus trichocarpa and it has been revised for Oryza sativa. They have also been compared to GTPase subunits involved in the cpSRP system. A distant homology has been revealed among Toc and cpSRP GTP-hydrolyzing proteins of A. thaliana, and repetitions of a GTPase domain were also found in cpSRP protein receptors, by means of HCA analysis.     

FbpA — A bacterial transferrin with more to offer

Background

Gram negative bacteria require iron for growth and virulence. It has been shown that certain pathogenic bacteria such as Neisseria gonorrhoeae possess a periplasmic protein called ferric binding protein (FbpA), which is a node in the transport of iron from the cell exterior to the cytosol.

Scope of review

The relevant literature is reviewed which establishes the molecular mechanism of FbpA mediated iron transport across the periplasm to the inner membrane.

Major conclusions

Here we establish that FbpA may be considered a bacterial transferrin on structural and functional grounds. Data are presented which suggest a continuum whereby FbpA may be considered as a naked iron carrier, as well as a Fe–chelate carrier, and finally a member of the larger family of periplasmic binding proteins.

General significance

An investigation of the molecular mechanisms of action of FbpA as a member of the transferrin super family enhances our understanding of bacterial mechanisms for acquisition of the essential nutrient iron, as well as the modes of action of human transferrin, and may provide approaches to the control of pathogenic diseases. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.     

Caveolin-rich lipid rafts of the plasma membrane of mature cerebellar granule neurons are microcompartments for calcium/reactive oxygen and nitrogen species cross-talk signaling

In previous works, we have shown that L-type voltage-operated calcium channels, N-methyl-d-aspartate receptors (NMDAr), neuronal nitric oxide synthase (nNOS) and cytochrome b₅ reductase (Cb₅R) co-localize within the same lipid rafts-associated nanodomains in mature cerebellar granule neurons (CGN). In this work, we show that the calcium transport systems of the plasma membrane extruding calcium from the cytosol, plasma membrane calcium pumps (PMCA) and sodium–calcium exchangers (NCX), are also associated with these nanodomains. All these proteins were found to co-immunoprecipitate with caveolin-1 after treatment with 25 mM methyl-β-cyclodextrin, a lipid rafts solubilizing agent. However, the treatment of CGN with methyl-β-cyclodextrin largely attenuated the rise of cytosolic calcium induced by l-glutamate through NMDAr. Fluorescence energy transfer imaging revealed that all of them are present in sub-microdomains of a size smaller than 200 nm, with a peripheral distribution of the calcium extrusion systems PMCA and NCX. Fluorescence microscopy images analysis revealed high calcium dynamic sub-microcompartments near the plasma membrane in fura-2-loaded CGN at short times after addition of l-glutamate. In addition, the close proximity between sources of nitric oxide (nNOS) and superoxide anion (Cb₅R) suggests that these nanodomains are involved in the fast and efficient cross-talk between calcium and redox signaling in neurons.     

Phenylketonuria as a protein misfolding disease: The mutation pG46S in phenylalanine hydroxylase promotes self-association and fibril formation

The missense mutation pG46S in the regulatory (R) domain of human phenylalanine hydroxylase (hPAH), associated with a severe form of phenylketonuria, generates a misfolded protein which is rapidly degraded on expression in HEK293 cells. When overexpressed as a MBP-G46S fusion protein, soluble and fully active tetrameric/dimeric forms are assembled and recovered in a metastable conformational state. When MBP is cleaved off, G46S undergoes a conformational change and self-associates with a lag phase and an autocatalytic growth phase (tetramers ? dimers), as determined by light scattering. The self-association is controlled by pH, ionic strength, temperature, protein concentration and the phosphorylation state of Ser16; the net charge of the protein being a main modulator of the process. A superstoichiometric amount of WT dimers revealed a 2-fold enhancement of the rate of G46S dimer self-association. Electron microscopy demonstrates the formation of higher-order oligomers and linear polymers of variable length, partly as a branching network, and partly as individual long and twisted fibrils (diameter ~ 145-300 Å). The heat-shock proteins Hsp70/Hsp40, Hsp90 and a proposed pharmacological PAH chaperone (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) partly inhibit the self-association process. Our data indicate that the G46S mutation results in a N-terminal extension of α-helix 1 which perturbs the wild-type α-β sandwich motif in the R-domain and promotes new intermolecular contacts, self-association and non-amyloid fibril formation. The metastable conformational state of G46S as a MBP fusion protein, and its self-association propensity when released from MBP, may represent a model system for the study of other hPAH missense mutations characterized by misfolded proteins.     

