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PubMed1990

Structural features of the cytoplasmic region of CD4 required for internalization.

CD4, the T cell surface antigen, is phosphorylated and internalized when T cells are activated or treated with a phorbol ester, PMA. The actual phosphorylation sites have been identified and the role of phosphorylation of each on CD4 internalization investigated. Seven different mutants, in each of which one, two or all three of the serine residues of the cytoplasmic region was modified to alanine(s) (CD4.SA mutants) and one mutant in which the whole amino acid sequence from Gln421 to the C-terminal Ile433 was changed (CD4.EP mutant) were constructed and used to determine the effect of phosphorylation on CD4 internalization. Ser408 was the most efficiently phosphorylated by PMA treatment, Ser415 next and Ser431 to a minor extent. The effect of mutation on internalization was well matched with the effect on extent of phosphorylation, i.e. Ser408 was the residue most important for internalization. However, complete inhibition of CD4 internalization was achieved only by mutating all three serine residues. Interestingly, the mutant CD4.EP in which Ser408 was present and phosphorylated was not measurably internalized, suggesting that phosphorylation of Ser408 induces CD4 internalization only when other structural features of the cytoplasmic domain remain intact. In addition, the data suggest the existence of an additional minor pathway for CD4 internalization which is phosphorylation independent.

Amino Acid Sequence↗
PubMed1994

Design and chemical synthesis of a neoprotein structural model for the cytoplasmic domain of a multisubunit cell-surface receptor: integrin alpha IIb beta 3 (platelet GPIIb-IIIa).

Integrins are a class of heterodimeric cell adhesion receptors involved in cell migration, cell anchorage, and cell-cell interactions. The cytoplasmic domains of integrins are of key importance in these activities. We have designed and chemically synthesized a 126 amino acid model protein (MP-1) containing both cytoplasmic tails of the platelet-derived integrin alpha IIb beta 3 covalently linked via a helical coiled coil. The coiled-coil tertiary structure was incorporated to mimic the membrane-spanning domain of the integrin and to act as a topological constraint fixing the two cytoplasmic tails in a parallel arrangement. This molecule, which contains two C-termini, was constructed by chemical dovetailing. The bromoacetylated and cysteinyl peptide synthons were unambiguously ligated through the formation of a thioether linkage. Ultraviolet circular dichroism (CD) spectroscopy has been performed on MP-1 and related compounds, confirming that a helical coiled coil is present within the MP-1 molecule. Significantly, the helicity apparently extends beyond the predicted amphiphilic region of MP-1. Fluorescence measurements suggest that a defined tertiary structure has formed by the association of the two cytoplasmic domains. We conclude that this is a practical design strategy for the study of the cytoplasmic domain of multisubunit cell-surface receptors.

Amino Acid Sequence↗
PubMed1997

The Drosophila gene fs(2)cup interacts with otu to define a cytoplasmic pathway required for the structure and function of germ-line chromosomes.

The Drosophila ovarian tumor gene (otu) encodes cytoplasmic proteins that are required in germ-line cells for cyst formation, nurse cell chromosome structure and egg maturation. We have analyzed a gene, fs(2)cup, that participates in many of the same processes and interacts with otu genetically. Both nurse cell and oocyte chromosomes require cup to attain a normal morphology. In addition, the gene is needed for the oocyte to grow normally by taking up materials transported from the nurse cells. The gene encodes a 1132-amino-acid protein containing a putative membrane-spanning domain. Cup protein (but not cup RNA) is transported selectively into the oocyte in germarial cysts, like the p104 Otu protein. It is strongly associated with large structures in the cytoplasm and perinuclear region of nurse cells and, like Otu, moves to the periphery of these cells in stages 9-10. Moreover, cup mutations dominantly disrupt meiotic chromosome segregation. We propose that cup, otu and another interacting gene, fs(2)B, take part in a common cytoplasmic pathway with multiple functions during oogenesis.

Amino Acid Sequence↗
PubMed2001

Solution structure of the dimeric cytoplasmic domain of syndecan-4.

