Heme transfer to the heme chaperone CcmE during cytochrome c maturation requires the CcmC protein, which may function independently of the ABC-transporter CcmAB

Cytochrome c maturation in Escherichia coli requires the ccm operon, which encodes eight membrane proteins (CcmABCDEFGH). CcmE is a periplasmic heme chaperone that binds heme covalently and transfers it onto apocytochrome c in the presence of CcmF, CcmG, and CcmH. In this work we addressed the functions of the ccmABCD gene products with respect to holo-CcmE formation and the subsequent ligation of heme to apocytochrome c. In the absence of the ccmABCD genes, heme is not bound to CcmE. We report that CcmC is functionally uncoupled from the ABC transporter subunits CcmA and CcmB, because it is the only Ccm protein that is strictly required for heme transfer and attachment to CcmE. Site-directed mutagenesis of conserved histidines inactivates the CcmC protein, which is in agreement with the hypothesis that this protein interacts directly with heme. We also present evidence that questions the role of CcmAB as a heme exporter; yet, the transported substrate remains unknown. CcmD was found to be involved in stabilizing the heme chaperone CcmE in the membrane. We propose a heme-trafficking pathway as part of a substantially revised model for cytochrome c maturation in E. coli.

Apical sorting of a voltage- and Ca2+-activated K+ channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation

The voltage- and Ca2+-activated K+ (KV,Ca) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel KV,Ca α-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., KV,Ca β-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.

Mutations of γ-aminobutyric acid and glycine receptors change alcohol cutoff: Evidence for an alcohol receptor?

Alcohols in the homologous series of n-alcohols increase in central nervous system depressant potency with increasing chain length until a “cutoff” is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the α subunit. We now demonstrate that these residues in the glycine α1 and the γ-aminobutyric acid ρ1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine α1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the γ-aminobutyric acid ρ1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.

A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae

The PKC1–MPK1 pathway in yeast functions in the maintenance of cell wall integrity and in the stress response. We have identified a family of genes that are putative regulators of this pathway. WSC1, WSC2, and WSC3 encode predicted integral membrane proteins with a conserved cysteine motif and a WSC1–green fluorescence protein fusion protein localizes to the plasma membrane. Deletion of WSC results in phenotypes similar to mutants in the PKC1–MPK1 pathway and an increase in the activity of MPK1 upon a mild heat treatment is impaired in a wscΔ mutant. Genetic analysis places the function of WSC upstream of PKC1, suggesting that they play a role in its activation. We also find a genetic interaction between WSC and the RAS–cAMP pathway. The RAS–cAMP pathway is required for cell cycle progression and for the heat shock response. Overexpression of WSC suppresses the heat shock sensitivity of a strain in which RAS is hyperactivated and the heat shock sensitivity of a wscΔ strain is rescued by deletion of RAS2. The functional characteristics and cellular localization of WSC suggest that they may mediate intracellular responses to environmental stress in yeast.

Polar residues drive association of polyleucine transmembrane helices

Although many polar residues are directly involved in transmembrane protein functions, the extent to which they contribute to more general structural features is still unclear. Previous studies have demonstrated that asparagine residues can drive transmembrane helix association through interhelical hydrogen bonding [Choma, C., Gratkowski, H., Lear, J. D. & DeGrado, W. F. (2000) Nat. Struct. Biol. 7, 161–166; and Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. & Engelman, D. M. (2000) Nat. Struct. Biol. 7, 154–160]. We have studied the ability of other polar residues to promote helix association in detergent micelles and in biological membranes. Our results show that polyleucine sequences with Asn, Asp, Gln, Glu, and His, residues capable of being simultaneously hydrogen bond donors and acceptors, form homo- or heterooligomers. In contrast, polyleucine sequences with Ser, Thr, and Tyr do not associate more than the polyleucine sequence alone. The results therefore provide experimental evidence that interactions between polar residues in the helices of transmembrane proteins may serve to provide structural stability and oligomerization specificity. Furthermore, such interactions can allow structural flexibility required for the function of some membrane proteins.

Chlorophyll and carotenoid binding in a simple red algal light-harvesting complex crosses phylogenetic lines

The membrane proteins of peripheral light-harvesting complexes (LHCs) bind chlorophylls and carotenoids and transfer energy to the reaction centers for photosynthesis. LHCs of chlorophytes, chromophytes, dinophytes, and rhodophytes are similar in that they have three transmembrane regions and several highly conserved Chl-binding residues. All LHCs bind Chl a, but in specific taxa certain characteristic pigments accompany Chl a: Chl b and lutein in chlorophytes, Chl c and fucoxanthin in chromophytes, Chl c and peridinin in dinophytes, and zeaxanthin in rhodophytes. The specificity of pigment binding was examined by in vitro reconstitution of various pigments with a simple light-harvesting protein (LHCaR1), from a red alga (Porphyridium cruentum), that normally has eight Chl a and four zeaxanthin molecules. The pigments typical of a chlorophyte (Spinacea oleracea), a chromophyte (Thallasiosira fluviatilis), and a dinophyte (Prorocentrum micans) were found to functionally bind to this protein as evidenced by their participation in energy transfer to Chl a, the terminal pigment. This is a demonstration of a functional relatedness of rhodophyte and higher plant LHCs. The results suggest that eight Chl-binding sites per polypeptide are an ancestral trait, and that the flexibility to bind various Chl and carotenoid pigments may have been retained throughout the evolution of LHCs.

