Complement C5a-Induced Changes in Neutrophil Morphology During Inflammation.

The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose- and time-dependently resulted in a rapid C5a receptor-1 (C5aR1)-dependent shape change, indicated by enhanced flow cytometric forward-scatter area values. Similar changes were observed after incubation with zymosan-activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high-resolution digital imaging revealed a C5a-induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a-induced shape changes were inhibited by pharmacological blockade of either the Cl-/HCO3--exchanger or the Cl- -channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP-P2X/Y-purinoceptor interaction was also involved in the C5a-induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a-dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin-cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.

Targeting of Pseudomonas aeruginosa in the bloodstream with bispecific monoclonal antibodies.

We examined the ability of a bispecific mAb reagent, consisting of a mAb specific for the primate erythrocyte complement receptor cross-linked with an anti-bacterial mAb, to target bacteria in the bloodstream in an acute infusion model in monkeys. In vitro studies demonstrated a variable level of complement-mediated binding (immune adherence) of Pseudomonas aeruginosa (strain PAO1) to primate E in serum. In vivo experiments in animals depleted of complement revealed that binding of bacteria to E was <1% before administration of the bispecific reagent, but within 5 min of its infusion, >99% of the bacteria bound to E. In complement-replete monkeys, a variable fraction of infused bacteria bound to E. This finding may have significant implications in the interpretation of animal models and in the understanding of bacteremias in humans. Treatment of these complement-replete monkeys with the bispecific reagent led to >99% binding of bacteria to E. Twenty-four-hour survival studies were conducted; several clinical parameters, including the degree of lung damage, cytokine levels, and liver enzymes in the circulation, indicate that the bispecific mAb reagent provides a degree of protection against the bacterial challenge.

A bispecific dsDNAxmonoclonal antibody construct for clearance of anti-dsDNA IgG in systemic lupus erythematosus.

High avidity anti-dsDNA IgG antibodies are believed to play an important role in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) and therefore attempts have been made to reduce the concentration of these antibodies in the bloodstream of SLE patients. Previously we reported the development of an antigen based heteropolymer (AHP), a bispecific complex prepared by using the avidin-biotin system to crosslink dsDNA to a mAb specific for the human erythrocyte (E) complement receptor. Our studies indicated that this AHP could bind anti-dsDNA antibodies to E and facilitate clearance of these autoantibodies from the circulation of a monkey without E destruction. Here we report an improved covalent crosslinking procedure and purification scheme in which the AHP construct is isolated by precipitation in 50% saturated ammonium sulfate. We used a dsDNA binding dye, PicoGreen, to demonstrate specificity of binding of dsDNA to E via the AHP. The efficacy of the AHP in binding IgG anti-dsDNA antibodies to E was demonstrated in a sensitive and quantitative assay, based on the time resolved fluorescence properties of europium-labeled anti-human IgG mAbs used to probe the E. We also used this assay to screen SLE patient and normal plasmas for levels of anti-dsDNA IgG. The results of this assay correlate very well with the Farr assay, and therefore this approach may be useful in the development of informative and specific assays for a variety of autoantibodies. Treatment of SLE plasmas with E-AHP under conditions close to physiological led to substantial reductions (> or = 90%) in anti-dsDNA titers. It should be possible to test these new AHP for their ability to target and safely remove IgG anti-dsDNA antibodies from the circulation in animal models.

Heteropolymer-mediated clearance of immune complexes via erythrocyte CR1: mechanisms and applications.

