Two distinct yolk lipoprotein complexes from Caenorhabditis elegans

The four yolk polypeptides of the nematode Caenorhabditis elegans are found in two types of lipoprotein particle: 12 S particles with Mr estimated at 450,000 and 8 S particles with Mr estimated at 250,000. Both types of particle contain approximately 8% phospholipids, 3% triglycerides, and 3% other lipids by mass. All four C. elegans yolk polypeptides can be found in either 12 or 8 S particles, depending upon the conditions of isolation. While the properties of the 12 and 8 S lipoprotein particles are consistent with a dimermonomer relationship, the asymmetric distribution of the yolk polypeptides between 12 and 8 S fractions suggests that at least two different oligomeric lipoprotein complexes are present in C. elegans embryos. In order to clarify the subunit composition of the C. elegans yolk lipoproteins, the patterns of polypeptides retained in immunoaffinity binding procedures by immunoglobulins of different antigenic specificities have been compared. When immunoaffinity binding is performed in the absence of sodium dodecyl sulfate, three C. elegans yolk proteins (yp170A, yp115, and yp88) are retained together by polyclonal immunoglobulins directed against either yp115 or yp88. A monoclonal immunoglobulin also retains these three proteins together. In contrast, a second monoclonal immunoglobulin retains only the fourth yolk protein (yp170B). Aggregate species, evidently reflecting the spontaneous formation of interchain disulfide bonds, indicate that yp170A and yp88 are physically associated, whereas yp170B self-associates in dimers. It is concluded that there are two distinct lipoprotein complexes in C. elegans: the A complex, which consists of yp170A, yp115, and yp88 and is essentially heterodimeric and the B dimer, a simple dimer of yp170B.     

The Caenorhabditis elegans vitellogenin gene family includes a gene encoding a distantly related protein.

While the nematode Caenorhabditis elegans is more primitive than most egg-laying organisms, it's vitellogenins, or yolk protein precursors, appear to be more complex. C. elegans oocytes accumulate two major classes of yolk proteins. The first consists of two polypeptides with an Mr of about 170,000 (yp170A and yp170B) encoded by a family of five closely related genes called vit-1 through vit-5. The second class consists of two smaller proteins with Mr values of 115,000 (yp115) and 88,000 (yp88) which are cut from a single precursor. Here we report the cloning and analysis of a single-copy gene (vit-6) that encodes this precursor. The lengths of the gene and its mRNA are about 5 X 10(3) base pairs. Like vit-1 through vit-5, vit-6 is expressed exclusively in adult hermaphrodites. Comparison of portions of the coding sequence indicates that vit-6 is distantly related to the vit-1 through vit-5 gene family. Thus, even though the two classes of yolk proteins are antigenically and physically distinct, they are encoded by a single highly diverged gene family.     

The antigenic interrelationship between the endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum Nichols strain. I. Treponemicidal activity of cross-reactive endoflagellar antibodies against T. pallidum

Treponemicidal activity against Treponema pallidum, Nichols strain, by anti-endoflagellar antibodies and the presence of antigenic interrelationships between the endoflagella of Treponema phagedenis biotype Reiter (TPR) and T. pallidum have been demonstrated. SDS-PAGE profiles of purified endoflagella from both organisms were similar, identifying five polypeptide bands for TPR (37,000, 33,000 doublet, 30,000, and 27,000 daltons) and five polypeptide bands for T. pallidum (35,000, 33,000 doublet, 30,000, and 27,000 daltons). Antiserum against TPR endoflagella identified identical bands on Western blots of TPR, T. pallidum, and the respective endoflagellar preparations. Western blots confirmed the presence of antibodies in normal human serum (NHS) against the 33,000 dalton treponemal endoflagellar proteins. The complement-dependent treponemicidal activity of NHS against T. pallidum was completely removed by absorption with purified TPR endoflagella. Furthermore, rabbit antisera against TPR endoflagella were reactive in the Treponema pallidum immobilization (TPI) test. These findings demonstrate that anti-endoflagellar antibodies are treponemicidal against T. pallidum. A possible mechanism for this activity is discussed in relation to the subsurface location of endoflagella.     