The syndecans, transmembrane proteoglycans which are involved in the organization of cytoskeleton and/or actin microfilaments, have important roles as cell surface receptors during cell-cell and/or cell-matrix interactions. Since previous studies indicate that the function of the syndecan-4 cytoplasmic domain is dependent on its oligomeric status, the conformation of the syndecan-4 cytoplasmic domain itself is important in the understanding of its biological roles. Gel filtration results show that the syndecan-4 cytoplasmic domain (4L) itself forms a dimer stabilized by ionic interactions between peptides at physiological pH. Commensurately, the NMR structures demonstrate that syndecan-4L is a compact intertwined dimer with a symmetric clamp shape in the central variable V region with a root-mean-square deviation between backbone atom coordinates of 0.95 A for residues Leu(186)-Ala(195). The molecular surface of the 4L dimer is highly positively charged. In addition, no intersubunit NOEs in membrane proximal amino acid resides (C1 region) have been observed, demonstrating that the C1 region is mostly unstructured in the syndecan-4L dimer. Interestingly, two parallel strands of 4L form a cavity in the center of the dimeric twist similar to our previously reported 4V structure. The overall topology of the central variable region within the 4L structure is very similar to that of 4V complexed with the phosphatidylinositol 4,5-bisphosphate; however, the intersubunit interaction mode is affected by the presence of C1 and C2 regions. Therefore, we propose that although the 4V region in the full cytoplasmic domain has a tendency for strong peptide--peptide interaction, it may not be enough to overcome the repulsion of the C1 regions of syndecan-4L.

Amino Acid Sequence↗
PubMed1996

Structure determination of the fourth cytoplasmic loop and carboxyl terminal domain of bovine rhodopsin.

PURPOSE: High resolution structural information is lacking for any member of the class of G-protein receptors. This dearth of structural information extends to virtually all integral membrane proteins. As part of an alternative approach to examining integral membrane protein structure, we are determining the structures of the extramembraneous domains of the G-protein receptor, rhodopsin. METHODS: The carboxyl terminal domain of bovine rhodopsin was synthesized, containing the last 43 amino acids of the protein sequence (rhoIVe). This sequence included the entire putative fourth cytoplasmic loop as well as a significant portion of helix seven, the transmembrane helix of this receptor to which the carboxyl terminal is attached. The solution structure of rhoIVe was determined by multidimensional 1H nuclear magnetic resonance. RESULTS: The structure contained a portion of alpha-helix corresponding to the top of transmembrane helix seven of the receptor. This allowed unambiguous docking of the carboxyl terminal domain to a model of the transmembrane domain. Helix seven is longer than suggested by hydropathy analysis. The structure also revealed the fourth cytoplasmic loop. The palmitoylation sites of rhodopsin are located near the deduced membrane surface. However, palmitoylation is not required for formation of this loop. CONCLUSIONS: The carboxyl terminal of rhodopsin forms a structural domain whose structure can be determined separately from the rest of the protein. This structure reveals the fourth cytoplasmic loop that had been suggested to exist based on the presence of palmitoylation sites in the carboxyl terminal domain. Determination of the structure of all of the cytoplasmic domains of rhodopsin in a manner that allows docking to the structure of the transmembrane domain should permit construction of the entire surface of rhodopsin that interacts with the G-protein, transducin. Additionally, the rhodopsin phosphorylation sites and mutations associated with certain autosomal dominant forms of retinitis pigmentosa can now be located in the three dimensional structure of the carboxyl terminal domain.

Amino Acid Sequence↗
PubMed1994

Quantitative ultrastructural study of acrylamide induced cytoplasmic desmosome-like structures in cultured rat keratinocytes.