Opsonization of particulate pathogens by antibodies and complement can lead to their binding to the complement receptor (CR1), specific for C3b, on primate erythrocytes (E). This process of immune adherence may play a role in immunologic defense by immobilizing bacteria and viruses, thus preventing them from leaving the bloodstream to invade susceptible tissue and organs. Immune adherence of C3b-opsonized and immune complexed pathogens to E may also facilitate their transfer to, and destruction by, fixed tissue macrophages. We have used mAbs specific for CR1 crosslinked with pathogen specific mAbs to generate heteropolymers (HP) which can bind a wide range of substrates to primate erythrocytes. Both prototype and bonafide pathogens bound to primate E via HP are handled in the circulation of non-human primates in a manner which appears to be virtually identical to the mechanism by which C3b-opsonized substrates bound to E CR1 are cleared. In this process of focused phagocytosis, Fc receptors on the phagocytic cell engage the E-bound complex, CR1 is removed by proteolysis, and the entire immune complex and CR1 are internalized while sparing the E. It may be possible to use HP to target pathogens in the bloodstream in a wide range of therapeutic applications.

Targeting of cancer cells with monoclonal antibodies specific for C3b(i).

PURPOSE: The goal of this research is to determine the feasibility of an immunotherapeutic approach based on the use of monoclonal antibodies (mAb) to target complement activation fragments on opsonized cancer cells. METHODS: We investigated whether treatment of LNCaP and C4-2 human prostate cancer cell lines with normal human serum would allow for deposition of sufficient amounts of the complement-activation protein C3b and its fragments [collectively referred to as C3b(i)] such that these proteins could serve as cancer-cell-associated antigens for targeting by mAb. Radioimmunoassays, flow cytometry, and magnetic purging with specific immunomagnetic beads were used for the analyses. RESULTS: In vitro opsonization of human prostate cancer cells with normal human serum resulted in deposition of C3b(i) in sufficient quantity (approx. 100,000 molecules/cell) for the cells to be targeted in a variety of protocols. We found that 51Cr-labeled and C3b(i)-opsonized cancer cells could be specifically purged at high efficiency (95%-99%) using anti-C3b(i) mAb covalently coupled to magnetic beads. Flow-cytometry experiments indicated that most normal white cells were not removed under similar conditions. Opsonization of cancer cells with sera from men with prostate cancer led to lower levels of cell-associated IgM and, subsequently, lower amounts of C3b(i) deposited than in normal subjects. Prototype experiments suggested that this deficiency could be corrected by addition of IgM from normal donor plasma. CONCLUSION: mAb directed against complement-activation products may provide new opportunities to deliver diagnostic and therapeutic agents selectively to cancer cells and tumor deposits. These opportunities may include ex vivo purging of C3b(i)-opsonized cancer cells prior to autologous bone marrow or stem cell transplantation.

Selective coupling of G protein beta gamma complexes to inhibition of Ca2+ channels.

Several mechanisms couple heterotrimeric guanine nucleotide-binding proteins (G proteins) to cellular effectors. Although alpha subunits of G proteins (Galpha) were the first recognized mediators of receptor-effector coupling, Gbetagamma regulation of effectors is now well known. Five Gbeta and 12 Ggamma subunit genes have been identified, suggesting through their diversity that specific subunits couple selectively to effectors. The molecular determinants of Gbetagamma-effector coupling, however, are not well understood, and most studies of G protein-effector coupling do not support selectivity of Gbetagamma action. To explore this issue further, we have introduced recombinant Gbetagamma complexes into avian sensory neurons and measured the inhibition of Ca(2+) currents mediated by an endogenous phospholipase Cbeta- (PLCbeta) and protein kinase C-dependent pathway. Activities of Gbetagamma in the native cells were compared with enzyme assays performed in vitro. We report a surprising selective activation of the PLCbeta pathway by Gbetagamma complexes containing beta(1) subunits, whereas beta(2)-containing complexes produced no activation. In contrast, when assayed in vitro, PLCbeta and type II adenylyl cyclase did not discriminate among these same Gbetagamma complexes, suggesting the possibility that additional cellular determinants confer specificity in vivo.

Facilitation of signal onset and termination by adenylyl cyclase.

