Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein.

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G protein) consists of a continuous stretch of 20 uncharged and mostly hydrophobic amino acids. We examined the effects of two mutations which change the amino acid sequence in this domain. These mutations were generated by oligonucleotide-directed mutagenesis of a cDNA clone encoding the G protein, and the altered G proteins were then expressed in animal cells. Replacement of an isoleucine residue in the center of this domain with a strongly polar but uncharged amino acid (glutamine) had no effect on membrane anchoring or transport of the protein to the cell surface. Replacement of this same isoleucine residue with a charged amino acid (arginine) generated a G protein that still spanned intracellular membranes but was not transported efficiently to the cell surface. The protein accumulated in the Golgi region in about 50% of the cells, and about 20% of the cells had detectable protein levels in a punctate pattern on the cell surface. In the remaining cells the protein accumulated in a vesicular pattern throughout the cytoplasm. Models which might explain the abnormal behavior of this protein are discussed.     

Use of green fluorescent protein fusions to analyse the N- and C-terminal signal peptides of GPI-anchored cell wall proteins in Candida albicans

Glycophosphatidylinositol (GPI)-anchored proteins account for 26-35% of the Candida albicans cell wall. To understand the signals that regulate these proteins' cell surface localization, green fluorescent protein (GFP) was fused to the N- and C-termini of the C. albicans cell wall proteins (CWPs) Hwp1p, Als3p and Rbt5p. C. albicans expressing all three fusion proteins were fluorescent at the cell surface. GFP was released from membrane fractions by PI-PLC and from cell walls by beta-glucanase, which implied that GFP was GPI-anchored to the plasma membrane and then covalently attached to cell wall glucans. Twenty and 25 amino acids, respectively, from the N- and C-termini of Hwp1p were sufficient to target GFP to the cell surface. C-terminal substitutions that are permitted by the omega rules (G613D, G613N, G613S, G613A, G615S) did not interfere with GFP localization, whereas some non-permitted substitutions (G613E, G613Q, G613R, G613T and G615Q) caused GFP to accumulate in intracellular ER-like structures and others (G615C, G613N/G615C and G613D/G615C) did not. These results imply that (i) GFP fusions can be used to analyse the N- and C-terminal signal peptides of GPI-anchored CWPs, (ii) the omega amino acid in Hwp1p is G613, and (iii) C can function at the omega+2 position in C. albicans GPI-anchored proteins.     

Exposed hydrophobic residues in human immunodeficiency virus type 1 Vpr helix-1 are important for cell cycle arrest and cell death

The human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein R (Vpr) is a major determinant for virus-induced G2/M cell cycle arrest and cytopathicity. Vpr is thought to perform these functions through the interaction with partner proteins. The NMR structure of Vpr revealed solvent exposed hydrophobic amino acids along helices 1 and 3 of Vpr, which could be putative protein binding domains. We previously showed that the hydrophobic patch along helix-3 was important for G2/M blockade and cytopathicity. Mutations of the exposed hydrophobic residues along helix-1 were found to reduce Vpr-induced cell cycle arrest and cell death as well. The levels of toxicity during virion delivery of Vpr correlated with G2/M arrest. Thus, the exposed hydrophobic amino acids in the amino-terminal helix-1 are important for the cell cycle arrest and cytopathicity functions of Vpr.     

Structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G) contains 20 uncharged and mostly hydrophobic amino acids. We created DNAs specifying G proteins with shortened transmembrane domains, by oligonucleotide-directed mutagenesis. Expression of these DNAs showed that G proteins containing 18, 16, or 14 amino acids of the original transmembrane domain assumed a transmembrane configuration and were transported to the cell surface. G proteins containing only 12 or 8 amino acids of this domain also spanned intracellular membranes, but their transport was blocked within a Golgi-like region in the cell. A G protein completely lacking the membrane-spanning domain accumulated in the endoplasmic reticulum and was secreted slowly. These experiments indicate that the size of the transmembrane domain is critical not only for membrane anchoring, but also for normal cell surface transport.     

Control of the Balb/c-3T3 cell cycle by nutrients and serum factors: analysis using platelet-derived growth factor and platelet-poor plasma.

Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractionated into a platelet-derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF-treated, competent cells into S phase. The nutrient independent, PDGF-modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density-arrested Balb/c-3T3 cells. The nutrient dependent, plasma-modulated event is located six hours prior to the G1/S phase boundary and corresponds in concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.     