Although present in many tumours, the mechanism of formation and the significance of the so-called 'cytoplasmic desmosome' (CD) remain speculative. Recently, we reported an in vitro model for the induction of CD in rat keratinocytes following acrylamide treatment. In the present study and based on quantitative and qualitative evidence, CD could be divided into two categories. The first comprised individually scattered desmosomes always located in the cortical cytoplasm and associated with tonofilament bundles. Their sizes were comparable with those of intercellular desmosomes (ID) in control cells. The second category comprised clusters of homogeneously small-size desmosomes that may be located deep in the cytoplasm and were not always associated with tonofilament bundles. Whilst the latter group may be formed de novo in the cytoplasm as a result of the acrylamide-induced inhibition of protein kinases, the first group may be the result of internalization of surface desmosomes by certain tonofilaments under tension. In order to assess this possibility, we examined tonofilament organization following wounding of the epithelial sheet. Injured cells exhibited spiral-form tonofilaments extending close to the cell membrane. This retraction could be justified ultrastructurally by direct association between tonofilaments and microfilaments.

Acrylamides↗
PubMed1979

[Temperature-dependent structural rearrangements in the cytoplasmic membranes of Escherichia coli cells].

By the freeze-etching method, in has been shown that E. coli plasma membranes undergo a structural transition in the range of temperatures within 0 and 20 degrees C which could be observed as redistribution of intramembrane particles with free-zone formation. The onset of temperature interval of this transition (20 degrees) well correlate with the break in the Arrhenius curves characterizing the cell membrane permeability for free nucleotides and for respiration intensity.

Cell Membrane↗
PubMed1981

Cytoplasmic actin in postsynaptic structures at the neuromuscular junction.

We used an antibody prepared against Aplysia (mollusc) body-wall actin that specifically reacts with certain forms of cytoplasmic actin in mammalian cells to probe for the presence of actin at the neuromuscular junction. Immunocytochemical studies showed that actin or an actinlike molecule is concentrated at neuromuscular junctions of normal and denervated adult rat muscle fibers. Actin is present at the neuromuscular junctions of fibers of developing diaphragm muscles as early as embryonic day 18, well before postsynaptic folds are formed. These results suggest that cytoplasmic actin may play a role in the clustering or stabilization of acetylcholine receptors at the neuromuscular junction.

Actins↗
PubMed1998

Induction of tyrosine aminotransferase of primary cultured rat hepatocytes depends on the organization of microtubules.

We investigated the relationship between the expression of tyrosine aminotransferase (TAT) and cytoskeletal systems of cultured rat hepatocytes by using serum-free culture conditions and changing three factors: 1) the concentration of calcium, 2) the dish-coating material, and 3) the cell-plating density. In hepatocytes in low-calcium medium, induction of TAT by dexamethasone and glucagon was maintained, although cell-cell adhesion was lost. Hepatocytes on Matrigel formed a nonspreading, spherical shape that provided them with the full extent of TAT activity without cell-cell adhesion. Hepatocytes plated on collagen at low cell density spread and changed shape, and the induction of TAT activity was markedly reduced. By using confocal laser-scanning microscopy, we analyzed the three-dimensional organization of cytoplasmic microtubules of hepatocytes maintaining the ability of TAT induction. Hepatocytes plated on collagen at low cell density possessed the radial filamentous structure of cytoplasmic microtubules. When the spherical shape of hepatocytes was maintained by cultivating cells on Matrigel, a ring-like structure of cytoplasmic microtubules beneath the plasma membrane was dominant. Moreover, the induction of TAT activity of hepatocytes in a standard culture system was strongly inhibited by the addition of 1 microM colchicine. These studies suggest that the organization of cytoplasmic microtubules may participate in the shape-related regulation of cell function.

Actins↗
PubMed1994

Electron microscopic evidence of a viral nature for osteoclast inclusions in Paget's disease of bone.

Circumstantial evidence from electron microscopic and immunological studies support the view that Paget's disease of bone represents a slow virus infection. However, there is only limited information available regarding its electron microscopic, enzyme and immunocytochemical characteristics. Two cases were studied using electron microscopy with particular emphasis on the inclusions in osteoclasts. Detailed ultrastructural and cytochemical studies including immuno-electron microscopy were performed. Some osteoclasts demonstrated specific virus-like structures composed of aggregations of microtubules in the nucleus and cytoplasm. The structures were easily digested by trypsin or protease, and were sensitive to RNase, which provided substantial evidence of a proteinaceous nature and inclusion of ribonucleic acid. Immunocytochemical examination identified binding of anti-respiratory syncytial virus and anti-measles virus antibodies in the tissue obtained from one of the two cases examined. The presence of viral antigens in structures in the cytoplasm of Pagetic osteoclasts supports the theory of paramyxovirus involvement in this disease.