The alpha subunit (Gsalpha) of the stimulatory heterotrimeric guanosine triphosphate binding protein (G protein) Gs activates all isoforms of mammalian adenylyl cyclase. Adenylyl cyclase (Type V) and its subdomains, which interact with Gsalpha, promoted inactivation of the G protein by increasing its guanosine triphosphatase (GTPase) activity. Adenylyl cyclase and its subdomains also augmented the receptor-mediated activation of heterotrimeric Gs and thereby facilitated the rapid onset of signaling. These findings demonstrate that adenylyl cyclase functions as a GTPase activating protein (GAP) for the monomeric Gsalpha and enhances the GTP/GDP exchange factor (GEF) activity of receptors.

How are immune complexes bound to the primate erythrocyte complement receptor transferred to acceptor phagocytic cells?

Immune complexes (IC) bound to the primate erythrocyte (E) complement receptor (CR1) are cleared from the circulation of primates and localized to phagocytic cells in the liver and spleen without E destruction. IC can be bound to E CRI either via C3b opsonization or with cross-linked mAb complexes (heteropolymers, HP) which contain a mAb specific for CRI and a mAb specific for an antigen. The long-term goal of our work is to apply the HP system to the treatment of human diseases associated with blood-borne pathogens. This review discusses the mechanism by which the E-bound IC are transferred to acceptor cells. Our studies in animal models as well as our in vitro investigations indicate that IC transfer is rapid (usually >90% in 10 min) and does not lead to lysis or phagocytosis of the E. Experiments with specific inhibitors and the use of IC prepared with Fab' fragments suggest that transfer depends mainly upon recognition by Fc receptors on the acceptor cell. Moreover, we find that IC release from the E is associated with a concerted loss of CR1, and is followed by uptake and internalization of the IC by the acceptor cell. We suggest that recognition and binding of the E-bound IC substrates by Fc receptors allows close contact between the E and acceptor cells, which in turn facilitates proteolysis of E CR1, presumably by a macrophage-associated protease. After proteolysis, the released IC are internalized by the macrophages.

Differential activity of the G protein beta5 gamma2 subunit at receptors and effectors.

The G protein beta5 subunit differs substantially in amino acid sequence from the other known beta subunits suggesting that beta gamma dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the beta5 subunit, recombinant beta5 gamma2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the gamma2 subunit (gamma2HF). The function of the pure beta5 gamma2HF dimers was examined in three assays: activation of pure phospholipase C-beta in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of alpha subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the beta5 gamma2HF dimer was compared with that of the beta1 gamma2HF dimer, which has demonstrated activity in these assays. The beta5 gamma2HF dimer activated phospholipase C-beta with a potency and efficacy similar to that of beta1 gamma2 or beta1 gamma2HF; however, it was markedly less effective than the beta1 gamma2HF or beta1 gamma2 dimer in its ability to activate type II adenylyl cyclase (EC50 of approximately 700 nM versus 25 nM). Both the beta5 gamma2HF and the beta1 gamma2HF dimers supported coupling of M1 muscarinic receptors to the Gq alpha subunit. The ETB receptor coupled effectively to both the Gi and Gq alpha subunits in the presence of the beta1 gamma2HF dimer. In contrast, the beta5 gamma2HF dimer only supported coupling of the Gq alpha subunits to the ETB receptor and did not support coupling of the Gi alpha subunit. These results suggest that the beta5 gamma2HF dimer binds selectively to Gq alpha subunits and does not activate the same set of effectors as dimers containing the beta1 subunit. Overall, the data support a specialized role for the beta5 subunit in cell signaling.

Phosphorylation of the G protein gamma12 subunit regulates effector specificity.

