Purification and characterization of proteoliaisin, a coordinating protein in fertilization envelope assembly

We report the purification and characterization of proteoliaisin, a protein that participates in the assembly of the sea urchin fertilization envelope. Proteoliaisin was purified from egg cortical granule exudate to greater than 99% homogeneity using chromatography on DEAE-Sepharose and on phenyl-Sepharose. Native proteoliaisin is a highly asymmetric protein (f/fo = 2.0) composed of a single Mr approximately 230,000 peptide. Its asymmetry was demonstrated both by analytical ultracentrifugation and by nondenaturing polyacrylamide gel electrophoresis, a novel analysis that detects molecular asymmetry in heterogeneous protein mixtures. Proteoliaisin is enriched in six amino acids: aspartic acid/asparagine, glutamic acid/glutamine, glycine, and cysteine, which account for over 50% of its mass. Nearly all of the cysteine residues are disulfide bonded. The protein contains a small proportion of aromatic amino acids with phenylalanine greater than tyrosine greater than tryptophan. At neutral pH its absorbance maximum is at 274.5 nm, with an extinction coefficient of 0.43 ml mg-1 cm-1. Proteoliaisin forms a 1:1 Ca2+-stabilized complex with ovoperoxidase, another component of the fertilization envelope, with Kd = 1.1 X 10(-6) M. Proteoliaisin, a constituent of the specialized echinoderm extracellular matrix called the fertilization envelope, has certain structural similarities to mammalian extracellular matrix proteins.     

Functional domains of proteoliaisin, the adhesive protein that orchestrates fertilization envelope assembly

Ovoperoxidase, the enzyme that hardens the sea urchin fertilization envelope, is inserted into the assembling extracellular matrix through the action of an intermediary protein, proteoliaisin (PLN). The domain structure of PLN, a large, rod-shaped protein that binds to ovoperoxidase and the vitelline layer, was examined by limited proteolytic cleavage. Purified proteolytic fragments of PLN were tested for their ability to bind ovoperoxidase, inhibit the binding of 125I-PLN to the vitelline layer, or act as substrates for the hardening reaction. Based on these results, the vitelline layer-binding domain can be placed near the amino terminus, followed by the binding site for ovoperoxidase; the distal two-thirds of the protein contain sites for ovoperoxidase-catalyzed dityrosine formation. The pentapeptide GRGDS (but not RGD) inhibited PLN-vitelline layer binding half-maximally at 0.2 mM. Moreover, PLN promoted adhesion of bovine aortic endothelial cells to plastic dishes, a process inhibited by GRGDS. Thus PLN is a new member of the adhesive protein family, the function of which is to coordinate the morphogenesis of a specific, rapidly assembled extracellular matrix.     

Assembly of the sea urchin fertilization membrane: isolation of proteoliaisin, a calcium-dependent ovoperoxidase binding protein

Fertilization of the sea urchin egg is accompanied by the assembly of an extracellular glycoprotein coat, the fertilization membrane. Assembly of the fertilization membrane involves exocytosis of egg cortical granules, divalent cation-mediated association of exudate proteins with the egg glycocalyx (the vitelline layer), and cross- linking of the assembled structure by ovoperoxidase, a fertilization membrane component derived from the cortical granules. We have identified and isolated a new protein, which we call proteoliaisin, that appears to be responsible for inserting ovoperoxidase into the fertilization membrane. Proteoliaisin is a 250,000-Mr protein that binds ovoperoxidase in a Ca2+-dependent manner, with half-maximal binding at 50 microM Ca2+. Other divalent cations are less effective (Ba2+, Mn2+, and Sr2+) or ineffective (Mg2+ and Cd2+) in mediating the binding interaction. Binding is optimal over the physiological pH range of fertilization membrane assembly (pH 5.5-7.5). Both proteoliaisin and ovoperoxidase are found in isolated, uncross-linked fertilization membranes. We have identified several macromolecular aggregates that are released from uncross-linked fertilization membranes after dilution into divalent cation-free buffer. One of these is an ovoperoxidase- proteoliaisin complex that is further disrupted only upon the addition of EGTA. These results suggest that a Ca2+-stabilized complex of ovoperoxidase and proteoliaisin forms one structural subunit of the fertilization membrane.     

Evidence for the existence of two assembly domains within the sea urchin fertilization envelope.