Amino acid substitutions at positions 242, 255 and 268 in rabies virus glycoprotein affect spread of viral infection

Rabies virus Nishigahara strain kills adult mice after intracerebral inoculation, whereas the derivative RC‐HL strain does not. It has previously been reported by us that the R(G 242/255/268) strain, in which amino acids at positions 242, 255 and 268 on the G protein have been replaced by those from the Nishigahara strain in the genetic background of the RC‐HL strain, kills adult mice. This indicates that these three amino acids of G protein are important for pathogenicity of the Nishigahara strain. In order to obtain insights into the mechanism by which these amino acids affect pathogenicity, in this study spread of viral infection and apoptosis‐inducing ability of the attenuated RC‐HL strain and the virulent R(G 242/255/268) strain were compared. RC‐HL infection spread less efficiently in the mouse brain than did R(G 242/255/268) infection. However, the apoptosis‐inducing abilities of both viruses were almost identical, as shown by both in vitro and in vivo experiments. It was demonstrated that cell‐to‐cell spread of RC‐HL strain was less efficient than that of R(G 242/255/268) strain in mouse neuroblastoma cells. These results indicate that the three amino acid substitutions affect efficiency of cell‐to‐cell spread but not apoptosis‐inducing ability, probably resulting in the distinct distributions of RC‐HL and R(G 242/255/268) strain‐infected cells in the mouse brain and, consequently, the different pathogenicities of these strains.     

G protein betagamma complex-mediated apoptosis by familial Alzheimer''s disease mutant of APP.

In familial Alzheimer's disease (FAD), three missense mutations, V642I, V642F and V642G, that co-segregate with the disease phenotype have been discovered in the 695 amino acid form of the amyloid precursor protein APP. Expression of these mutants causes a COS cell NK1 clone to undergo pertussis toxin-sensitive apoptosis in an FAD trait-linked manner by activating the G protein Go, which consists of G alpha(o) and G betagamma subunits. We investigated which subunit was responsible for the induction of apoptosis by V642I APP in NK1 cells. In the same system, expression of mutationally activated G alpha(o) or G alpha(i) induced little apoptosis. Apoptosis by V642I APP was antagonized by the overexpression of the carboxy-terminal amino acids 495-689 of the beta-adrenergic receptor kinase-1, which blocks the specific functions of G betagamma. Co-transfection of G beta2gamma2 cDNAs, but not that of other G beta(x)gamma(z) (x = 1-3; z = 2, 3), induced DNA fragmentation in a manner sensitive to bcl-2. These data implicate G betagamma as a cell death mediator for the FAD-associated mutant of APP.     

Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of alpha3 chain

The basement membrane protein laminin-5 promotes cell adhesion and migration. The carboxyl-terminal G3 domain in the alpha3 chain is essential for the unique activity of laminin-5. To investigate the function of the G3 domain, we prepared various recombinant laminin-5 forms with a partially deleted or mutated G3 domain. The deletion of the carboxyl-terminal 28 amino acids (region III) markedly decreased the cell adhesion activity with a slight loss of the cell motility activity toward BRL and EJ-1 cells. This change was attributed to the loss of Lys-Arg-Asp sequence. Further deletion of 83 amino acids (region II) led to almost complete loss of the cell motility activity. All charged amino acid residues tested in this region were not responsible for the activity loss. These results suggest that the G3 domain contains two distinct regions that differently regulate cell adhesion and migration. Analysis of laminin-5 receptors showed that integrins alpha3beta1, alpha6beta1, and alpha6beta4 had different but synergistic effects on cell adhesion and migration on laminin-5. However, the structural change of the G3 domain appeared not to change integrin specificity. The present study demonstrates that the G3 domain in laminin-5 plays a central role to produce different biological effects on cells.     