Although the G protein betagamma dimer is an important mediator in cell signaling, the mechanisms regulating its activity have not been widely investigated. The gamma12 subunit is a known substrate for protein kinase C, suggesting phosphorylation as a potential regulatory mechanism. Therefore, recombinant beta1 gamma12 dimers were overexpressed using the baculovirus/Sf9 insect cell system, purified, and phosphorylated stoichiometrically with protein kinase C alpha. Their ability to support coupling of the Gi1 alpha subunit to the A1 adenosine receptor and to activate type II adenylyl cyclase or phospholipase C-beta was examined. Phosphorylation of the beta1 gamma12 dimer increased its potency in the receptor coupling assay from 6.4 to 1 nM, changed the Kact for stimulation of type II adenylyl cyclase from 14 to 37 nM, and decreased its maximal efficacy by 50%. In contrast, phosphorylation of the dimer had no effect on its ability to activate phospholipase C-beta. The native beta1gamma10 dimer, which has 4 similar amino acids in the phosphorylation site at the N terminus, was not phosphorylated by protein kinase C alpha. Creation of a phosphorylation site in the N terminus of the protein (Gly4 --> Lys) resulted in a beta1 gamma10G4K dimer which could be phosphorylated. The activities of this beta gamma dimer were similar to those of the phosphorylated beta1 gamma12 dimer. Thus, phosphorylation of the beta1 gamma12 dimer on the gamma subunit with protein kinase C alpha regulates its activity in an effector-specific fashion. Because the gamma12 subunit is widely expressed, phosphorylation may be an important mechanism for integration of the multiple signals generated by receptor activation.

The G protein beta5 subunit interacts selectively with the Gq alpha subunit.

The diversity in the heterotrimeric G protein alpha, beta, and gamma subunits may allow selective protein-protein interactions and provide specificity for signaling pathways. We examined the ability of five alpha subunits (alphai1, alphai2, alphao, alphas, and alphaq) to associate with three beta subunits (beta1, beta2, and beta5) dimerized to a gamma2 subunit containing an amino-terminal hexahistidine-FLAG affinity tag (gamma2HF). Sf9 insect cells were used to overexpress the recombinant proteins. The hexahistidine-FLAG sequence does not hinder the function of the beta1gamma2HF dimer as it can be specifically eluted from an alphai1-agarose column with GDP and AlF4-, and purified beta1gamma2HF dimer stimulates type II adenylyl cyclase. The beta1gamma2HF and beta2gamma2HF dimers immobilized on an anti-FLAG affinity column bound all five alpha subunits tested, whereas the beta5gamma2HF dimer bound only alphaq. The ability of other alpha subunits to compete with the alphaq subunit for binding to the beta5gamma2HF dimer was tested. Addition of increasing amounts of purified, recombinant alphai1 to the alphaq in a Sf9 cell extract did not decrease the amount of alphaq bound to the beta5gamma2HF column. When G proteins in an extract of brain membranes were activated with GDP and AlF4- and deactivated in the presence of equal amounts of the beta1gamma2HF or beta5gamma2HF dimers, only alphaq bound to the beta5gamma2HF dimer. The alphaq-beta5gamma2HF interaction on the column was functional as GDP, and AlF4- specifically eluted alphaq from the column. These results indicate that although the beta1 and beta2 subunits interact with alpha subunits from the alphai, alphas, and alphaq families, the structurally divergent beta5 subunit only interacts with alphaq.

Activation of the calcium-permeable cation channel CD20 by alpha subunits of the Gi protein.

When the calcium-permeable cation channel CD20 is expressed in Balb/c 3T3 cells, it is activated by insulin-like growth factor-I (IGF-I) via the IGF-I receptor (Kanzaki, M., Nie, L., Shibata, H., and Kojima, I. (1997) J. Biol. Chem. 272, 4964-4969). The present study was conducted to investigate the role of G proteins in the regulation of the CD20 channel. In the excised patch clamp mode, activation of the CD20 channel by IGF-I required GTP, Mg2+, and ATP in the bath solution, and removal of either GTP or ATP attenuated the activation. Non-hydrolyzable ATP could substitute for ATP, and guanyl-5'-yl thiophosphate blocked the activation of the channel by IGF-I. The CD20 channel was also activated by guanosine 5'-3-O-(thio)triphosphate, and ATP was not required for the activation. Addition of a preparation of Gi/Go holoprotein purified from bovine brain activated the CD20, and the beta-adrenergic receptor kinase peptide did not affect the number of channel openings induced by the G protein. The CD20 channel was stimulated by the GTP-bound form of recombinant Gi2 alpha subunit purified from Sf9 cells. The Gi3 alpha subunit was less effective, and the Gi1 alpha subunit had no effect. Purified recombinant beta1gamma2 subunits did not affect the activity of the channel. Finally, IGF-I-induced activation of CD20 was inhibited by an antibody against Gi2 alpha subunit. These findings indicate that the CD20 channel expressed in Balb/c 3T3 cells is activated by the IGF-I receptor via the alpha subunits of heterotrimeric G proteins.