The sea urchin fertilization envelope (FE) is a complex, macromolecular aggregate assembled by the addition of cortical granule secretions to the vitelline layer. The completed, trilaminar structure has a dense layer sandwiched between surface coats of paracrystalline material. Two cortical granule enzymes, ovoperoxidase and protease, and a cell surface transglutaminase are required for the assembly process. We have examined, by quick-freeze, deep-etch, rotary-shadow electron microscopy, the effects of inhibiting each of these enzymes upon FE assembly. These experiments reveal two domains within the FE, distinguishable by their enzymatic requirements for proper maturation. The first domain consists of the microvillar casts which require both protease and transglutaminase activities to obtain a normal paracrystalline coat. The second domain comprises the regions between casts and appears to mature by ovoperoxidase-mediated cross-linking of paracrystalline material to the envelope.     

Superoxide peroxidase activity of ovoperoxidase, the cross-linking enzyme of fertilization

Ovoperoxidase, an enzyme secreted by the eggs of the sea urchin Stronglycocentrotus purpuratus upon activation, catalyzes the formation of dityrosine residues in the fertilization envelope. This cross-linking reaction requires extracellular H2O2, which is produced by the egg during the cyanide-insensitive "respiratory burst" of fertilization. While investigating the possibility that the sea urchin oxidase might generate O2- as a precursor to H2O2, we discovered that ovoperoxidase possessed O2- degrading activity. Ovoperoxidase catalyzed the breakdown of O2- in a reaction that was sensitive to inhibition by catalase, indicating a requirement for H2O2. High concentrations of either O2- or H2O2 inhibited the O2- degrading activity of ovoperoxidase, as did the peroxidase inhibitors aminotriazole, azide, and phenylhydrazine. When ovoperoxidase was heated at 56 degrees C, it lost O2- degrading activity in parallel with peroxidase activity. In contrast, the copper-chelating agent diethyldithiocarbamate, which completely inactivated CuZn superoxide dismutase, failed to affect ovoperoxidase. The requirement for H2O2 and the inhibition by aminotriazole, azide, and phenylhydrazine support the hypothesis that ovoperoxidase catalyzes the breakdown of O2- by a peroxidative mechanism. Ovoperoxidase may play a role in protecting the developing embryo from oxidants derived from O2-.     

Ovoperoxidase assembly into the sea urchin fertilization envelope and dityrosine crosslinking

Ovoperoxidase, the enzyme implicated in hardening the extracellular coat of the fertilized sea urchin egg, is inserted into the assembling uncrosslinked (soft) fertilization membrane via specific interactions with a protein, proteoliaisin (P. Weidman, E. Kay, and B. M. Shapiro (1985). J. Cell. Biol. 100, 938-946), and the vitelline scaffold. Dityrosine crosslinks introduced by ovoperoxidase have been postulated to harden the assembled structure from such indirect data as the discovery of dityrosine in hard fertilization membranes (Foerder and B. M. Shapiro (1977). Proc. Natl. Acad. Sci. USA 74, 4214-4128; H. G. Hall (1978). Cell 15, 343-355). In this report, we show directly that soft fertilization membranes (SFM) contain no dityrosine residues but acquire these crosslinks in vitro only during hardening. In vitro hardening alters the susceptibility of the fertilization membrane to disruption in cation-depleted solutions and in detergent; the kinetics of these phenomena are all similar to those of hardening in vivo. Ovoperoxidase substrates were identified as a class of high-molecular-weight proteins of SFM by polyacrylamide gel electrophoresis after in vitro hardening or after an ovoperoxidase-catalyzed radioiodination reaction. The specificity of ovoperoxidase for particular substrates decreased once it was no longer associated with these polypeptides within the SFM. Moreover, after disruption of the SFM, ovoperoxidase had an increased capacity to iodinate an exogenous protein, myoglobin. These data suggest that assembly of ovoperoxidase into a specific locus within the soft fertilization membrane provides a regulatory mechanism to guarantee the crosslinking of only certain appropriately juxtaposed tyrosyl residues in the assembled structure.     

