Segregation of two spectrin isoforms: polarized membrane-binding sites direct polarized membrane skeleton assembly

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and beta spectrin are recruited to sites of cell-cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (alpha beta H), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and alpha beta spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, alpha beta spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, alpha beta H spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell-cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.     

Molecular basis of spectrin deficiency in beta spectrin Durham. A deletion within beta spectrin adjacent to the ankyrin-binding site precludes spectrin attachment to the membrane in hereditary spherocytosis

We describe a spectrin variant characterized by a truncated beta chain and associated with hereditary spherocytosis. The clinical phenotype consists of a moderate hemolytic anemia with striking spherocytosis and mild spiculation of the red cells. We describe the biochemical characteristics of this truncated protein which constitutes only 10% of the total beta spectrin present on the membrane, resulting in spectrin deficiency. Analysis of reticulocyte cDNA revealed the deletion of exons 22 and 23. We show, using Southern blot analysis, that this truncation results from a 4.6-kb genomic deletion. To elucidate the basis for the decreased amount of the truncated protein on the membrane and the overall spectrin deficiency, we show that (a) the mutated gene is efficiently transcribed and its mRNA abundant in reticulocytes, (b) the mutant protein is normally synthesized in erythroid progenitor cells, (c) the stability of the mutant protein in the cytoplasm of erythroblasts parallels that of the normal beta spectrin, and (d) the abnormal protein is inefficiently incorporated into the membrane of erythroblasts. We conclude that the truncation within the beta spectrin leads to inefficient incorporation of the mutant protein into the skeleton despite its normal synthesis and stability. We postulate that this misincorporation results from conformational changes of the beta spectrin subunit affecting the binding of the abnormal heterodimer to ankyrin, and we provide evidence based on binding assays of recombinant synthetic peptides to inside-out-vesicles to support this model.     

Spectrin localization in osteoclasts: immunocytochemistry, cloning, and partial sequencing

The presence of spectrin was demonstrated in chick osteoclasts by Western blotting and light and electron microscopic immunolocalization. Additionally, screening of a chick osteoclast cDNA library revealed the presence of alpha-spectrin. Light microscope level immunocytochemical staining of osteoclasts in situ revealed spectrin staining throughout the cytoplasm with heavier staining found at the marrow-facing cell margin and around the nuclei. Confocal microscopy of isolated osteoclasts plated onto a glass substrate showed that spectrin encircled the organelle-rich cell center. Nuclei and cytoplasmic inclusions were also stained and the plasma membrane was stained in a nonuniform, patchy distribution corresponding to regions of apparent membrane ruffling. Ultracytochemical localization showed spectrin to be found at the plasma membrane and distributed throughout the cytoplasm with especially intense staining of the nuclear membrane and filaments within the nuclear compartment.     

Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms alpha, gamma, epsilon, zeta and lambda only the conventional isoforms PKC alpha and gamma responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC alpha and PKC gamma toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC gamma was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC alpha redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage.     

An essential role for autophosphorylation in the dissociation of activated protein kinase C from the plasma membrane

The cellular localization of protein kinase C (PKC) is intimately associated with the regulation of its biological activity. Previously we have demonstrated that the redistribution of PKC to the plasma membrane in response to physiological stimuli is followed by a rapid returning of PKC back to the cytoplasm (Feng, X., Zhang, J., Barak, L. S., Meyer, T., Caron, M. G., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 10755-10762). Although the process of PKC membrane targeting has been extensively studied, the molecular mechanism underlying the dissociation of membrane-bound PKC remains unclear. In the present study, by examining the dynamic distribution of wild-type PKC betaII and its kinase-deficient mutant (K371R), we demonstrate that kinase activity is required for PKC membrane dissociation. Moreover, the inability of PKC betaII(K371R) to dissociate from the plasma membrane in cells overexpressing wild-type PKC betaII suggests that autophosphorylation activity of the kinase might be essential for its membrane dissociation. This was further supported by mutational analysis of two in vivo autophosphorylation sites on PKC betaII. The replacement of Ser660 or Thr641 by alanine (S660A or T641A) was found to synergistically reduce the reversal of PKC betaII membrane translocation, whereas the replacement of the same amino acids by glutamic acid (S660E or T641E), an amino acid commonly used to mimic phosphate, results in mutants behaving similar to wild-type PKC betaII. These findings point to an essential role for autophosphorylation in the dissociation of activated PKC from the plasma membrane and suggest that, like PKC membrane translocation, the returning of PKC to the cytoplasm after its activation is also delicately regulated.     

