p185, a product of the neu proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity.

The neu oncogene was originally identified in cell lines derived from rat neuroectodermal tumors. neu is related to but distinct from the c-erbB gene, which encodes the epidermal growth factor (EGF) receptor. neu encodes a protein, designated p185, that is serologically related to the EGF receptor. Identification of the normal homolog of p185 encoded by the neu proto-oncogene enabled us to compare the product of the neu proto-oncogene with the mutated version specified by the neu oncogene and with the EGF receptor. The normal form of p185 was structurally similar to its transforming counterpart, indicating that activation of the neu oncogene did not cause major structural alterations in the gene product. Both normal and transforming forms of p185 were associated with tyrosine kinase activity, supporting the idea that normal p185 functions as a growth factor receptor. p185 differed both structurally and functionally from the EGF receptor. p185 and the EGF receptor had distinct electrophoretic mobilities when synthesized under normal culture conditions or in the presence of tunicamycin. EGF did not stimulate increased turnover of p185 and did not bind quantitatively to p185. A number of other growth factors failed to stimulate degradation of p185 or tyrosine phosphorylation of p185 and are therefore unlikely to be ligands for p185.     

In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p.

The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.     

The inner nuclear membrane protein p58 associates in vivo with a p58 kinase and the nuclear lamins.

p58, also referred to as the lamin B receptor, is an intrinsic protein of the inner nuclear membrane that binds in vitro to lamin B. Previous studies have demonstrated that p58 is phosphorylated in vivo and removal of its phosphate moieties affects lamin B binding. Using affinity-purified antipeptide antibodies, we have now immunoisolated p58 from bird erythrocyte lysates under isotonic, non-denaturing conditions. Analysis of the immunopurified material shows that five distinct proteins are tightly and specifically associated with p58. Two of these polypeptides can be identified as nuclear lamins A and B. The immunoisolate also contains a kinase activity that phosphorylates p58 in vivo and in vitro, exclusively at serine residues, as indicated by phosphoamino acid analysis and two-dimensional phosphopeptide mapping. Cell fractionation experiments and in vitro phosphorylation assays demonstrate that the p58 kinase resides in the nuclear envelope and is distinct from protein kinase A and cdc2 kinase, for both of which p58 is an in vitro substrate. These data suggest that p58 is interacting in vivo with a p58 kinase and the nuclear lamins.     

The Sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins.

SEC13 encodes a 33 kDa protein that participates in vesicle budding from the endoplasmic reticulum (ER). In order to purify a functional form of Sec13p, a SEC13-dihydrofolate reductase (mouse) fusion gene (SEC13:DHFR) was constructed that complements both sec13 temperature sensitive and null mutations. Methotrexate-agarose affinity chromatography facilitated the purification of two forms of the Sec13-dhfrp fusion protein: a monomeric form and a high molecular weight complex. The complex form consists of two subunits: Sec13-dhfrp and a 150 kDa protein (p150). Native immunoprecipitation experiments confirm that Sec13p exists in a complex with p150 in wild type cells. Functional analysis supports a role for both subunits in protein transport. Vesicle budding from the ER in a cell-free reaction is inhibited by Fab antibody fragments directed against either Sec13p or p150. The purified Sec13-dhfrp/p150 complex, but not the Sec13-dhfrp monomer, in combination with two other pure protein fractions (Sar1p and a Sec23/Sec24 protein complex) satisfies the requirement for cytosol in a cell-free vesicle budding reaction. The vesicles formed with the purified protein fractions are competent to fuse with the Golgi and are biochemically distinct from the ER membrane fraction from which they derive.     

Purification,Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase

An NADH dehydrogenase activity from red beet (Beta vulgaris L.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays L. B73) mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other less prevalent proteins. Polyclonal antibodies prepared against the 58-kD protein of red beet roots were found to immunoprecipitate the NAD(P)H dehydrogenase activity. The antibodies cross-reacted to similar proteins in mitochondria from a number of plant species but not to rat liver mitochondrial proteins. The polyclonal antibodies were used in conjunction with maize mitochondrial fractionation to show that the 58-kD protein was likely part of a protein complex loosely associated with the membrane fraction. A membrane-impermeable protein cross-linking agent was used to further show that the majority of the 58-kD protein was located on the outer surface of the inner mitochondrial membrane or in the intermembrane space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD protein doublet. The NAD(P)H dehydrogenase activity was not affected by ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or CaCl2, was stimulated somewhat (21%) by flavin mononucleotide, was inhibited by p-chloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containing platanetin (61%).     

A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH.