Conformational control of ovoperoxidase catalysis in the sea urchin fertilization membrane

The ovoperoxidase-catalyzed oxidation of iodide has been investigated as a function of pH for the homogeneous enzyme and for ovoperoxidase incorporated into several forms of the egg fertilization membrane. The pH dependent hysteresis previously observed in purified ovoperoxidase (Deits, T. L., Shapiro, B. M. (1985) J. Biol. Chem. 260, 7882-7888) is entirely absent in ovoperoxidase incorporated into the mature fertilization membrane, where the enzyme is bound noncovalently in vivo. The pH activity profile of ovoperoxidase incorporated into the mature fertilization membrane closely resembles the profile observed only transiently in purified ovoperoxidase subjected to a rapid downward pH shift. These observations can be accounted for by our previously presented mechanism for ovoperoxidase hysteresis (ibid.). We hypothesize that ovoperoxidase, upon incorporation into the fertilization membrane, is restricted to a limited subset of the conformational states available to the purified enzyme. This matrix-dependent conformational restriction is a novel control mechanism that serves to enhance the catalytic activity of ovoperoxidase upon its assembly into the fertilization membrane and thereby modulates ovoperoxidase catalysis in the vicinity of the developing egg.     

Reproduction in mice: The fate of spermatozoa not involved in fertilization

Mechanisms were sought through which the control of preimplantation mouse embryo development by spermatozoa might be effected. A potential route for the transmission of sperm-dependent stimuli to C3HeB/FeJ females was uncovered. It was found that within 24–48 hr after artificial insemination with spermatozoa, in which the DNA had been labeled with tritiated thymidine, a minimum of 9% of the radioactivity was transported across the uterine walls. It was deposited among the maternal tissues in a pattern that differed from the patterns of isotope distribution obtained when either free tritiated thymidine or Escherichia coli cells containing DNA labeled with tritiated thymidine were used instead of labeled sper-matozoa. In sperm-treated animals the ovaries, the adrenals, and a mesenteric lymph node exhibited strikingly large accumulations of radioactivity. The heart, spleen, and uterus manifested lesser accumulations of label, but were higher than liver, kidney, lung, brain, muscle, and intestine. The specific activity of the lymph node was found to decrease during the 12–72-hr period following insemination. This result led to the hypothesis that the lymphatic system could serve as a route for the dissemination, to maternal tissues, of radioactivity originally associated with spermatozoa deposited in the uterus. Heat-inactivated spermatozoa, which have the potential for facilitating the first cleavage of fertilized embryos, exhibited a distribution pattern indistinguishable from untreated spermatozoa. Sperm protein kinase was found to survive the heat inactivation of spermatozoa. This stability was interpreted as being compatible with the kinase functioning as an intermediary in the transmission of sperm-dependent stimuli that control preimplantation embryo development in mice.     

Deep-etch visualization of proteins involved in clathrin assembly

Assembly proteins were extracted from bovine brain clathrin-coated vesicles with 0.5 M Tris and purified by clathrin-Sepharose affinity chromatography, then adsorbed to mica and examined by freeze-etch electron microscopy. The fraction possessing maximal ability to promote clathrin polymerization, termed AP-2, was found to be a tripartite structure composed of a relatively large central mass flanked by two smaller mirror-symmetric appendages. Elastase treatment quantitatively removed the appendages and clipped 35 kD from the molecule's major approximately 105-kD polypeptides, indicating that the appendages are made from portions of these polypeptides. The remaining central masses no longer promote clathrin polymerization, suggesting that the appendages are somehow involved in the clathrin assembly reaction. The central masses are themselves relatively compact and brick-shaped, and are sufficiently large to contain two copies of the molecule's other major polypeptides (16- and 50-kD), as well as two copies of the approximately 70-kD protease-resistant portions of the major approximately 105-kD polypeptides. Thus the native molecule seems to be a dimeric, bilaterally symmetrical entity. Direct visualization of AP-2 binding to clathrin was accomplished by preparing mixtures of the two molecules in buffers that marginally inhibit AP-2 aggregation and cage assembly. This revealed numerous examples of AP-2 molecules binding to the so-called terminal domains of clathrin triskelions, consistent with earlier electron microscopic evidence that in fully assembled cages, the AP's attach centrally to inwardly-directed terminal domains of the clathrin molecule. This would place AP-2s between the clathrin coat and the enclosed membrane in whole coated vesicles. AP-2s linked to the membrane were also visualized by enzymatically removing the clathrin from brain coated vesicles, using purified 70 kD, uncoating ATPase plus ATP. This revealed several brick-shaped molecules attached to the vesicle membrane by short stalks. The exact stoichiometry of APs to clathrin in such vesicles, before and after uncoating, remains to be determined.     