Purification of a calcium-activated neutral proteinase from bovine brain

A calcium-activated neutral proteinase (CANP) resolved into three components has been partially purified from bovine brain. The method of isolation has resulted in 22,000, 7,100, and 8,000-fold purification for CANP I, II and III respectively. All three fractions require Ca2+ for activation. The characterization of the purified CANP I has shown that it is activated by 250 microM Ca2+ and the enzyme loses its activity when incubated in the presence of Ca2+ without substrate. Mg2+ is ineffective. The enzyme degrades neurofilament triplet proteins, tubulin and casein efficiently. The myelin basic protein is hydrolyzed after longer incubation. Bovine serum albumin and histones are unaffected. The enzyme is active at pH 5.5 to 9.0 with optimum between pH 7.5 and 8.5. It has a Km of 1.8 X 10(-7) M for the 69,000 dalton neurofilament protein. The enzyme is inhibited by sulphydryl blocking reagents and also by EGTA, leupeptin and E-64c. The SDS-PAGE analysis of the enzyme fractions has shown a major band at 66-68,000 daltons and two minor bands at 60,000 and 48-50,000 daltons for CANP I; a major band at 48-50,000 daltons and a minor band at 30-32,000 daltons for CANP II and a predominant doublet at 30-32,000 daltons with a minor band at 48-50,000 daltons for CANP III. The degradation of neurofilament proteins suggests that the CANP(s) may be involved in the turnover of these proteins.     

Compositional studies of myofibrils from rabbit striated muscle

The localization of high-molecular-weight (80,000-200,000-daltons) proteins in the sarcomere of striated muscle has been studied by coordinated electron-microscopic and sodium dodecyl sulfate (SDS) gel electrophoretic analysis of native myofilaments and extracted and digested myofibrils. Methods were developed for the isolation of thick and thin filaments and of uncontracted myofibrils which are devoid of endoproteases and membrane fragments. Treatment of crude myofibrils with 0.5% Triton X-100 results in the release of a 110,000-dalton component without affecting the myofibrillar structure. Extraction of uncontracted myofibrils with a relaxing solution of high ionic strength results in the complete disappearance of the A band and M line. In this extract, five other protein bands in addition to myosin are resolved on SDS gels: bands M 1 (190,000 daltons) and M 2 (170,000 daltons), which are suggested to be components of the M line; M 3 (150,000 daltons), a degradation product; and a doublet M 4, M 5 (140,000 daltons), thick-filament protein having the same mobility as C protein. Extraction of myofibrils with 0.15% deoxycholate, previously shown to remove Z-line density, releases a doublet Z 1, Z 2 (90,000 daltons) with the same mobility as alpha-actinin, as well as proteins of 60,000 daltons and less, and small amounts of M 1, M 2, M 4, and M 5; these proteins were not extracted with 0.5% Triton X-100. The C, M-line, and Z-line proteins and/or their binding to myofibrils are very sensitive to tryptic digestion, whereas the M 3 (150,000 daltons) component and an additional band at 110,000 daltons are products of proteolysis. Gentle treatment of myofibrils with an ATP relaxing solution results in the release of thick and thin myofilaments which can be pelleted by 100,000-g centrifugation. These myofilaments lack M-and Z-line structure when examined with the electron microscope, and their electrophoretograms are devoid of the M 1, M 2, Z 1, and Z 2 bands. The M 4, M 5 (C-protein doublet), and M 3 bands, however, remain associated with the filaments.     

Phosphorylation polymorphism in the yolk protein 2 of Drosophila hawaiiensis

An intraspecific polymorphism in the electrophoretic migration pattern of the yolk proteins in D. hawaiiensis was established and characterized. The polymorphism includes yolk protein migration patterns of two, three, or four bands by polyacrylamide gel electrophoresis. Peptide mapping analysis demonstrates that in the two band migration pattern YP2 comigrates with YP3, whereas in the three and four band migration patterns YP2 migrates between YP1 and YP3 in addition to comigrating with YP3. It further demonstrates that the top two bands of the four band migration pattern consists of YP1. Phosphatase treatment of the yolk proteins establishes that the different electrophoretic migration patterns of YP2 are caused by different degrees of phosphorylation. It is suggested that the YP1 polymorphism is caused by a yp1 gene modification and that the YP2 polymorphism is caused by two different post-translational processing paths.     

The nucleotide sequence of the gene coding for Drosophila melanogaster yolk protein 3

The entire sequence of the Drosophila melanogaster yolk protein 3 (YP3) gene (yp3), including 1822 nucleotides (nt) of 5'- and 834 nt of 3'-flanking DNA, has been determined. In addition, the 5' and 3' ends of the mRNA and the two introns have been mapped. The predicted amino acid sequence of YP3 has considerable homology (43%) to the other two yolk proteins of D. melanogaster. The nucleotide sequence of yp3 was compared to the other two yolk protein genes which have the same developmental pattern of expression. In addition to extensive homology between the protein coding regions, we found two small regions of homology between yp3 flanking sequences and a segment of DNA required for normal expression of the yolk protein 1 gene in adult female fat bodies.     

Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain

A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.     

Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte

The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme (receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor.     

76 and 14 kDa polypeptides, two major components released from amphibian urinary bladder epithelium. Purification and characterization

Exocytotic processes play a major role in the hormonal control of water permeability in the amphibian urinary bladder. Different treatments such as antidiuretic hormone (ADH) stimulation, incubation with phorbol ester or mild detergent and mechanical stretch of the bladder, consistently induce a liberation of two major polypeptides of 76 and 14 kDa molecular mass into the luminal medium. Each of these polypeptides represents 3 to 5% of the total protein of epithelial cell homogenates and 20 to 50% of the released material. Proportions of released 76 kDa polypeptide in urinary bladders of toads (Bufo marinus) and frogs (Rana esculenta) were similar but, in the frog extracts, two bands ("doublet") were resolved at the level of 76 kDa. In high performance liquid chromatography (HPLC), using gel filtration and ion exchange chromatography, the frog 76 kDa protein was resolved into two polypeptides of 80,000 to 100,000 and 60,000 to 80,000 daltons while the 14 kDa protein included two polypeptides, each with a molecular mass of approximately 14,000 daltons. Isoelectric focusing of the material released during a mechanical stretch of the tissue ("stretch extract") or of isolated purified proteins from the frog urinary bladder showed that the 14 kDa polypeptides were resolved in two major groups of polypeptides, one in the range of pH 7.4 to 7.8, the other at pH 5.6. The lower band of the 76 kDa doublet also comprised some diffuse bands (5.0 less than pI less than 5.2) while the other polypeptide of the doublet presented a sharp band at pH 6.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Caenorhabditis elegans CPI-2a cystatin-like inhibitor has an essential regulatory role during oogenesis and fertilization

In the present study, we characterized a sterile cpi-2a(ok1256) deletion mutant in Caenorhabditis elegans and showed that CPI-2a has an essential regulatory role during oogenesis and fertilization. We have also shown that the CPI2a inhibitor and both Ce-CPL-1 and Ce-CPZ-1 enzymes are present in the myoepithelial sheath surrounding germ cells, oocytes, and embryos as well as in the yolk granules within normal oocytes. Staining of mutant worms with anti-yolk protein antibodies has indicted that the proteins are not present in the mature oocytes. Moreover, green fluorescent protein expression was absence or reduced in cpi-2a/yp170:gfp mutant oocytes, although it was expressed in one of the successfully developed embryos. Based on these results, we hypothesize that the sterility in cpi-2a(ok1256) mutant worms is potentially caused by two possible mechanisms: 1) defects in the uptake and/or processing of yolk proteins by the growing oocytes and 2) indirect induction of defects in cell-cell signaling that is critical for promoting germ line development, oocyte maturation, ovulation, and fertilization. A defect in any of these processes would have detrimental effects on the development of normal embryos and consequently normal production of progenies as we observed in cpi-2a mutant worms. This is the first study that demonstrates the expression of cysteine proteases and their endogenous inhibitor in the gonadal sheath cells surrounding germ cells and oocytes, which indirectly have established their potential involvement in proteolytic processing of molecules within the gonadal sheath cells, such as components of the extracellular matrix or the cytoskeletal proteins, which are essential for proper cell-cell signaling activities of the gonadal sheath cells during normal maturation and ovulation processes.     

Purification and properties of yolk protein factor I, a sequence-specific DNA-binding protein from Drosophila melanogaster

We report the purification and some of the biochemical properties of yolk protein factor I (YPF1). This protein binds to a specific site in the yolk protein 1 gene (yp1) of Drosophila melanogaster. YPF1 has been purified to 95% homogeneity and consists of a heterodimer of two subunits with molecular weights 85,000 and 69,000. The protein is highly asymmetric with a frictional ratio of 1.56 which leads to calculated dimensions of 510 x 51 A when modeled as a prolate ellipsoid of revolution. It binds the yp1 DNA site with a protein/DNA stoichiometry of 1:1. Binding to that site is essentially irreversible with a dissociation rate constant of koff less than or equal to 2 x 10(-7) s-1, which gives the complex a dissociation half-life of approximately 55 days. The measured apparent second order association rate constant is 4 x 10(8) M-1 s-1 resulting in a calculated equilibrium dissociation constant of KD less than or equal to 5 x 10(-16) M. YPF1 also has a 10(8) selectivity for the yp1 site over poly(dA).poly(dT) (KDapp = 2 x 10(-8) M(nucleotide].     