A glycoprotein encoded by the UL1 gene of herpes simplex virus type 1 (HSV-1) was detected in infected cells with antipeptide sera. The UL1 gene has previously been implicated in virus-induced cell fusion (S. Little and P. A. Schaffer, Virology 112:686-697, 1981). Two protein species, a 30-kDa precursor form and a 40-kDa mature form of the glycoprotein, both of which were modified with N-linked oligosaccharides, were observed. This novel glycoprotein is the 10th HSV-1 glycoprotein to be described and was named glycoprotein L (gL). A complex was formed between gL and gH, a glycoprotein known to be essential for entry of HSV-1 into cells and for virus-induced cell fusion. Previously, it had been reported that gH expressed in the absence of other viral proteins was antigenically abnormal, not processed, and not expressed at the cell surface (U.A. Gompels and A. C. Minson, J. Gen. Virol. 63:4744-4755, 1989; A. J. Forrester, V. Sullivan, A. Simmons, B. A. Blacklaws, G. L. Smith, A. A. Nash, and A. C. Minson, J. Gen. Virol. 72:369-375, 1991). However, gH coexpressed with gL by using vaccinia virus recombinants was antigenically normal, processed normally, and transported to the cell surface. Similarly, gL was dependent on gH for proper posttranslational processing and cell surface expression. These results suggest that it is a hetero-oligomer of gH and gL which is incorporated into virions and transported to the cell surface and which acts during entry of virus into cells.     

The 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs. Interaction with actin.

In the preceding paper [Golsteyn & Waisman (1989) Biochem. J. 257, 809-815] an EGTA-stable, Ca2+-binding heterodimer comprised of a 50 kDa protein and actin called '50K-A' was identified in the unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. In the present paper we have documented the binding of 50K-A to DNAase I and the effect of 50K-A on the kinetics of actin polymerization. When 50K-A was added to pyrene-labelled rabbit skeletal-muscle actin and the salt concentration increased, the initial rate of actin polymerization was inhibited by a very low molar ratio of 50K-A to actin. Furthermore, the steady-state level of G-actin was increased in the presence of 50K-A, suggesting that 50K-A caps the preferred end of actin polymer, shifting the steady-state concentration to that of the non-preferred end. Dilution of F-actin to below its critical concentration into 50K-A resulted in a much slower rate of depolymerization, consistent with capping of the preferred end. In contrast with the Ca2+-dependent binding to DNAase, the effect of 50K-A on the kinetics of actin assembly and disassembly was Ca2+-independent. These results suggest that 50K-A is a novel actin-binding protein with some similarities to the severin/fragmin/gelsolin family of F-actin-capping proteins.     

The crystal and molecular structure of cytosine-glycyl-glycine-copper(II) complex,a biologically important ternary coordination complex

The title compound was prepared and studied to gain some insight into the structural basis for the protein-nucleic acid-metal ion interaction. The crystal structure has been determined from three-dimensional diffractometer X-ray data using Cu Kα radiation. The crystals are monoclinic, space group $\text{P2}{}_1\text{c}$, with cell dimension; a=10.642(1)Å, b=8.081(1)Å, c=17.792(1)Å, β=124.29(1)^o, z=4. Amino and amide nitrogen, carboxyl O(8) of glycylglycine, N(3) of cytosine and O(2) of adjacent cytosine molecule coordinate to the central copper ion to form a square pyramid. An additional weak interaction in complex molecule between copper and O(2) of cytosine is also observed. The complex molecules are held together by hydrogen- and coordination-bonds in crystalline state.     

B lymphocyte antigen receptors (mIg) are non-covalently associated with a disulfide linked, inducibly phosphorylated glycoprotein complex.

T and B lymphocyte antigen receptors exhibit single transmembrane spanning regions and very short, three to five amino acid, C-terminal cytoplasmic tails. Ligation of these receptors leads, apparently through GTP binding protein activation, to rapid stimulation of a polyphosphoinositide specific phosphodiesterase (PPI-PDE). T lymphocyte antigen receptors (alpha beta) are coupled to PPI-PDE via a receptor associated complex of membrane proteins, designated CD3. Although an analogous transducer complex is presumed to exist in B cells, no such structure has been defined. We utilized in vitro [32P]phosphorylation to identify and characterize a membrane immunoglobulin (mIg) associated phosphoprotein complex which appears to represent a B cell analog of CD3. The phosphoprotein complex consists of three N-glycosylated polypeptides which occur as disulfide linked dimers, non-covalently associated with mIg. The complex associated with mIgM (pp32, pp34 and pp37 subunits) differs from that associated with mIgD (pp33, pp34 and pp37 subunits), and the isotype specific phosphoprotein (pp32 or pp33) appears to exist as a disulfide linked heterodimer with either pp34 or pp37. Aluminum fluoride stimulates phosphorylation of all of the subunits, and at least one of the proteins is phosphorylated on a tyrosine residue(s).     