Domains of the Rsp5 Ubiquitin-Protein Ligase Required for Receptor-mediated and Fluid-Phase Endocytosis

Yeast Rsp5p and its mammalian homologue, Nedd4, are hect domain ubiquitin-protein ligases (E3s) required for the ubiquitin-dependent endocytosis of plasma membrane proteins. Because ubiquitination is sufficient to induce internalization, E3-mediated ubiquitination is a key regulatory event in plasma membrane protein endocytosis. Rsp5p is an essential, multidomain protein containing an amino-terminal C2 domain, three WW protein-protein interaction domains, and a carboxy-terminal hect domain that carries E3 activity. In this study, we demonstrate that Rsp5p is peripherally associated with membranes and provide evidence that Rsp5p functions as part of a multimeric protein complex. We define the function of Rsp5p and its domains in the ubiquitin-dependent internalization of the yeast α-factor receptor, Ste2p. Temperature-sensitive rsp5 mutants were unable to ubiquitinate or to internalize Ste2p at the nonpermissive temperature. Deletion of the entire C2 domain had no effect on α-factor internalization; however, point mutations in any of the three WW domains impaired both receptor ubiquitination and internalization. These observations indicate that the WW domains play a role in the important regulatory event of selecting phosphorylated proteins as endocytic cargo. In addition, mutations in the C2 and WW1 domains had more severe defects on transport of fluid-phase markers to the vacuole than on receptor internalization, suggesting that Rsp5p functions at multiple steps in the endocytic pathway.

Oxaloacetate Transport into Plant Mitochondria1

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of 14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.

A Transmembrane Form of the Prion Protein Contains an Uncleaved Signal Peptide and Is Retained in the Endoplasmic Reticululm

Although there is considerable evidence that PrPSc is the infectious form of the prion protein, it has recently been proposed that a transmembrane variant called CtmPrP is the direct cause of prion-associated neurodegeneration. We report here, using a mutant form of PrP that is synthesized exclusively with the CtmPrP topology, that CtmPrP is retained in the endoplasmic reticulum and is degraded by the proteasome. We also demonstrate that CtmPrP contains an uncleaved, N-terminal signal peptide as well as a C-terminal glycolipid anchor. These results provide insight into general mechanisms that control the topology of membrane proteins during their synthesis in the endoplasmic reticulum, and they also suggest possible cellular pathways by which CtmPrP may cause disease.

Structure and function in rhodopsin: Mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants

Retinitis pigmentosa (RP) point mutations in both the intradiscal (ID) and transmembrane domains of rhodopsin cause partial or complete misfolding of rhodopsin, resulting in loss of 11-cis-retinal binding. Previous work has shown that misfolding is caused by the formation of a disulfide bond in the ID domain different from the native Cys-110–Cys-187 disulfide bond in native rhodopsin. Here we report on direct identification of the abnormal disulfide bond in misfolded RP mutants in the transmembrane domain by mass spectrometric analysis. This disulfide bond is between Cys-185 and Cys-187, the same as previously identified in misfolded RP mutations in the ID domain. The strategy described here should be generally applicable to identification of disulfide bonds in other integral membrane proteins.

Solution 19F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

19F nuclear Overhauser effects (NOEs) between fluorine labels on the cytoplasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution 19F NMR spectra of fluorine-labeled rhodopsin in detergent micelles were described, demonstrating the applicability of this technique to studies of tertiary structure in the cytoplasmic domain. To quantitate tertiary contacts we have applied a transient one-dimensional difference NOE solution 19F NMR experiment to this system, permitting assessment of proximities between fluorine labels specifically incorporated into different regions of the cytoplasmic face. Three dicysteine substitution mutants (Cys-140–Cys-316, Cys-65–Cys-316, and Cys-139–Cys-251) were labeled by attachment of the trifluoroethylthio group through a disulfide linkage. Each mutant rhodopsin was prepared (8–10 mg) in dodecylmaltoside and analyzed at 20°C by solution 19F NMR. Distinct chemical shifts were observed for all of the rhodopsin 19F labels in the dark. An up-field shift of the Cys-316 resonance in the Cys-65–Cys-316 mutant suggests a close proximity between the two residues. When analyzed for 19F-19F NOEs, a moderate negative enhancement was observed for the Cys-65–Cys-316 pair and a strong negative enhancement was observed for the Cys-139–Cys-251 pair, indicating proximity between these sites. No NOE enhancement was observed for the Cys-140–Cys-316 pair. These NOE effects demonstrate a solution 19F NMR method for analysis of tertiary contacts in high molecular weight proteins, including membrane proteins.