Genetic studies of spectrin: new life for a ghost protein

Spectrin, together with actin and a number of other accessory proteins, forms a submembrane cytoskeletal network in the human erythrocyte ghost. Through an elegant combination of structural, biochemical, and genetic studies, spectrin was shown to be an important determinant of erythrocyte shape and membrane stability. Genetic studies of a novel nonerythroid spectrin (beta H) in Drosophila and Caenorhabditis elegans now reveal that spectrin can influence the shape and stability of whole organisms. Nonerythroid spectrins are proposed to have roles in cell adhesion, establishment of cell polarity, and attachment of other cytoskeletal structures to the plasma membrane. The phenotypes of the beta H spectrin mutations provide an exciting biological context in which to evaluate these roles and perhaps to uncover new ones.     

Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons

Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in alpha, beta, and gamma adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant alpha adducin. The mutant alpha adducin was no longer concentrated at the cell membrane at sites of cell-cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant alpha adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant alpha adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.     

Complexes of the alpha1C and beta subunits generate the necessary signal for membrane targeting of class C L-type calcium channels

In the present study, we investigated the role of channel subunits in the membrane targeting of voltage-dependent L-type calcium channel complexes. We co-expressed the calcium channel pore-forming alpha1C subunit with different accessory beta subunits in HEK-tsA201 cells and examined the subcellular localization of the channel subunits by immunohistochemistry using confocal microscopy and whole-cell radioligand binding studies. While the pore-forming alpha1C subunit exhibited perinuclear staining when expressed alone, and several of the wild-type and mutant beta subunits also exhibited intracellular staining, co-expression of the alpha1C subunit with either the wild-type beta2a subunit, a palmitoylation-deficient beta2a(C3S/C4S) mutant or three other nonpalmitoylated beta isoforms (beta1b, beta3, and beta4 subunits) resulted in the redistribution of both the alpha1C and beta subunits into clusters along the cell surface. Furthermore, the redistribution of calcium channel complexes to the plasma membrane was observed when alpha1C was co-expressed with an N- and C-terminal truncated mutant beta2a containing only the central conserved regions. However, when the alpha1C subunit was co-expressed with an alpha1 beta interaction-deficient mutant, beta2aBID-, we did not observe formation of the channels at the plasma membrane. In addition, an Src homology 3 motif mutant of beta2a that was unable to interact with the alpha1C subunit also failed to target channel complexes to the plasma membrane. Interestingly, co-expression of the pore-forming alpha1C subunit with the largely peripheral accessory alpha2 delta subunit was ineffective in recruiting alpha1C to the plasma membrane, while co-distribution of all three subunits was observed when beta2a was co-expressed with the alpha1C and alpha2 delta subunits. Taken together, our results suggested that the signal necessary for correct plasma membrane targeting of the class C L-type calcium channel complexes is generated as a result of a functional interaction between the alpha1 and beta subunits.     

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (betaISigma*) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of betaI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of alpha- and beta-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of beta-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular-tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin-ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.     

Identification of a novel ankyrin isoform (AnkG190) in kidney and lung that associates with the plasma membrane and binds alpha-Na, K-ATPase

Ankyrins are a family of adapter molecules that mediate linkages between integral membrane and cytoskeletal proteins. Such interactions are crucial to the polarized distribution of membrane proteins in transporting epithelia. We have cloned and characterized a novel 190-kDa member of this family from a rat kidney cDNA library, which we term AnkG190 based on the predicted size and homology with the larger neuronal AnkG isoform. AnkG190 displays a unique 31-residue amino terminus, a repeats domain consisting of 24 repetitive 33-residue motifs, a spectrin binding domain, and a truncated regulatory domain. Probes derived from the unique amino terminus hybridize to an 8-kilobase message exclusively in kidney and lung and specifically to the kidney outer medullary collecting ducts by in situ hybridization. Transfections of Madin-Darby canine kidney and COS-7 epithelial cell lines with a full-length AnkG190 construct result in (a) expression at the lateral plasma membrane, (b) functional assembly with the cytoskeleton, and (c) interaction with at least one membrane protein, the Na,K-ATPase. Two independent Na,K-ATPase binding domains on AnkG190 are demonstrated as follows: one within the distal 12 ankyrin repeats, and a second site within the spectrin binding domain. Thus, ankyrins may interact with integral membrane proteins in a pleiotropic manner that may involve complex tertiary structural determinants.     