Investigation of cis-acting sequences regulating expression of the gene encoding yolk protein 3 in Drosophila melanogaster.

Summary The regulatory sequences leading to the ovarian and fat body expression of yolk proteins 1 and 2 (YP1 and 2) of Drosophila melanogaster have been characterised in some detail. These genes (yp1 and yp2) share many enhancer elements, and some important regulatory sequences lie within the coding regions. We have begun to investigate the cis-regulation of the gene encoding yolk protein 3 (yp3). We describe a system for P element transformation using the complete and unaltered yp3 gene rather than reporter genes and describe sequences conferring correct expression in the ovary and carcass.     

Immuno-EM localization of GFP-tagged yolk proteins in C. elegans using microwave fixation.

Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures.     

Effects of precocene II on female-specific hemolymph polypeptides in Oncopeltus fasciatus

Using polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, two major polypeptides of 200,000 and 170,000 daltons were detected in the hemolymph of mature female Oncopeltus fasciatus, but they were not found in the hemolymph of males or newly emerged females. Those polypeptides constituted the two major bands of early vitellogenic oocytes; however, they were absent from the yolk of mature eggs. The slower-migrating band (200,000 daltons) appears to correspond to a vitellogenic protein already identified in O. fasciatus, whose synthesis has been suggested to be independent of juvenile hormone (JH). Treatment of newly emerged adult females with the corpus allatum cytotoxin precocene II prevented the appearance of the female-specific bands and induced an important accumulation of other proteins in the hemolymph. Yolk deposition was also inhibited in those animals. Topical application of JH to precocene-treated females restored the appearance of the 200,000 and 170,000 dalton polypeptides in the hemolymph. These results suggest that JH is required for the synthesis of female-specific polypeptides in O. fasciatus.     

Identification of fodrin as a major calmodulin-binding protein in postsynaptic density preparations

A major protein of postsynaptic densities (PSDs), a doublet of 230,000 and 235,000 Mr that becomes enriched in PSDs after treatment of synaptic membranes with 0.5% Triton X-100, has been found to be identical to fodrin (Levine, J., and M. Willard, 1981, J. Cell Biol. 90:631) by the following criteria. The upper bands of the PSD doublet and purified fodrin (alpha-fodrin) were found to be identical since both bands (a) co-migrated on SDS gels, (b) reacted with antifodrin, (c) bound calmodulin, and (d) had identical peptide maps after Staphylococcus aureus protease digestion. The lower bands of the PSD doublet and of purified fodrin (beta-fodrin) were found to be identical since both bands co-migrated on SDS gels and both had identical peptide maps after S. aureus protease digestion. The binding of calmodulin to alpha-fodrin was confirmed by cross-linking azido-125I-calmodulin to fodrin before running the protein on SDS gels. No binding of calmodulin to beta-fodrin was observed with either the gel overlay or azido- calmodulin techniques. A second calmodulin binding protein in the PSD has been found to be the proteolytic product of alpha-fodrin. This band (140,000 Mr), which can be created by treating fodrin with chymotrypsin, both binds calmodulin and reacts with antifodrin.     

Identification, synthesis, and characterization of the yolk polypeptides of Plodia interpunctella

The mature eggs of Plodia interpunctella were found to contain four major polypeptides. These yolk polypeptides (YPs) were found to have approximate molecular weights of 153,000 daltons (YP1), 69,000 daltons (YP2), 43,000 daltons (YP3), and 33,000 daltons (YP4) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, we found YP1 was resolved by a 5% polyacrylamide gel into two separate polypeptides of 153,000 and 147,000 daltons. All of the YPs could be labeled in vivo or in vitro with [35S]-methionine. Yolk peptide 1 and YP3 were synthesized by fat body of pharate adult and adult females and secreted into the hemolymph. Yolk peptide 2 and YP4 were synthesized and secreted into incubation medium by ovaries that contained vitellogenic oocytes, but these polypeptides were not found in the hemolymph. Fat bodies of males synthesized and secreted an immunoprecipitable polypeptide similar to YP3 as well as immunoprecipitable polypeptides larger than 200,000 daltons that had no counterparts in the oocytes. Peptide mapping by protease digestion showed each YP to be cleaved into unique fragments, suggesting that no precursor-product relationship exists between the YPs. Ion exchange chromatography and gel permeation chromatography separated that yolk proteins into two groups with approximate molecular weights of 462,000 and 264,000 daltons. By resolving these peaks on SDS-PAGE, it was found that YP1 and YP3 formed the 462,000-dalton yolk protein and YP2 and YP4 formed the 264,000-dalton yolk protein.