An essential amino acid residue in the protein translocation channel revealed by targeted random mutagenesis of SecY

The SecY/Sec61α family of membrane proteins are the central subunits of the putative protein translocation channel. We introduced random mutations into a segment of Escherichia coli SecY within its cytoplasmic domain 5, which was shown previously to be important for the SecA-dependent translocation activity. Mutations were classified into those retaining function and those gaining a dominant-interfering ability caused by a loss of function. These analyses showed that Arg-357, Pro-358, Gly-359, and Thr-362 are functionally important; Arg-357, conserved in almost all organisms, was identified as an indispensable residue.

Transcriptional and posttranslational plasticity and the generation of inflammatory pain

Inflammatory pain manifests as spontaneous pain and pain hypersensitivity. Spontaneous pain reflects direct activation of specific receptors on nociceptor terminals by inflammatory mediators. Pain hypersensitivity is the consequence of early posttranslational changes, both in the peripheral terminals of the nociceptor and in dorsal horn neurons, as well as later transcription-dependent changes in effector genes, again in primary sensory and dorsal horn neurons. This inflammatory neuroplasticity is the consequence of a combination of activity-dependent changes in the neurons and specific signal molecules initiating particular signal-transduction pathways. These pathways phosphorylate membrane proteins, changing their function, and activate transcription factors, altering gene expression. Two distinct aspects of sensory neuron function are changed as a result of these processes, basal sensitivity, or the capacity of peripheral stimuli to evoke pain, and stimulus-evoked hypersensitivity, the capacity of certain inputs to generate prolonged alterations in the sensitivity of the system. Posttranslational changes largely alter basal sensitivity. Transcriptional changes both potentiate the system and alter neuronal phenotype. Potentiation occurs as a result of the up-regulation in the dorsal root ganglion of centrally acting neuromodulators and simultaneously in the dorsal horn of their receptors. This means that the response to subsequent inputs is augmented, particularly those that induce stimulus-induced hypersensitivity. Alterations in phenotype includes the acquisition by A fibers of neurochemical features typical of C fibers, enabling these fibers to induce stimulus-evoked hypersensitivity, something only C fiber inputs normally can do. Elucidation of the molecular mechanisms responsible provides new opportunities for therapeutic approaches to managing inflammatory pain.

Afipia felis induces uptake by macrophages directly into a nonendocytic compartment

Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease. A. felis can survive and multiply in several mammalian cell types, including macrophages, but the precise intracellular compartmentalization of A. felis-containing phagosomes is unknown. Here, we demonstrate that, in murine macrophages, most A. felis-containing phagosomes exclude lysosomal tracer loaded into macrophage lysosomes before, as well as endocytic tracer loaded after, establishment of an infection. Established Afipia-containing phagosomes possess neither early endosomal marker proteins [early endosome antigen 1 (EEA1), Rab5, transferrin receptor, trytophane aspartate containing coat protein (TACO)] nor late endosomal or lysosomal proteins [cathepsin D, β-glucuronidase, vacuolar proton-pumping ATPase, rab7, mannose-6-phosphate receptor, vesicle-associated membrane protein 8, lysosome-associated membrane proteins LAMP-1 and LAMP-2]. Those bacteria that will be found in a nonendosomal compartment enter the macrophage via an EEA1-negative compartment, which remains negative for LAMP-1. The smaller subpopulation of afipiae whose phagosomes will be part of the endocytic system enters into an EEA1-positive compartment, which also subsequently acquires LAMP-1. Killing of Afipia or opsonization with immune antibodies leads to a strong increase in the percentage of A. felis-containing phagosomes that interact with the endocytic system. We conclude that most phagosomes containing A. felis are disconnected from the endosome–lysosome continuum, that their unusual compartmentalization is decided at uptake, and that this compartmentalization requires bacterial viability.

High Expression of the Tonoplast Aquaporin ZmTIP1 in Epidermal and Conducting Tissues of Maize1

Aquaporins are integral membrane proteins of the tonoplast and the plasma membrane that facilitate the passage of water through these membranes. Because of their potentially important role in regulating water flow in plants, studies documenting aquaporin gene expression in specialized tissues involved in water and solute transport are important. We used in situ hybridization to examine the expression pattern of the tonoplast aquaporin ZmTIP1 in different organs of maize (Zea mays L.). This tonoplast water channel is highly expressed in the root epidermis, the root endodermis, the small parenchyma cells surrounding mature xylem vessels in the root and the stem, phloem companion cells and a ring of cells around the phloem strand in the stem and the leaf sheath, and the basal endosperm transfer cells in developing kernels. We postulate that the high level of expression of ZmTIP1 in these tissues facilitates rapid flow of water through the tonoplast to permit osmotic equilibration between the cytosol and the vacuolar content, and to permit rapid transcellular water flow through living cells when required.