Isoprenylation of C-terminal cysteine in a G-protein gamma subunit

The predicted amino acid sequences for the Gi alpha 1 and G gamma 6 subunits of brain heterotrimeric G-proteins both contain C-terminal Cys-A-A-X elements (A is an aliphatic residue and X is any amino acid). This domain represents the site of Cys thioether modification by isoprenoids in p21ras, nuclear lamins, and fungal mating factors. We now show that G gamma 6, translated in reticulocyte lysate, is efficiently labeled with the isoprenoid precursor, [3H]mevalonate. Alteration of the sequence of G gamma 6 so that a Gly was substituted for Cys in the C-terminal Cys-A-A-X element rendered the protein incapable of undergoing isoprenoid modification. In contrast to G gamma 6, the Gi alpha 1 subunit did not appear to undergo isoprenylation when translated in reticulocyte lysate. Transient expression of the protein in COS cells, which were able to isoprenylate the p21 product of transfected H-ras, also failed to demonstrate isoprenylation of Gi alpha 1. The modification of the gamma subunit by a hydrophobic moiety may have important implications for the assembly of the brain G-protein beta gamma complexes into the cell membrane.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

G-protein beta gamma dimers. Membrane targeting requires subunit coexpression and intact gamma C-A-A-X domain.

Attachment of heterotrimeric G-proteins to the inner face of the plasma membrane is fundamental to their role as signal transducers by allowing interaction with both receptors and effectors. Certain G-protein alpha subunits are anchored to the membrane by covalent myristoylation. The beta gamma complex is required for G-protein interaction with receptors and is independently membrane associated through an unknown mechanism. A series of carboxyl-terminal modifications including isoprenylation which may contribute to membrane attachment has been identified recently in G-protein gamma subunits. Expression and membrane targeting of beta and gamma subunits were examined in COS cells. The expression of either subunit was found to require cotransfection with both beta and gamma cDNAs. Mutation of the carboxyl-terminal cysteine residue of gamma shown to undergo isoprenylation and carboxymethyl-esterification preserved beta gamma expression but blocked isoprenylation and membrane attachment. These results implicate the carboxyl-terminal processing of G-protein gamma subunits and beta coexpression as necessary and sufficient for membrane targeting of the beta gamma complex.     

A genetic and biochemical analysis of petite mutations in yeast

A series of spontaneous cytoplasmic petite mutants was isolated from a grande strain of Saccharomyces cerevisiae doubly marked with the cytoplasmically inherited determinants to erythromycin and oligomycin resistance. The petites were characterized with regard to the genetic stability of these antibiotic resistance markers and to their degree of suppressivity. No relation was found between the genetic instability of a petite mutant and the degree of suppressivity exhibited by that mutant. Three petites of 19.4%, 57.4% and 90.4% suppressivity were selected and their mitochondrial DNA characterized with regard to molecular weight, buoyant density in analytical cesium chloride density gradients, and the percentage of the total cellular DNA represented by the mitochondrial DNA. From these results it appears that the molecular weight of the mitochondrial DNA of the petite strains examined is the same as that shown by the parental grande strain, regardless of the degree of suppressivity exhibited.     

Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling.

The STE4 gene, which encodes the beta subunit of the mating response G-protein in the yeast Saccharomyces cerevisiae, was subjected to a saturation mutagenesis using 'doped' oligodeoxynucleotides. We employed a genetic screen to select dominant-negative STE4 mutants, which when overexpressed from the GAL1 promoter, interfered with the signalling function of the wild type protein. The identified inhibitory amino acid alterations define two small regions that are crucially involved in transmitting the mating signal from G beta to downstream components of the signalling pathway. These results underline the positive signalling role of yeast G beta and assign for the first time the positive signalling function of a G-protein beta subunit to specific structural features.     