Reconstitution of bovine A1 adenosine receptors and G proteins in phospholipid vesicles: betagamma-subunit composition influences guanine nucleotide exchange and agonist binding.

We have studied the interactions of purified A1 adenosine receptors and G proteins reconstituted into phospholipid vesicles to investigate how the betagamma composition of G protein heterotrimers influences coupling. Recombinant hexahistidine-tagged bovine A1 adenosine receptors were expressed in Sf9 cells and purified to homogeneity by sequential chromatography over heparin-sepharose, xanthine amino congener-agarose, and nickel-nitrilotriacetic acid columns. These receptors were reconstituted with pure recombinant G proteins of defined subunit composition. Receptor-G protein complexes containing alphai2 and beta1gamma2 or beta1gamma3 and stimulated with the agonist, (R)-phenylisopropyladenosine, exchange guanine nucleotide 2-3 times more rapidly than do complexes containing beta1gamma1. This difference is not overcome by increasing the concentration of betagamma subunits. Receptor-G protein complexes containing beta1gamma1 also bind less of the agonist, [125I]-iodoaminobenzyladenosine (125I-ABA), than do complexes containing beta1gamma3. Kinetic experiments show that 125I-ABA dissociates 2-fold more rapidly from receptor-G protein complexes containing beta1gamma1 than from complexes containing the other betagamma subunits. The affinity of the interaction between immobilized Galphai2 subunits and beta1gamma1 or beta1gamma2 measured with an optical biosensor in the absence of receptor is similar. Taken together, these data implicate the gamma-subunit in influencing the interaction between the A1 adenosine receptor and G proteins.

Reconstitution of recombinant bovine A1 adenosine receptors in Sf9 cell membranes with recombinant G proteins of defined composition.

We investigated the coupling of A1 adenosine receptors to recombinant G proteins. Recombinant baculoviruses were used to express bovine A1 adenosine receptors in Sf9 insect cells that lack endogenous adenosine receptors. Binding parameters for recombinant receptors expressed in Sf9 cell membranes using the antagonist radioligand [125I]BW-A844U ([125I]8-cyclopentyl-3-iodoaminophenethyl-1-propylxanthine) are Bmax = 2-5 pmol/mg of protein and K(D) = 0.53 +/- 0.12 nM. In competition assays, the potency order of agonists is (R)-phenylisopropyladenosine > (S)-phenylisopropyladenosine > 5'-N-ethylcarboxamidoadenosine, properties characteristic of native bovine A1 adenosine receptors. The agonist radioligand 125I-N6-4-aminobenzyladenosine binds to two affinity states of the recombinant A1 adenosine receptors with K(D) values of 0.09 and 10.4 nM. The high affinity binding site represents <10% of total sites and is increased 7-fold on reconstitution with both alpha and betagamma G protein subunits but not with either subunit alone; thus, exogenous alpha and betagamma subunits do not functionally interact with endogenous Sf9 betagamma and alpha subunits, respectively. Four different alpha subunits (alpha i1, alpha i2, alpha i3, and alpha o) and six different beta gamma subunits (beta1gamma1, beta1gamma2, beta1gamma3, beta2gamma2, beta2gamma3, and bovine brain betagamma)) increased GTP-sensitive, high affinity agonist binding. The results indicate that bovine A1 adenosine receptors couple equally well to G protein alpha i and alpha o subunits in combination with betagamma subunits containing the beta1 or beta2 subunits and gamma2 or gamma3 subunits. G protein heterotrimers that contain the beta1gamma1 dimer couple with similar potency but reduced efficacy to A1 adenosine receptors.