Short- and medium-chain fatty acids enhance the cell surface expression and transport capacity of the bile salt export pump (BSEP/ABCB11)

The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We previously reported that 4-phenylbutyrate (4PBA), an approved drug for urea cycle disorders, is a promising agent for intrahepatic cholestasis because it increases both the cell surface expression and the transport capacity of BSEP. In the present study, we searched for effective compounds other than 4PBA by focusing on short- and medium-chain fatty acids, which have similar characteristics to 4PBA such as their low-molecular-weight and a carboxyl group. In transcellular transport studies using Madin–Darby canine kidney (MDCK) II cells, all short- and medium-chain fatty acids tested except for formate, acetate, and hexanoic acid showed more potent effects on wild type (WT) BSEP-mediated [³H]taurocholate transport than did 4PBA. The increase in WT BSEP transport with butyrate and octanoic acid treatment correlated with an increase in its expression at the cell surface. Two PFIC2-type variants, E297G and D482G BSEP, were similarly affected with both compounds treatment. The prolonged half-life of cell surface-resident WT BSEP was responsible for this increased octanoic acid-stimulated transport, but not for that of butyrate. In conclusion, short- and medium-chain fatty acids have potent effects on the increase in WT and PFIC2-type BSEP-mediated transport in MDCK II cells. Although both short- and medium-chain fatty acids enhance the transport capacity of WT and PFIC2-type BSEP by inducing those expressions at the cell surface, the underlying mechanism seems to differ between fatty acids.     

Interactions of fluorinated surfactants with diphtheria toxin T-domain: testing new media for studies of membrane proteins

The principal difficulty in experimental exploration of the folding and stability of membrane proteins (MPs) is their aggregation outside of the native environment of the lipid bilayer. To circumvent this problem, we recently applied fluorinated nondetergent surfactants that act as chemical chaperones. The ideal chaperone surfactant would 1), maintain the MP in solution; 2), minimally perturb the MP's structure; 3), dissociate from the MP during membrane insertion; and 4), not partition into the lipid bilayer. Here, we compare how surfactants with hemifluorinated (HFTAC) and completely fluorinated (FTAC) hydrophobic chains of different length compare to this ideal. Using fluorescence correlation spectroscopy of dye-labeled FTAC and HFTAC, we demonstrate that neither type of surfactant will bind lipid vesicles. Thus, unlike detergents, fluorinated surfactants do not compromise vesicle integrity even at concentrations far in excess of their critical micelle concentration. We examined the interaction of surfactants with a model MP, DTT, using a variety of spectroscopic techniques. Site-selective labeling of DTT with fluorescent dyes indicates that the surfactants do not interact with DTT uniformly, instead concentrating in the most hydrophobic patches. Circular dichroism measurements suggest that the presence of surfactants does not alter the structure of DTT. However, the cooperativity of the thermal unfolding transition is reduced by the presence of surfactants, especially above the critical micelle concentration (a feature of regular detergents, too). The linear dependence of DTT's enthalpy of unfolding on the surfactant concentration is encouraging for future application of (H)FTACs to determine the stability of the membrane-competent conformations of other MPs. The observed reduction in the efficiency of Förster resonance energy transfer between donor-labeled (H)FTACs and acceptor-labeled DTT upon addition of lipid vesicles indicates that the protein sheds the layer of surfactant during its bilayer insertion. We discuss the advantages of fluorinated surfactants over other types of solubilizing agents, with a specific emphasis on their possible applications in thermodynamic measurements.     

Recent advances in quantitative colocalization analysis: Focus on neuroscience

Quantitative colocalization analysis is an advanced digital imaging tool to characterize the spatial expression of molecules of interest in immunofluorescence images obtained using confocal microscopes. It began from simple pixel counting and, with introduction of specialized algorithms, transformed into a powerful image analyzing technique capable of identifying the exact locations of various molecules in tissues and cells and describing their subtle changes in dynamics. Applications of quantitative colocalization in the field of neuroscience proved to be particularly informative by helping to obtain observations not otherwise achievable using other techniques. In this article, we review the background and applicability of quantitative colocalization with special focus on neuroscience research.     

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.