The G-protein gamma subunit gpc-1 of the nematode C.elegans is involved in taste adaptation

Caenorhabditis elegans has two heterotrimeric G-protein gamma subunits, gpc-1 and gpc-2. Although GPC-1 is specifically expressed in sensory neurons, it is not essential for the detection of odorants or salts. To test whether GPC-1 is involved in sensory plasticity, we developed a water soluble compound adaptation assay. The behaviour of wild-type animals in this assay confirms that prolonged exposure to salts can abolish chemo-attraction to these compounds. This process is time and concentration dependent, partly salt specific and reversible. In contrast, gpc-1 mutant animals show clear deficits in their ability to adapt to NaAc, NaCl and NH4Cl, but normal wild-type adaptation to odorants. Two other loci previously implicated in odorant adaptation, adp-1 and osm-9, are also involved in adaptation to salts. Our finding that G proteins, OSM-9 and ADP-1 are involved in taste adaptation offer the first molecular insight into this process.     

Mitochondrial oligomycin-resistance mutations affecting the proteolipid subunit of the mitochondrial adenosine triphosphatase.

Two independently isolated oligomycin resistant mutants of Saccharomyces cerevisiae have been studied. The oligomycin resistance is conferred in each case by a single mutation at an oliA locus. In both strains the proteolipid subunit of the mitochondrial ATPase (subunit 9) shows an apparent increase in molecular weight as judged by its mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Variable effects are seen on other subunits. These results suggest that oliA loci may play some role in the determination of proteolipid ATPase subunit.     

Biochemical and genetic analysis of Streptococcus mutans alpha-galactosidase.

The aga gene coding for alpha-galactosidase in Streptococcus mutans was detected in a recombinant gene library constructed in phage lambda. The gene was subcloned into plasmid vectors and shown to specify a novel protein of Mr 80,000. Characterization of alpha-galactosidase from S. mutans and from recombinant Escherichia coli expressing aga indicated that the enzyme functions as a tetramer. The amino acid composition of the alpha-galactosidase, deduced from nucleotide sequencing of aga, gave a predicted Mr of 82,022 and revealed regions of homology to alpha-galactosidases encoded by the E. coli Raf plasmids and by Bacillus stearothermophilus. Inactivation of the aga gene in S. mutans resulted in loss of all alpha-galactosidase activity and abolished the ability to ferment melibiose; alpha-glucosidase activity was also lost, due to an indirect effect on the dexB gene.     

Biochemical analysis of genetic recombination in eukaryotes.

Recent studies concerning molecular mechanisms of genetic recombination in eukaryotes are reviewed. Since many of these studies have focused on the testable predictions arising from the hybrid DNA theory of genetic recombination, this theory is summarised. Experiments to determine the time of meiotic crossing-over and the structure of the synaptonemal complex which facilitates meiotic crossing-over are described. Investigations of DNA nicking and repair events implicated in recombination are discussed. Properties of proteins which may facilitate hybrid DNA formation, and biochemical evidence for hybrid DNA formation are presented. Finally, a nuclease which has been implicated in gene conversion is described.     

Biochemical and genetic characterization of the structure of yeast ornithine decarboxylase

The ornithine decarboxylase gene of S. cerevisiae encodes a predicted protein of approximately 53 kD highly homologous with the ornithine decarboxylase of other species. However, the native enzyme has been reported as an 86 kD protein. Our molecular sieve analysis indicated a Mr = 110,000 for the native enzyme. SDS-PAGE analysis of [H3]-alpha-difluoromethylornithine labelled enzyme demonstrated a subunit Mr of approximately 50 kD and suggested the native enzyme is a dimer. Genetic analyses support this conclusion. The complementary, ornithine decarboxylase deficient mutations spe 1A and spe 1B were mapped to the enzyme structural gene by linkage analysis and gene conversion mapping. This demonstrated that the mutations exhibit intragenic complementation which suggests protein-protein interactions and an oligomeric structure for the yeast enzyme. We conclude that yeast ornithine decarboxylase is a dimeric enzyme of 53 kD subunits.     

Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L)

The BCL-2 family includes both proapoptotic (e.g., BAX and BAK) and antiapoptotic (e.g., BCL-2 and BCL-X(L)) molecules. The cell death-regulating activity of BCL-2 members appears to depend on their ability to modulate mitochondrial function, which may include regulation of the mitochondrial permeability transition pore (PTP). We examined the function of BAX and BCL-X(L) using genetic and biochemical approaches in budding yeast because studies with yeast suggest that BCL-2 family members act upon highly conserved mitochondrial components. In this study we found that in wild-type yeast, BAX induced hyperpolarization of mitochondria, production of reactive oxygen species, growth arrest, and cell death; however, cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Coexpression of BCL-X(L) prevented all BAX-mediated responses. We also assessed the function of BCL-X(L) and BAX in the same strain of Saccharomyces cerevisiae with deletions of selected mitochondrial proteins that have been implicated in the function of BCL-2 family members. BAX-induced growth arrest was independent of the tested mitochondrial components, including voltage-dependent anion channel (VDAC), the catalytic beta subunit or the delta subunit of the F(0)F(1)-ATP synthase, mitochondrial cyclophilin, cytochrome c, and proteins encoded by the mitochondrial genome as revealed by [rho(0)] cells. In contrast, actual cell killing was dependent upon select mitochondrial components including the beta subunit of ATP synthase and mitochondrial genome-encoded proteins but not VDAC. The BCL-X(L) protection from either BAX-induced growth arrest or cell killing proved to be independent of mitochondrial components. Thus, BAX induces two cellular processes in yeast which can each be abrogated by BCL-X(L): cell arrest, which does not require aspects of mitochondrial biochemistry, and cell killing, which does.     

F0F1-ATPase gamma subunit mutations perturb the coupling between catalysis and transport.

We introduced mutations to test the function of the conserved amino-terminal region of the gamma subunit from the Escherichia coli ATP synthase (F0F1-ATPase). Plasmid-borne mutant genes were expressed in an uncG strain which is deficient for the gamma subunit (gamma Gln-14-->end). Most of the changes, which were between gamma Ile-19 and gamma Lys-33, gamma Asp-83 and gamma Cys-87, or at gamma Asp-165, had little effect on growth by oxidative phosphorylation, membrane ATPase activity, or H+ pumping. Notable exceptions were gamma Met-23-->Arg or Lys mutations. Strains carrying these mutations grew only very slowly by oxidative phosphorylation. Membranes prepared from the strains had substantial levels of ATPase activity, 100% compared with wild type for gamma Arg-23 and 65% for gamma Lys-23, but formed only 32 and 17%, respectively, of the electrochemical gradient of protons. In contrast, other mutant enzymes with similar ATPase activities (including gamma Met-23-->Asp or Glu) formed H+ gradients like the wild type. Membranes from the gamma Arg-23 and gamma Lys-23 mutants were not passively leaky to protons and had functional F0 sectors. These results suggested that substitution by positively charged side chains at position 23 perturbed the energy coupling. The catalytic sites of the mutant enzymes were still regulated by the electrochemical H+ gradient but were inefficiently coupled to H+ translocation in both ATP-dependent H+ pumping and delta mu H+ driven ATP synthesis.     

The RACK1 ortholog Asc1 functions as a G-protein beta subunit coupled to glucose responsiveness in yeast

According to the prevailing paradigm, G-proteins are composed of three subunits, an alpha subunit with GTPase activity and a tightly associated betagamma subunit complex. In the yeast Saccharomyces cerevisiae there are two known Galpha proteins (Gpa1 and Gpa2) but only one Gbetagamma, which binds only to Gpa1. Here we show that the yeast ortholog of RACK1 (receptor for activated protein kinase C1) Asc1 functions as the Gbeta for Gpa2. As with other known Gbeta proteins, Asc1 has a 7-WD domain structure, interacts directly with the Galpha in a guanine nucleotide-dependent manner, and inhibits Galpha guanine nucleotide exchange activity. In addition, Asc1 binds to the effector enzyme adenylyl cyclase (Cyr1), and diminishes the production of cAMP in response to glucose stimulation. Thus, whereas Gpa2 promotes glucose signaling through elevated production of cAMP, Asc1 has opposing effects on these same processes. Our findings reveal the existence of an unusual Gbeta subunit, one having multiple functions within the cell in addition to serving as a signal transducer for cell surface receptors and intracellular effectors.     