Production of mouse ES cells homozygous for Cdk5-phosphorylated site mutation in c-Src alleles

c-Src-null mutants have not provided a full understanding of the cellular functions of c-Src, reflecting the functional redundancy among Src family members. c-Src is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and Cdk5 at Ser75 in the unique amino terminal c-Src-specific domain. The specific roles of c-Src may be assessed by establishing mouse embryonic stem (ES) cells homozygous for a point mutation at Ser75. Mammalian homozygous cultured cells with a point mutation, however, have not yet been produced by gene targeting. Here we show an efficient procedure for producing ES cell clones bearing a homozygous Ser75 to Asp mutation in the c-src gene. This procedure was developed by combining two previously reported strategies: our procedure for introducing a point mutation into one allele with no exogenous sequence, and the high-geneticin (G418) selection procedure for introducing a mutation into both alleles. The mutant clones expressed the same levels of c-Src protein and autophosphorylation activity as wild-type cells, but the mutant c-Src was not phosphorylated on Ser75 during mitosis. This procedure is feasible for generating cells homozygous for a subtle mutation in most genes, and is expected to be applicable to other somatic cell lines.     

Amino Acids and mTOR Mediate Distinct Metabolic Checkpoints in Mammalian G1 Cell Cycle

Objective

In multicellular organisms, cell division is regulated by growth factors (GFs). In the absence of GFs, cells exit the cell cycle at a site in G1 referred to as the restriction point (R) and enter a state of quiescence known as G0. Additionally, nutrient availability impacts on G1 cell cycle progression. While there is a vast literature on G1 cell cycle progression, confusion remains – especially with regard to the temporal location of R relative to nutrient-mediated checkpoints. In this report, we have investigated the relationship between R and a series of metabolic cell cycle checkpoints that regulate passage into S-phase.

Methods

We used double-block experiments to order G1 checkpoints that monitor the presence of GFs, essential amino acids (EEAs), the conditionally essential amino acid glutamine, and inhibition of mTOR. Cell cycle progression was monitored by uptake of [³H]-thymidine and flow cytometry, and analysis of cell cycle regulatory proteins was by Western-blot.

Results

We report here that the GF-mediated R can be temporally distinguished from a series of late G1 metabolic checkpoints mediated by EAAs, glutamine, and mTOR – the mammalian/mechanistic target of rapamycin. R is clearly upstream from an EAA checkpoint, which is upstream from a glutamine checkpoint. mTOR is downstream from both the amino acid checkpoints, close to S-phase. Significantly, in addition to GF autonomy, we find human cancer cells also have dysregulated metabolic checkpoints.

Conclusion

The data provided here are consistent with a GF-dependent mid-G1 R where cells determine whether it is appropriate to divide, followed by a series of late-G1 metabolic checkpoints mediated by amino acids and mTOR where cells determine whether they have sufficient nutrients to accomplish the task. Since mTOR inhibition arrests cells the latest in G1, it is likely the final arbiter for nutrient sufficiency prior to committing to replicating the genome.     

A competitive labeling method for the determination of the chemical properties of solitary functional groups in proteins.

The properties of the functional groups in a protein can be used as built-in-probes of the structure of the protein. We have developed a general procedure whereby the ionization constant and chemical reactivity of solitary functional groups in proteins may be determined. The method may be applied to the side chain of histidine, tyrosine, lysine, and cysteine, as well as to the amino terminus of the protein. The method, which is an extension of the competitive labeling technique using [3H]- and [14C]1-fluoro-2,4-dinitrobenzene (N2ph-F) in a double-labeling procedure, is rapid and sensitive. Advantage is taken of the fact that after acid hydrolysis of a dinitrophenylated protein, a derivative is obtained which must be derived from a unique position in the protein. The method has been applied to the solitary histidine residue of lysozyme, alpha-lytic protease, and Streptomyces griseus (S.G.) trypsin, as well as to the amino terminus of the latter protein. The following parameters were obtained for reaction with N2ph-F at 20 degrees C in 0.1 N KCl: the histidine of hen egg-white lysozyme, pKa of 6.4 and second-order velocity constant of 0.188 M-1 min-1; the histidine of alpha-lytic protease, pKa of 6.5 and second-order velocity constant of 0.0235 M-1 min-1; the histidine of S.G. trypsin, pKa of 6.5 and second-order velocity constant of 0.0328 M-1 min-1; the valyl amino terminus of S.G. trypsin, pKa of 8.1 and second-order velocity constant of 0.403 M-1 min-1. In addition, the results obtained provide clues as to the microenvironments of these functional groups, and indicate that the proteins studied undergo pH-dependent conformational changes which affect the microenvironment, and hence the chemical reactivity of these groups.     

Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains.