Forces involved in the assembly and stabilization of membrane proteins.

Hydrophobic organization: Determination of the structure of the bacterial photosynthetic reaction center, bacterial porins, and bacteriorhodopsin allows a comparison of the basic structural features of integral membrane proteins. Structure parameters of membrane- and water-soluble proteins are surprisingly similar, given the different dielectric environments, except for the polarity of residues on the protein surface. Hydrophobic and electrostatic forces: 1) Intramembrane helix-helix interactions that are sensitive to small structure changes can dictate assembly of membrane proteins, as indicated by reconstitution of bacteriorhodopsin from proteolytic fragments and specific dimer formation of the human erythrocyte sialoglycoprotein glycophorin A. 2) Electrostatic interactions have an important role in determining the trans-membrane orientation of integral membrane proteins of the bacterial inner membrane, as expressed by the "positive-inside" rule for the distribution of basic residues on the cis relative to the trans side of the membrane-spanning alpha-helices. The use of this charge asymmetry rule, in conjunction with a hydrophobicity algorithm for prediction of membrane-spanning domains, allows accurate prediction of the folding patterns of such polypeptides across the membrane. A role of electrostatic interactions in assembly and maintenance of the structure of oligomeric integral membrane protein complexes is also implied by the separation and extrusion from the membrane, at high pH, of the major hydrophobic subunits of the cytochrome b6f complex from the chloroplast thylakoid membrane. It is inferred that the hydrophobic helix-helix interactions between the subunits of this complex, whose function is electron transfer and proton translocation, are relatively weak compared to those in bacteriorhodopsin.     

Localization of TraC, a protein involved in assembly of the F conjugative pilus.

TraC is one of the proteins encoded by the F transfer region of the F conjugative plasmid which is required for the assembly of F pilin into the mature F pilus structure. Overproduction of this protein from the plasmid pKAS2, which carries only traC, resulted in the formation of inclusion bodies from which soluble TraC was purified. When small amounts of TraC were produced from pKAS2, the protein was localized to the cytoplasm by using anti-TraC antibodies. Similar analysis of a set of TraC-alkaline phosphatase fusion proteins localized all of these fusion proteins to the cytoplasm. However, when TraC was expressed from the F plasmid, much of it appeared associated with the bacterial membrane fraction. Under these conditions, TraC does not appear to be part of the tip of the F pilus, as neither anti-TraC antibodies nor purified TraC had any effect on the infection of F-containing bacteria by the filamentous bacteriophage f1. These data suggest that TraC is normally associated with the membrane through interactions with other proteins specified by the tra region. This interaction may be via the carboxyl-terminal region of the TraC protein, as a mutant TraC protein containing an Arg-Cys substitution at amino acid 811 exhibits an interaction with the membrane weaker than that of the wild-type protein in the presence of the other Tra proteins.     

Auxiliary proteins involved in the assembly and sustenance of photosystem II

Chloroplast proteins that regulate the biogenesis, performance and acclimation of the photosynthetic protein complexes are currently under intense research. Dozens, possibly even hundreds, of such proteins in the stroma, thylakoid membrane and the lumen assist the biogenesis and constant repair of the water splitting photosystem (PS) II complex. During the repair cycle, assistance is required at several levels including the degradation of photodamaged D1 protein, de novo synthesis, membrane insertion, folding of the nascent protein chains and the reassembly of released protein subunits and different co-factors into PSII in order to guarantee the maintenance of the PSII function. Here we review the present knowledge of the auxiliary proteins, which have been reported to be involved in the biogenesis and maintenance of PSII.     

Ran及其结合与调控蛋白在核膜装配过程中的调控作用

核膜在细胞周期中呈现高度的动态性：在细胞分裂的前中期,核膜崩解并分散到细胞质中;在细胞分裂的后期,核膜开始在染色体的表面重新装配,最终形成完整的核膜结构。近期的研究发现,Ran GTP酶、物质转运蛋白importinβ、内层核膜蛋白LBR（lamin B receptor）以及核孔复合体蛋白nucleoporins在核膜重建的过程中起关键性调控作用,并受到细胞周期调控因子p34cdc2激酶的调节。LBR是一个八次跨膜的膜蛋白,主要定位于内层核膜。在细胞分裂的早期,随着核膜崩解,LBR与核膜崩解而生成的小膜泡一起分散到细胞质中;在细胞分裂的后期,通过LBR与importinβ相互结合,含有LBR的膜泡被importinβ携带至染色质的表面参与核膜重建。目前已知p34cdc2激酶对LBR与importinβ介导的核膜重建起重要调控作用。Nucleoporins是核孔复合体主要组分。随核膜崩解,核孔复合体解聚成nucleoporins,分散到细胞质中,或结合到其他亚细胞成分上。细胞分裂后期,核孔复合体伴随核膜装配而组装。     