Antigens, Viral↗
PubMed2000

Structure of a murine cytoplasmic serine hydroxymethyltransferase quinonoid ternary complex: evidence for asymmetric obligate dimers.

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.

Animals↗
PubMed1960

Electron microscopy of the centrifuged sea urchin egg, with a note on the structure of the ground cytoplasm.

Centrifuged, unfertilized eggs of the sea urchin, Arbacia punctulata, have been studied with the electron microscope. Subcellular particles were stratified by centrifuging living cells, known to be normally fertilizable, for five minutes at 3,000 g. The layered subcellular particles, including cortical granules, 16 mmicro RNP particles, pigment, yolk, mitochondria, and oil droplets, possess characteristic ultrastructural features by which they may be identified in situ. The clear zone contains 16 mmicro particles, most of them freely dispersed, scattered mitochondria, and a few composite structures made up of annulate lamellae in parallel layers or in association with dense, spherical aggregates of the RNP particles. Free 16 mmicro particles are found, in addition, throughout the cell, in the interstices between the stratified larger particles. They show a tendency to form ramifying aggregates resulting from certain types of injury to the cell. A few vesicular structures, found mainly in the clear zone, have attached RNP particles, and appear to be related to the ER of tissue cells. Other vesicles, bounded by smooth membranes, are found throughout the cell. These are extremely variable in size, number, and distribution; their total number appears to depend upon conditions of fixation. It is suggested that limited formation of such structures is a normal property of the ground cytoplasm in this cell, but that fixed cells with very large numbers of smooth surfaced vesicles have produced the latter as a response to chemical injury. A model of the ground cytoplasm is proposed whose aim is to reconcile the rheological behavior of the living cell with the ultrastructural features observed.

Animals↗
PubMed2004

Longitudinal diffusion in retinal rod and cone outer segment cytoplasm: the consequence of cell structure.

Excitation signals spread along photoreceptor outer segments away from the site of photon capture because of longitudinal diffusion of cGMP, a cytoplasmic second messenger. The quantitative features of longitudinal diffusion reflect the anatomical structure of the outer segment, known to be profoundly different in rod and cone photoreceptors. To explore how structural differences affect cytoplasmic diffusion and to assess whether longitudinal diffusion may contribute to the difference in signal transduction between photoreceptor types, we investigated, both theoretically and experimentally, the longitudinal diffusion of small, hydrophilic molecules in outer segments. We developed a new theoretical analysis to explicitly compute the longitudinal diffusion constant, Dl, in terms of outer segment structure. Using time-resolved fluorescence imaging we measured Dl of Alexa488 and lucifer yellow in intact, single cones and validated the theoretical analysis. We used numerical simulations of the theoretical model to investigate cGMP diffusion in outer segments of various species. At a given time interval, cGMP spreads further in rod than in cone outer segments of the same dimensions. Across all species, the spatial spread of cGMP at the peak of the dim light photocurrent is 3-5 microm in rod outer segments, regardless of their absolute size. Similarly the cGMP spatial spread is 0.7-1 microm in cone outer segments, independently of their dimensions.

Animals↗
PubMed1990

Three-dimensional structure of the mammalian cytoplasmic ribosome.