G protein gamma subunits with altered prenylation sequences are properly modified when expressed in Sf9 cells.

The gamma subunits of heterotrimeric G proteins undergo post-translational prenylation and carboxylmethylation after formation of the betagamma dimer, modifications that are essential for alpha-betagamma, betagamma-receptor, and betagamma-effector interactions. We have determined the specific prenyl group present on the beta1gamma1, beta1gamma2, and beta1gamma3 dimers purified from baculovirus-infected Sf9 cells by specific binding to G protein alpha subunits immobilized on agarose. These recombinant dimers undergo the same post-translational modifications determined for gamma1 and gamma2 isolated from mammalian tissues. Furthermore, infection of Sf9 cells with a recombinant baculovirus encoding an alteration of the gamma1 CaaX sequence (gamma1-S74L) resulted in geranylgeranylation of the resulting gamma1 subunit, and alteration of the gamma2 CaaX sequence to CAIS (gamma2-L71S) resulted in farnesylation. Both of these altered gamma subunits were able to associate stably with beta1, and the resulting betagamma dimer bound tightly to alpha-agarose and eluted specifically with aluminum fluoride. These results indicate that Sf9 insect cells properly process the CaaX motif in G protein gamma subunits and are a useful model system to study the role of prenylation in the protein-protein interactions in which the betagamma subunits participate.

Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors.

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein alpha subunits and by the composition of the betagamma dimers. To determine the role of the gamma subunit prenyl modification in this interaction, the CaaX motifs in the gamma1 and gamma2 subunits were altered to direct modification with different prenyl groups, recombinant betagamma dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the betagamma dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the alpha subunit. The beta1gamma1 dimer (modified with farnesyl) was significantly less effective than beta1gamma2 (modified with geranylgeranyl) in either assay. The beta1gamma1-S74L dimer (modified with geranylgeranyl) was nearly as effective as beta1gamma2 in either assay. The beta1gamma2-L71S dimer (modified with farnesyl) was significantly less active than beta1gamma2. Using 125I-labeled betagamma subunits, it was determined that native and altered betagamma dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the gamma subunit is an important determinant of the interaction between receptors and G protein gamma subunits.

Inactivation of raf-1 by a protein-tyrosine phosphatase stimulated by GTP and reconstituted by Galphai/o subunits.

A membrane-associated form of Raf-1 in v-Ras transformed NIH 3T3 cells can be inactivated by protein phosphatases regulated by GTP. Herein, a distinct protein-tyrosine phosphatase (PTPase) in membrane preparations from v-Ras transformed NIH 3T3 cells was found to be activated by guanyl-5'-yl imidodiphosphate (GMPPNP) and was identified as an effector for pertussis toxin (PTx)-sensitive G-protein alpha subunits. PTPase activation was blocked by prior treatment of cells with PTx. PTPase activation by GTP, but not GMPPNP, was transient. A GMPPNP-stimulated PTPase (PTPase-G) co-purified with Galphai/o subunits during Superose 6 and Mono Q chromatography. PTPase-G activity in Superose 6 fractions from GDP-treated membranes was reconstituted by activated Galphai/o, but not G beta gamma, subunits. PTPase-G may contribute to GMPPNP-stimulated inactivation of Raf-1 in v-Ras cell membranes because Raf-1 inactivation was PTx-sensitive and PTPase-G inactivated exogenous Raf-1.

The Saccharomyces cerevisiae VMA6 gene encodes the 36-kDa subunit of the vacuolar H(+)-ATPase membrane sector.