Sea urchin fertilization envelope: Uncoupling of cortical granule exocytosis from envelope assembly and isolation of an envelope intermediate from Strongylocentrotus purpuratus embryos

The sea urchin fertilization envelope (FE) is an extraembryonic coat which develops from the egg vitelline envelope (VE) and the secreted paracrystalline protein fraction of the cortical granules at fertilization. The FE undergoes further developmental changes postinsemination which are characterized by changes in envelope permeability, solubility in reducing and denaturing solvents, and morphology. We have developed a procedure to uncouple cortical granule exocytosis from assembly of the paracrystalline protein fraction onto the VE template. Egg suspensions were inseminated in normal seawater and diluted into Ca²⁺- and Mg²⁺-free seawater at 15 sec postinsemination. Phase-contrast and electron microscopic observations showed that the embryos formed a normally elevated, extremely thin envelope through which the cortical granule exudate permeated. Secretion studies showed that eggs which were diluted into divalent ion-free seawater postinsemination secreted as much protein into the surrounding seawater as eggs which had their VEs removed prior to the experiment. We have termed the envelope elevated in divalent ion-free seawater the VE1 and we believe that it is the VE structural component of the FE based on its thickness and morphology. VE1s were isolated by gentle physical means and the preparations appeared to be greater than 80% pure based on radioactive mixing experiments and on malate dehydrogenase and glucose-6-phosphate dehydrogenase marker studies. VE1s were at least 80% soluble based on extraction of radioiodinated preparations with reducing and denaturing solvents. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of VE1s showed eight major polypeptides which ranged from 30,500 to 270,000 in molecular weight.     

Ultrastructural changes during fertilization envelope assembly in Lytechinus pictus eggs revealed by quick-freeze,deep-etch electron microscopy

Morphological features of fertilization envelope assembly in egges from the sea urchin Lytechinus pictus were examind in platinum replicas of samples quick-frozen, deep-etched, and rotary-shadowed at various times after insemination. Unfertilized eggs are surrounded by the vitelline layer, a glycocalyx, which faith-fully follows the contours of the microvillus-studded egg surface. The vitelline layer is secured to the plasma membrane below via a series of short projections called vitelline posts. The vitelline matrix itself is an elaborate meshwork of uniformly sized filaments, which are decorated in places with globular particles. At fertilization, the vitelline layer elevates off the egg surface and by 1 min after insemination appears as a thin, airy network of fibers. In contrast to Strongylocentrotus purpuratus, impressions of the underlying microvilli are not retained in this species. The vitelline template appears to become filled in by the deposition of amorphous secretory material between 1 and 5 min after fertilization. This smooth, amorphous layer is then coated with a thin sheet of paracrystalline material. Paracrystalline coating is incomplete at 5 min, but by 20 min after insemination the coat is complete, consisting of ordered parallel rows of roset-telike particles.     

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.     

Localization of synaptic proteins involved in neurosecretion in different membrane microdomains

A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Ca(v)2.1 (P/Q-type) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Ca(v)2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-beta-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery.     

N-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity

West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein. Addition of the E protein glycosylation site in a lineage II strain that lacked this site increased SVP production. Similar results were obtained in the context of either reporter virus particles (RVPs) or infectious lineage II WNV. RVPs or virions bearing combinations of glycosylated and nonglycosylated forms of prM and E could infect mammalian, avian, and mosquito cells (BHK-21, QT6, and C6/36, respectively). Those particles lacking glycosylation on the E protein were modestly more infectious per genome copy on BHK-21 and QT6 cells, while this absence greatly enhanced the infection of C6/36 cells. Thus, glycosylation of WNV prM and E proteins can affect the efficiency of virus release and infection in a manner that is cell type and perhaps species dependent. This suggests a multifaceted role for envelope N-linked glycosylation in WNV biology and tropism.