ADP ribosylation factor regulates spectrin binding to the Golgi complex

Homologues of two major components of the well-characterized erythrocyte plasma-membrane-skeleton, spectrin (a not-yet-cloned isoform, betaI Sigma* spectrin) and ankyrin (AnkG119 and an approximately 195-kDa ankyrin), associate with the Golgi complex. ADP ribosylation factor (ARF) is a small G protein that controls the architecture and dynamics of the Golgi by mechanisms that remain incompletely understood. We find that activated ARF stimulates the in vitro association of betaI Sigma* spectrin with a Golgi fraction, that the Golgi-associated betaI Sigma* spectrin contains epitopes characteristic of the betaI Sigma2 spectrin pleckstrin homology (PH) domain known to bind phosphatidylinositol 4,5-bisphosphate (PtdInsP2), and that ARF recruits betaI Sigma* spectrin by inducing increased PtdInsP2 levels in the Golgi. The stimulation of spectrin binding by ARF is independent of its ability to stimulate phospholipase D or to recruit coat proteins (COP)-I and can be blocked by agents that sequester PtdInsP2. We postulate that a PH domain within betaI Sigma* Golgi spectrin binds PtdInsP2 and acts as a regulated docking site for spectrin on the Golgi. Agents that block the binding of spectrin to the Golgi, either by blocking the PH domain interaction or a constitutive Golgi binding site within spectrin's membrane association domain I, inhibit the transport of vesicular stomatitis virus G protein from endoplasmic reticulum to the medial compartment of the Golgi complex. Collectively, these results suggest that the Golgi-spectrin skeleton plays a central role in regulating the structure and function of this organelle.     

Translocation of phosducin in living neuroblastoma x glioma hybrid cells (NG 108-15) monitored by red-shifted green fluorescent protein

Activation of G protein-coupled receptors triggers translocation of certain proteins from cytoplasm to cell membrane located targets. One of these cytosolic proteins is phosducin (Phd) which has been described to compete with G protein-coupled receptor kinases for Gbetagamma dimers attached to the cell membrane, thereby attenuating desensitization of activated receptors. These features of protein redistribution prompted us to examine whether stimulation of membrane associated E-prostaglandin receptors coupled to Gs causes Phd to migrate towards the plasma membrane. We made use of enhanced green fluorescence protein (EGFP), a reporter protein, to follow redistribution of Phd both by means of confocal microscopy and biochemical techniques in living neuronal NG 108-15 hybrid cells challenged with prostaglandin E1 (PGE1). The cells were transiently transfected to express Phd fused to the C-terminus of EGFP, or to express EGFP only. Overexpression of the proteins is implied by FACS analysis as well as by western blot technique, and the functional integrity of EGFP-tagged Phd was confirmed by its ability to elevate cAMP accumulation. Time-lapse imaging of single living cells by means of confocal microscopy revealed that exposure to prostaglandin causes EGFP/Phd, which is evenly spread throughout the cell, to relocate towards the membrane within few minutes. Fluorescence associated with the cell nucleus displayed little rearrangement. The principle finding that prostaglandin triggers translocation of Phd from cytosol to the cell periphery was verified with membranes prepared from EGFP/Phd expressing cells. We found maximal concentrations of membrane associated fluorescent material 5 to 7 min upon prostaglandin exposure. The present study reports for living NG 108-15 hybrid cells that PGE1 stimulation causes cytosolic Phd to translocate towards the membrane, where it is believed to bind to G protein subunits such as Gbetagamma and Galphas.     

Beta-galactoside-binding protein (beta GBP) alters the cell cycle, up-regulates expression of the alpha- and beta-chains of the IFN-gamma receptor, and triggers IFN-gamma-mediated apoptosis of activated human T lymphocytes

In this paper, the effects of beta-galactoside binding protein (beta GBP), the LGALS1 gene product, on the cell cycle progression and expansion of activated human T lymphocytes were studied. Beta GBP drastically inhibits the IL-2 induced proliferation of PHA-activated T lymphocytes as well as the IL-2 independent proliferation of malignant T lymphocytes by arresting them in the S and G2/M phases of the cell cycle. In addition, beta GBP up-regulates the expression of both the alpha- and the beta-chains of the IFN-gamma R on activated T lymphocyte membrane. None of these effects depend on sugar binding: saturating amounts of lactose do not affect the cell cycle block nor IFN-gamma R up-modulation. The increased expression of both chains renders beta GBP-treated T lymphoblasts sensitive to IFN-y-induced apoptosis. Taken as a whole, these findings suggest that beta GBP plays an important immunoregulatory role by switching off T lymphocyte effector functions. They also provide the first evidence of up-modulation of IFN-gamma R expression on T lymphocytes by a negative cell growth regulator.     