The yeast vacuolar membrane proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral catalytic, and integral membrane domains. At least eight proteins cofractionate with purified preparations of the enzyme including 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa polypeptides (Kane, P.M., Yamashiro, C.T., and Stevens, T.H. (1989a) J. Biol. Chem. 264, 19236-19244). We took a reverse genetic approach to clone the structural gene for the 36-kDa subunit of the V-ATPase, VMA6, vma6 null mutants displayed growth characteristics typical of other vma mutants including sensitivity to media buffered at neutral pH or media containing 100 mM Ca2+. Vacuolar acidification was defective in vma6 cells and isolated vacuolar membrane preparations contained no detectable V-ATPase activity. The VMA6 gene encodes a hydrophilic polypeptide of 345 amino acids (predicted molecular mass 39.8-kDa). We present evidence that the VMA6 gene product (Vma6p) is a non-integral membrane component of the membrane pore domain and is required for V-ATPase complex assembly. Vma6p was removed from wild type vacuolar membranes by strong chaotropic agents such as alkaline Na2CO3 or 5M urea, which did not remove integral membrane polypeptides. In yeast cells lacking the integral membrane portion of the V-ATPase complex, Vma6p was unable to stably associate with vacuolar membranes. Conversely, in mutants specifically lacking Vma6p, components of the V-ATPase integral membrane domain were destabilized, and peripheral subunits failed to assemble onto vacuolar membranes. These results are discussed in the context of a developing model for V-ATPase assembly in yeast.

Protein splicing of the yeast TFP1 intervening protein sequence: a model for self-excision.

Protein splicing is the protein analogue of RNA splicing in which the central portion (spacer) of a protein precursor is excised and the amino- and carboxy-terminal portions of the precursor reconnected. The yeast Tfp1 protein undergoes a rapid protein splicing reaction to yield a spliced 69 kDa polypeptide and an excised 50 kDa spacer protein. We have demonstrated that the 69 kDa species arises by reformation of a bona fide peptide bond. Deletion analyses indicate that only sequences in the central spacer protein of the Tfp1 precursor are critical for the protein splicing reaction. A fusion protein in which only the Tfp1 spacer domain was inserted into an unrelated protein also underwent efficient splicing, demonstrating that all of the information required for protein splicing resides within the spacer domain. Alteration of Tfp1p splice junction residues blocked or kinetically impaired protein splicing. A protein splicing model is presented in which asparagine rearrangement initiates the self-excision of the spacer protein from the Tfp1 precursor. The Tfp1 spacer protein belongs to a new class of intervening sequences that are excised at the protein rather than the RNA level.

Isolation of vacuolar membrane H(+)-ATPase-deficient yeast mutants; the VMA5 and VMA4 genes are essential for assembly and activity of the vacuolar H(+)-ATPase.

The vacuolar membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae is a multisubunit enzyme complex composed of an integral membrane V0 sector, and a peripherally associated V1 sector. Deletion of one of several structural genes for vacuolar H(+)-ATPase subunits was previously demonstrated to prevent proper assembly of the remaining V1 subunits onto the vacuolar membrane (Kane, P.M., Kuehn, M.C., Howald-Stevenson, I., and Stevens, T.H. (1992) J. Biol. Chem. 267, 447-454). A genetic screen was designed to identify new genes whose products were essential for the synthesis, assembly, and/or function of the yeast vacuolar H(+)-ATPase. Mutants were identified based on phenotypes associated with vacuolar membrane H(+)-ATPase loss of function (vma), including an inability to grow on media buffered at neutral pH. Representatives in five complementation groups were identified, including four novel mutant vma5, vma21, vma22, and vma23, all of which were defective in vacuolar ATPase enzyme activity. We report here the characterization of two genes, VMA4 and VMA5, that encode peripheral subunits of the vacuolar H(+)-ATPase. We determined that VMA5 encodes the 42-kDa subunit of the vacuolar H(+)-ATPase. The VMA4 gene, originally described by Foury (Foury, F. (1990) J. Biol. Chem. 265, 18554-18560), was determined to encode the 27-kDa subunit of the purified yeast vacuolar H(+)-ATPase. Characterization of the vma5 and vma4 mutants revealed that the 42- and 27-kDa subunits are essential for the assembly of the peripheral membrane portion of the H(+)-ATPase onto the vacuolar membrane.