PKC zeta is a molecular switch in signal transduction of TNF-alpha, bifunctionally regulated by ceramide and arachidonic acid.

Tumor necrosis factor (TNF-alpha) stimulates a number of signal transduction pathways in which phospholipases produce lipid second messengers. However, the immediate molecular targets of these messengers, in particular those of ceramide and arachidonic acid (AA) and their role in TNF signaling are not well defined. In this study we investigated the relationship of ceramide and AA in regulating an atypical PKC isozyme, PKC zeta. U937 cells responding to TNF-alpha treatment with NF kappa B activation displayed enhanced phosphorylation of PKC zeta, which is already detectable 30 s after stimulation. [14C]ceramide specifically binds to and regulates kinase activity of PKC zeta in a biphasic manner. Binding studies indicate high and low affinity binding with bmax values of 60 and 600 nM and Kd values of 7.5 and 320 nM respectively. At ceramide concentrations as low as 0.5 nM an up to 4-fold increase in autophosphorylation is obtained, which, at concentrations > 60 nM, again declines to basal levels. Interestingly, AA competes for ceramide binding and inhibits basal and ceramide-stimulated PKC zeta kinase activity at < 100 nM. Metabolism of [14C]ceramide in cells is slow and is inhibited in the presence of equimolar concentrations of lyso-phosphatidylcholine. Based on the bifunctional modulation of PKC zeta by the lipid messengers ceramide and AA, a model of TNF signal pathways is suggested in which PKC zeta takes a central position, acting as a molecular switch between mitogenic and growth inhibitory signals of TNF-alpha.     

Purification, some properties, and tissue distribution of a major lysosome-associated membrane glycoprotein (r-lamp-2) of rat liver.

We previously purified and characterized a major lysosomal membrane glycoprotein (r-lamp-1) from rat liver [Akasaki et al. (1990) Chem. Pharm. Bull. 38, 2766-2770]. The present study describes the purification of another major lysosomal membrane glycoprotein (r-lamp-2) from rat liver and compares the tissue distribution of r-lamp-1 and r-lamp-2 in rats. R-lamp-2 was purified to apparent electrophoretic homogeneity from rat liver by a simple method with a protein yield of approximately 4.0 micrograms/g wet weight of liver. The purification procedure includes: preparation of tritosomal membranes, extraction of tritosomal membranes with Lubrol PX, wheat germ agglutinin (WGA)-Sepharose affinity chromatography, and monoclonal antibody-Sepharose affinity chromatography. R-lamp-2 exhibited an Mr of 96,000 on SDS-PAGE and had an acidic pI of less than 3.5. R-lamp-2 contained 52.3% carbohydrates. Its carbohydrate moieties were composed of numerous sialyl complex type N-linked oligosaccharides and small amounts of O-linked oligosaccharides. Both r-lamp-1 and r-lamp-2 were detected in all rat tissues examined by immunoblot analyses, while their apparent molecular weights differed among the tissues. Immunological quantitative analysis showed that the protein concentrations of r-lamp-2 were consistently lower than those of r-lamp-1 in all the tissues tested. There was a significant correlation with a regression coefficient of 0.86 in the tissue distribution between r-lamp-1 and r-lamp-2. A good correlation was also observed in the tissue distribution between acid phosphatase and r-lamp-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Murine leukemia virus (T-8)-transformed cells: identification of a precursor polyprotein containing gag gene-coded proteins (p15 and p12) and a nonstructural component.

Mink cells nonproductively transformed by the T-8 strain of mink cell focus-inducing virus express two type C viral amino terminal gag gene-coded structural proteins, p15 and p12, in the form of a 90,000 to 110,000 molecular weight polyprotein that lacks detectable immunological reactivity with other known type C virus-coded translational products. The observation concurs with the previous demonstration of similar high-molecular-weight precursor polyproteins in cell lines nonproductively transformed by either of two other mammalian sarcoma viruses also limited in virus-coded structural protein expression to p15 and p12.     

The fate of dsRNA in the nucleus: a p54(nrb)-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs

How do cells discriminate between selectively edited mRNAs that encode new protein isoforms, and dsRNA-induced, promiscuously edited RNAs that encode nonfunctional, mutant proteins? We have developed a Xenopus oocyte model system which shows that a variety of hyperedited, inosine-containing RNAs are specifically retained in the nucleus. To uncover the mechanism of inosine-induced retention, HeLa cell nuclear extracts were used to isolate a multiprotein complex that binds specifically and cooperatively to inosine-containing RNAs. This complex contains the inosine-specific RNA binding protein p54(nrb), the splicing factor PSF, and the inner nuclear matrix structural protein matrin 3. We provide evidence that one function of the complex identified here is to anchor hyperedited RNAs to the nuclear matrix, while allowing selectively edited mRNAs to be exported.     