The spike glycoprotein G of vesicular stomatitis virus (VSV) induces membrane fusion at low pH. We used linker insertion mutagenesis to characterize the domain(s) of G glycoprotein involved in low-pH-induced membrane fusion. Two or three amino acids were inserted in frame into various positions in the extracellular domain of G, and 14 mutants were isolated. All of the mutants expressed fully glycosylated proteins in COS cells. However, only seven mutant G glycoproteins were transported to the cell surface. Two of these mutants, D1 and A6, showed wild-type fusogenic properties. The mutant A2 had a temperature-sensitive defect in the transport of the mutant G glycoprotein to the cell surface. The other four mutants, H2, H5, H10, and A4, although present in cell surface, failed to induce cell fusion when cells expressing these mutant glycoproteins were exposed to acidic pH. These four mutant G proteins could form trimers, indicating that the defect in fusion was not due to defective oligomerization. One of these mutations, H2, is within a region of conserved, uncharged amino acids that has been proposed as a possible fusogenic sequence. The mutation in H5 was about 70 amino acids downstream of the mutation in H2, while mutations in H10 and A4 were about 300 amino acids downstream of the mutation in H2. Conserved sequences were also noted in the H10 and A4 segment. The results suggest that in the case of VSV G glycoprotein, the fusogenic activity may involve several spatially separated regions in the extracellular domain of the protein.     

Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene

We have cloned DNA fragments from Bacillus subtilis 168S into Escherichia coli, which produced a lytic zone on an agar medium containing B. subtilis cell wall. Sequencing of the fragments showed the presence of an open reading frame (ORF) which encodes a polypeptide of 272 amino acids with a molecular mass of 29919 Da. The deduced amino acid sequence showed considerable homology with that of the cell wall hydrolase gene of Bacillus sp. (Potvin, C., Leclerc, D., Tremblay, G., Asselin, A. & Bellemare, G. (1988). Molecular and General Genetics 214, 241-248). Accordingly, the gene was designated cwlA, for cell wall lysis. The N-terminal amino acid sequence of cwlA gene product prepared from a E. coli clone was AIKVVKNLVSKSKYGLKCPN, which is consistent with that of the deduced sequence starting from Ala at second position from the initiation codon of the cwlA gene. A presumed sigma A promoter and a rho-independent terminator were found upstream and downstream of the ORF, respectively. A chloramphenicol-resistance determinant integrated into the ORF was mapped by PBS1 transduction, which indicated the gene sequence dnaE-aroD-cwlA.     

Characterization of a novel 350-kilodalton nuclear phosphoprotein that is specifically involved in mitotic-phase progression.

A gene assigned to human chromosome 1q32-41 encodes a novel protein of 3,113 amino acids containing an internal tandem repeat of 177 amino acids. The protein, which we have named "mitosin," was identified by direct binding to purified retinoblastoma protein in vitro with a region distantly related to the retinoblastoma protein-binding site of E2F-1. Mitosin is expressed throughout S, G2, and M phases of the cell cycle but is absent in G1. Its localization is dramatically reorganized from a rather homogeneous nuclear distribution in S phase to paired dots at the kinetochore/centromere region, to the spindle apparatus, and then to the midbody during M-phase progression. This spatial reorganization coincides closely with the temporal phosphorylation patterns of mitosin. Overexpression of N-terminally truncated mutants blocks cell cycle progression mainly at G2/M. These results suggest that mitosin may play an important role in mitotic-phase progression.     

Processing, surface expression, and immunogenicity of carboxy-terminally truncated mutants of G protein of human respiratory syncytial virus.