Geographical distribution of dengue and socioeconomic factors in an urban locality in southeastern Brazil]

The hydrochloric acid secreting parietal cells of the human stomach mucosa have been shown to express anion exchanger 2 (AE2). AE2 is restricted to the basolateral membrane domain and is responsible for the basolateral uptake of Cl- and release of HCO3-. It is unknown which mechanism is responsible for the basolateral positioning of AE2 in parietal cells. We raised the question whether AE2 might be immobilized at the cell surface by linkage via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we communicate two observations that support this hypothesis, namely that in parietal cells ankyrin is localized with AE2 along the basolateral cell surface and, secondly, that purified erythrocyte ankyrin binds to the in vitro-translated cytoplasmic domain of AE2. We conclude from these observations that AE2 in parietal cells might be linked via ankyrin to the basolateral membrane cytoskeleton and that this type of linkage might play a role in immobilizing AE2 in a non-random fashion along the basolateral membrane domain.     

Pth1/Vam3p is the syntaxin homolog at the vacuolar membrane of Saccharomyces cerevisiae required for the delivery of vacuolar hydrolases

We constructed hybrid proteins containing a plant alpha-galactosidase fused to various C-terminal moieties of the hypoxic Srp1p; this allowed us to identify a cell wall-bound form of Srp1p. We showed that the last 30 amino acids of Srp1p, but not the last 16, contain sufficient information to signal glycosyl-phosphatidylinositol anchor attachment and subsequent cell wall anchorage. The cell wall-bound form was shown to be linked by means of a beta1,6-glucose-containing side-chain. Pmt1p enzyme is known as a protein-O-mannosyltransferase that initiates the O-glycosidic chains on proteins. We found that a pmt1 deletion mutant was highly sensitive to zymolyase and that in this strain the alpha-galactosidase-Srp1 fusion proteins, an alpha-galactosidase-Sed1 hybrid protein and an alpha-galactosidase-alpha-agglutinin hybrid protein were absent from both the membrane and the cell wall fractions. However, the plasma membrane protein Gas1p still receives its glycosyl-phosphatidylinositol anchor in pmt1 cells, and in this mutant strain an alpha-galactosidase-Cwp2 fusion protein was found linked to the cell wall but devoid of beta1,6-glucan side-chain, indicating an alternative mechanism of cell wall anchorage.     

Selective regulation of protein kinase C isoenzymes by oleic acid in human platelets

Cis-unsaturated fatty acids activate soluble protein kinase C (PKC) in vitro and in intact platelets. The following studies were conducted to determine the effects of oleate on individual isoenzymes of PKC in human platelets. Human platelets were found to contain predominantly PKC alpha, beta I, beta II, and delta with minor immunoreactivity for PKC epsilon, zeta, and eta. In intact platelets, sodium oleate caused a time-dependent redistribution of PKC alpha, beta II, and delta from cytosol to membrane fractions with little effects on PKC beta I. On the other hand, PMA and thrombin induced translocation of all four isoenzymes of PKC. In vitro, oleate partially activated (50% of Vmax) purified calcium-dependent PKC (alpha, beta I, and beta II) with an EC50 of 50 microM whereas it fully activated (100% of Vmax) purified calcium-independent PKC (predominantly delta) with an EC50 of 5 microM. The selective effects of oleate on PKC isoenzymes were investigated in platelet cytosol which contains endogenous PKC and its physiologic substrates. Under these conditions, oleate potently activated calcium-independent PKC causing the phosphorylation of the 40-kDa substrate. Activation of calcium-dependent isoforms occurred only at higher concentrations of oleate. Thus, oleate activates multiple isoenzymes of PKC with predominant effects on calcium-independent PKC.     

alpha-Spectrin is required for ovarian follicle monolayer integrity in Drosophila melanogaster