Purification and characterization of a complex between cloacin and its immunity protein isolated from Enterobacter cloacae (Clo DF13). Dissociation and reconstitution of the complex.

Cell of Enterobacter cloacae (Clo DF13) produce a bacteriocin which is characterized by its very effective killing activity against sensitive bacteria. Purification and characterization of the excreted bacteriocin has revealed that this bacteriocin consists of an equimolar complex of two plasmid-specific gene products: the cloacin and its inhibitor the immunity protein. Dissociation of the complex by treatment with sodium dodecylsulfate induces the endonucleolytic activity of the cloacin but strongly reduces the killing activity. The purified complex possesses no activity in vitro. Both cloacin and immunity protein isolated from the complex were functionally identical to cloacin and immunity protein purified from the bacteriocinogenic cells by other methods. Reconstitution of the complex results in a partial restoration of killing activity.     

The receptor-associated protein (RAP) interacts with several resident proteins of the endoplasmic reticulum including a glycoprotein related to actin

The receptor-associated protein (RAP) is a chaperone found primarily in the endoplasmic reticulum (ER) that plays a necessary role in the folding and exocytic trafficking of members of the LDL receptor gene family including megalin and the LDL receptor-related protein (LRP). Recently, RAP has been shown to interact with a growing number of proteins including several that are unrelated to the LDL receptor family as well as new members of this rapidly expanding family. Based on these observations, we have applied chemical crosslinking procedures to identify additional novel RAP-interacting proteins, and thereby better characterize the scope of RAP's ER-related function. In this study, we have identified eight proteins with molecular weights of 32, 35, 46, 55, 70, 95, 170, and 200 kDa that interact with endogenous RAP. These proteins were found to associate with RAP in multiple cell types from different species, suggesting that their expression and interactions with RAP are ubiquitous. Results of pulse-chase experiments show that most of the proteins remain sensitive to endoglycosidase-H digestion, and also remain stably associated with RAP over an extended period, suggesting that they are ER resident proteins. All of the RAP-associated proteins appear to be largely soluble as they partition into the aqueous phase following TX-114 detergent extraction. Sequence analysis and immunoblotting of the 46-kDa RAP-associated glycoprotein (gp46) shows that it is structurally and immunologically related to actin. If gp46 is also functionally related to actin as an intracellular structural protein, it may represent a novel component of the putative ER matrix.     

Purification and functional reconstitution of a truncated human Na(+)/glucose cotransporter (SGLT1) expressed in E. coli.

A truncated human Na(+)/glucose cotransporter (C(5), residues 407-664) was expressed and purified from Escherichia coli using a GST fusion vector and glutathione affinity chromatography. The truncated transporter (C(5)) was cleaved from GST-C(5) by Factor Xa proteolysis and purified by gel filtration chromatography. Up to 1 mg of purified GST-C(5) was obtained from 1 l bacterial culture. Reconstitution of both GST-C(5) and C(5) proteins into lipid vesicles resulted in 2.5-fold higher initial uptake rates of [(3)H]D-glucose into C(5)-proteoliposomes than into liposomes. Transport was stereospecific, saturable, and inhibited by phloretin. These properties are similar to those obtained for C(5) in Xenopus laevis oocytes, and provide additional evidence that the five C-terminal transmembrane helices in SGLT1 form the sugar translocation pathway.     

The MA (p15) and p12 regions of the gag gene are sufficient for the pathogenicity of the murine AIDS virus.

Inoculation of the replication-defective retrovirus DEF27 (BM5d), packaged as an amphotropic virus pseudotype, into C57BL/6J mice leads to development of murine AIDS. Disease development showed a long incubation period (20 to 24 weeks), was associated with amplification of the BM5d provirus in splenocytes and lymph nodes, and was independent of the presence of exogenous or endogenous replication-competent helper viruses. However, both the onset of disease and amplification of the defective provirus were significantly enhanced by coinfection with the replication-competent B-cell-tropic ecotropic helper virus BM5e. The part of the BM5d viral genome that was essential for the pathogenicity was determined by making precisely engineered alterations in the reading frame of the gag and pol genes of BM5d proviral DNA and examining the ability of the altered amphotropic BM5d pseudotypes to induce the disease in C57BL/6J mice. The results show that expression of the MA (p15) and p12 regions of the gag gene is sufficient for pathogenicity of the BM5d retrovirus.