Posttranslational processing and cell surface expression were examined for three C-terminally truncated mutants of the G protein of respiratory syncytial virus expressed from engineered cDNAs. The truncated mutants, encoded by cDNAs designated G71, G180, and G230, contained the N-terminal 71, 180, and 230 amino acids, respectively, of the 298-amino-acid G protein. To facilitate detection of G71, which reacted inefficiently with G-specific antisera, we constructed a parallel set of cDNAs, designated G71/13, G180/13, and G230/13, to encode the same truncated species with the addition of a C-terminal 13-amino-acid reporter peptide which could be detected efficiently with an antipeptide serum. G71, G180, and G230 were expressed as species of Mr 7,500, 48,000, and 51,000, respectively, compared with 84,000 for parental G protein. The proteins encoded by G180 and G230, like parental G protein, contained both N-linked and O-linked carbohydrate. Also, the protein encoded by G71/13 appeared to be O glycosylated, showing that even this highly truncated form contained the structural information required to target the protein for O glycosylation. As for parental G protein, the estimated Mrs of the proteins encoded by G180 and G230 were approximately twice the calculated molecular weight of the polypeptide chain. Experiments with monensin showed that most of this difference between the calculated and observed Mr was due to posttranslational processing in or beyond the trans-Golgi compartment, presumably owing to the addition of carbohydrate or aggregation into dimers or both. Like parental G protein, all three truncated forms accumulated abundantly at the cell surface, and in each case the C terminus was extracellular. Thus, the N-terminal 71 amino acids of the G protein contained all the structural information required for efficient membrane insertion and cell surface expression, whereas the extracellular domain was dispensable for these activities. Cotton rats were immunized with recombinant vaccinia viruses expressing the G71, G180, G230, or parental G protein to compare their abilities to induce serum antibodies and resistance to challenge virus replication. The G71 and G180 recombinants failed to induce significant levels of G-specific antibodies or resistance to challenge, whereas the immunogenicity of G230 equaled or exceeded that of parental G protein. This suggested that the C-terminal 68 amino acids of the 236-amino-acid ectodomain do not contribute to the major epitope(s) of the G protein that is involved in inducing protective immunity.     

Polarity of neutral amino acid transport and characterization of a broad specificity transport activity in a kidney epithelial cell line, MDCK

The Madin-Darby canine kidney (MDCK) cell line was investigated with respect to the cellular polarity of amino acid transport in early confluent versus late confluent cultures. Early confluent cultures could take up amino acids from the apical and the basolateral sides of the cell layer via amino acid transport Systems A, ASC, and L. However, in late confluent cultures the activities of Systems A and L were clearly localized to the basolateral surface of the cell monolayer. In addition to the presence of systems A, ASC, and L, a novel activity, measurable under conditions used for quantitating System ASC, was found to be active in the apical membrane of these cells. This transporter, termed System G (for general), recognized basic and neutral amino acids with high affinity and acidic amino acids with lower affinity. System G exhibited broad substrate specificity, strict cation specificity, and a broad pH optimum with maximal activity at acidic pH. The activity of System G was relatively low after growth in serum-containing medium but was induced in a defined medium. Induction of System G activity was dependent upon the presence of prostaglandin E1. The broad substrate specificity, low pH optimum, and Na+ dependence suggest that System G may function in apical membranes as an energy-dependent transport route during reabsorption of amino acids from the kidney tubule lumen.     

Purification and characterization of epidermal G2- and G1-chalones]

Highly purified epidermal G1- and G2-chalones from rat skin inhibit the entering of epidermocytes to S and M phases of cell cycle respectively. Their biological activity is characterized by tissue-specificity and not by species-specificity. Both of them are tissue-specific glycoproteins as for their antigenic properties. Molecular weight of G1-chlone is 21 000, G2-chalone--34 000, isoelectric point (pH) 5.55 and 5.85 respectively. G2-chalone is the fastest as compared to G1-chalone in 5% acrylamide gel electrophoresis, pH 8.3. When injected in rabbits, G2-chalone produced monospecific antibodies which have no cross-reactivity with G1-chalone. The amino acid composition of both chalones and immunofluorescent localization of G2-chalone in epidermal tisues are given.     

A reversible arrest point in the late G1 phase of the mammalian cell cycle

The effects of two different cell cycle inhibitors on the proliferation of human lymphoblastoid cells have been analyzed by flow cytometric techniques. Mimosine, a plant amino acid, reversibly blocks the cell cycle at a point which occurs roughly 2 h before the arrest mediated by aphidicolin, an inhibitor of DNA polymerase alpha activity, which defines the G1/S phase boundary. The levels of thymidine kinase mRNA, which increase at the onset of S phase, are higher in cells blocked with aphidicolin than in cells treated with mimosine whereas the opposite results are obtained in the case of p53 mRNA levels, which are known to be maximal in the late G1 phase. These results indicate that mimosine inhibits cell cycle traverse in the late G1 phase prior to the onset of DNA synthesis and identifies a previously undefined reversible cell cycle arrest point.