To understand the role of the spectrin-based membrane skeleton in generating epithelial polarity, we characterized the distribution of membrane skeletal components in Drosophila ovarian follicle cells and in somatic clones of mutant cells that lack alpha-spectrin. Immunolocalization data reveal that wild-type follicle cells contain two populations of spectrin heterodimers: a network of alphabeta heterodimers concentrated on the lateral plasma membrane and an alphabetaH population targeted to the apical surface. Induction of somatic clones lacking alpha-spectrin leads to follicle cell hyperplasia. Surprisingly, elimination of alpha-spectrin from follicle cells does not appear to prevent the assembly of conventional beta-spectrin and ankyrin at the lateral domain of the follicle cell plasma membrane. However, the alpha-subunit is essential for the correct localization of betaH-spectrin to the apical surface. As a consequence of disrupting the apical membrane skeleton a distinct sub population of follicle cells undergoes unregulated proliferation which leads to the loss of monolayer organization and disruption of the anterior-posterior axis of the oocyte. These results suggest that the spectrin-based membrane skeleton is required in a developmental pathway that controls follicle cell monolayer integrity and proliferation.     

Cis-activation of L1-mediated ankyrin recruitment by TAG-1 homophilic cell adhesion

Neural cell adhesion molecules (CAMs) of the immunoglobulin (Ig) superfamily mediate not only cell aggregation but also growth cone guidance and neurite outgrowth. In this study we demonstrate that two neural CAMs, L1-CAM and TAG-1, induce the homophilic aggregation of Drosophila S2 cells but are unable to interact with each other when expressed on different cells (trans-interaction). However, immunoprecipitations from cells co-expressing L1-CAM and TAG-1 showed a strong cis-interaction between the two molecules in the plane of the plasma membrane. TAG-1 is linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor and therefore is unable to directly interact with cytoplasmic proteins. In contrast, L1-CAM-mediated homophilic cell adhesion induces the selective recruitment of the membrane skeleton protein ankyrin to areas of cell contact. Immunolabeling experiments in which S2 cells expressing TAG-1 were mixed with cells co-expressing L1-CAM and TAG-1 demonstrated that the homophilic interaction between TAG-1 molecules results in the cis-activation of L1-CAM to bind ankyrin. This TAG-1-dependent recruitment of the membrane skeleton provides an example of how GPI-anchored CAMs are able to transduce signals to the cytoplasm. Furthermore, such interactions might ultimately result in the recruitment and the activation of other signaling molecules at sites of cell contacts.     

Isocitrate lyase localisation in Saccharomyces cerevisiae cells

The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm. This protein was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation. This conclusion is supported by experiments carried out by damaging the protoplast plasma membrane with DEAE-dextran, by differential centrifugation of osmotically lysed protoplast and by using the green fluorescent protein (GFP) of Aequorea victoria as a reporter fusion tag to localise the subcellular compartment to which isocitrate lyase is targeted.     

Structure of the ankyrin-binding domain of alpha-Na,K-ATPase

The ankyrin 33-residue repeating motif, an L-shaped structure with protruding beta-hairpin tips, mediates specific macromolecular interactions with cytoskeletal, membrane, and regulatory proteins. The association between ankyrin and alpha-Na,K-ATPase, a ubiquitous membrane protein critical to vectorial transport of ions and nutrients, is required to assemble and stabilize Na,K-ATPase at the plasma membrane. alpha-Na,K-ATPase binds both red cell ankyrin (AnkR, a product of the ANK1 gene) and Madin-Darby canine kidney cell ankyrin (AnkG, a product of the ANK3 gene) utilizing residues 142-166 (SYYQEAKSSKIMESFK NMVPQQALV) in its second cytoplasmic domain. Fusion peptides of glutathione S-transferase incorporating these 25 amino acids bind specifically to purified ankyrin (Kd = 118 +/- 50 nM). The three-dimensional structure (2.6 A) of this minimal ankyrin-binding motif, crystallized as the fusion protein, reveals a 7-residue loop with one charged hydrophilic face capping a double beta-strand. Comparison with ankyrin-binding sequences in p53, CD44, neurofascin/L1, and the inositol 1,4,5-trisphosphate receptor suggests that the valency and specificity of ankyrin binding is achieved by the interaction of 5-7-residue surface loops with the beta-hairpin tips of multiple ankyrin repeat units.