A three-dimensional reconstruction of the 80 S ribosome from rabbit reticulocytes has been calculated from low-dose electron micrographs of a negatively stained single-particle specimen. At 37 A resolution, the precise orientations of the 40 S and 60 S subunits within the monosome can be discerned. The translational domain centered on the upper portion of the subunit/subunit interface is quite open, allowing considerable space between the subunits for interactions with the non-ribosomal macromolecules involved in protein synthesis. Further, the cytosolic side of the monosome is strikingly more open than the membrane-attachment side, suggesting a greater ease of communication with the cytoplasm, which would facilitate the inwards and outwards diffusion of a number of ligands. Although the 60 S subunit portion of the 80 S structure shows essentially all of the major morphological features identified for the eubacterial 50 S large subunit, it appears to possess a region of additional mass that evidently accounts for the more ellipsoidal form of the eukaryotic subunit.

Animals↗
PubMed2006

Secondary structure of a KCNE cytoplasmic domain.

Type I transmembrane KCNE peptides contain a conserved C-terminal cytoplasmic domain that abuts the transmembrane segment. In KCNE1, this region is required for modulation of KCNQ1 K(+) channels to afford the slowly activating cardiac I(Ks) current. We utilized alanine/leucine scanning to determine whether this region possesses any secondary structure and to identify the KCNE1 residues that face the KCNQ1 channel complex. Helical periodicity analysis of the mutation-induced perturbations in voltage activation and deactivation kinetics of KCNQ1-KCNE1 complexes defined that the KCNE1 C terminus is alpha-helical when split in half at a conserved proline residue. This helical rendering assigns all known long QT mutations in the KCNE1 C-terminal domain as protein facing. The identification of a secondary structure within the KCNE1 C-terminal domain provides a structural scaffold to map protein-protein interactions with the pore-forming KCNQ1 subunit as well as the cytoplasmic regulatory proteins anchored to KCNQ1-KCNE complexes.

Amino Acid Sequence↗
PubMed1995

Behavior of interchromatin granules during the cell cycle.

We investigated at the ultrastructural level, by different cytochemical and immunocytological approaches, the behavior of interchromatin granules (IGs) during interphase and mitosis in two cell lines (HEp-2 and Ehrlich tumor cells). Identical results were found in all two cell types. In interphase cells, IGs group into irregular clusters of varying size. They are frequently associated with coiled bodies and homogeneous fibrillar bodies. Analysis of serial sections reveals that IG clusters occupy distinct regions within the nucleoplasm. During prophase, the aggregation of granules in these clusters gives rise to compact, spherical, granular structures. These disperse in the mitotic cytoplasm at the breakdown of the nuclear envelope. At early telophase, some of them come into close contact with the periphery of reforming nuclei. IG clusters reappear in the daughter nuclei only after the chromosomes have decondensed during late telophase. Concomitantly, the cytoplasmic granular structures disappear. During the cell cycle, IG are silver-stainable and EDTA-positive. They are also constantly labeled by the polyadenylate nucleotidyl transferase-immunogold technique for detecting RNA. These results support the view that IGs persist throughout the whole cell cycle.

Acetylation↗
PubMed1996

Protein structural change at the cytoplasmic surface as the cause of cooperativity in the bacteriorhodopsin photocycle.

The effects of excitation light intensity on the kinetics of the bacteriorhodopsin photocycle were investigated. The earlier reported intensity-dependent changes at 410 and 570 nm are explained by parallel increases in two of the rate constants, for proton transfers to D96 from the Schiff base and from the cytoplasmic surface, without changes in the others, as the photoexcited fraction is increased. Thus, it appears that the pKa of D96 is raised by a cooperative effect within the purple membrane. This interpretation of the wild-type kinetics was confirmed by results with several mutant proteins, where the rates are well separated in time and a model-dependent analysis is unnecessary. Based on earlier results that demonstrated a structural change of the protein after deprotonation of the Schiff base that increases the area of the cytoplasmic surface, and the effects of high hydrostatic pressure and lowered water activity on the photocycle steps in question, we suggest that the pKa of D96 is raised by a lateral pressure that develops when other bacteriorhodopsin molecules are photoexcited within the two-dimensional lattice of the purple membrane. Expulsion of no more than a few water molecules bound near D96 by this pressure would account for the calculated increase of 0.6 units in the pKa.

Bacteriorhodopsins↗