Biochemical and biophysical evidence for gamma 2 subunit association with neuronal voltage-activated Ca2+ channels.

A novel gene (Cacng2; gamma(2)) encoding a protein similar to the voltage-activated Ca(2+) channel gamma(1) subunit was identified as the defective gene in the epileptic and ataxic mouse, stargazer. In this study, we analyzed the association of this novel neuronal gamma(2) subunit with Ca(2+) channels of rabbit brain, and the function of the gamma(2) subunit in recombinant neuronal Ca(2+) channels expressed in Xenopus oocytes. Our results showed that the gamma(2) subunit and a closely related protein (called gamma(3)) co-sedimented and co-immunoprecipitated with neuronal Ca(2+) channel subunits in vivo. Electrophysiological analyses showed that gamma(2) co-expression caused a significant decrease in the current amplitude of both alpha(1B)(alpha(1)2.2)-class (36.8%) and alpha(1A)(alpha(1)2.1)-class (39.7%) Ca(2+) channels (alpha(1)beta(3)alpha(2)delta). Interestingly, the inhibitory effects of the gamma(2) subunit on current amplitude were dependent on the co-expression of the alpha(2)delta subunit. In addition, co-expression of gamma(2) or gamma(1) also significantly decelerates the activation kinetics of alpha(1B)-class Ca(2+) channels. Taken together, these results suggest that the gamma(2) subunit is an important constituent of the neuronal Ca(2+) channel complex and that it down-regulates neuronal Ca(2+) channel activity. Furthermore, the gamma(2) subunit likely contributes to the fine-tuning of neuronal Ca(2+) channels by counterbalancing the effects of the alpha(2)delta subunit.     

Biochemical and genetic analysis of a maltopentaose-producing amylase from an alkaliphilic gram-positive bacterium.

Two amylases have been purified from the culture fluid of an alkaliphilic bacterium. Amylase A-60 consists of a single type of polypeptide chain of 60 kDa and exhibits an alpha-amylase-type of starch cleavage. Amylase A-180 is approximately 180 kDa in size, represents the largest exoenzyme so far identified in prokaryotes and in the initial enzyme reaction cleaves starch exclusively to maltopentaose. A-60 and A-180 are immunologically unrelated enzymes. The structural gene for amylase A-180 has been cloned and its nucleotide sequence was determined. An open reading frame was identified for a putative protein of 182 kDa whose amino-terminal sequence, deduced from the nucleotide sequence, was identical in 23 out of 25 positions to that determined for the protein. The amino-terminus of the mature protein, at the gene level, is preceded by a sequence segment showing all the characteristics of a signal peptide from Gram-positive bacteria. Analysis of the deduced amino acid sequence revealed that the 70-kDa N-terminal part is similar to classical alpha-amylases. The C-terminal part contains three repeated sequence blocks of 99 amino acid residues each which are also present in two bacterial beta-amylases. It appears, therefore, that A-180 has arisen by gene fusion events.     

Biochemical properties of the ras-related YPT protein in yeast: a mutational analysis.

Using site-directed mutagenesis, the ras-related and essential yeast YPT1 gene was changed to generate proteins with amino acid exchanges within conserved regions. Bacterially produced wild-type proteins were used for biochemical studies in vitro and were found to have properties very similar to mammalian ras proteins. Gene replacement allowed the study of physiological consequences of the mutations in yeast cells. Lys21----Met and Asn121----Ile substitutions rendered the protein incapable of binding GTP and caused lethality. Ser17----Gly and Ala65----Thr substitutions slightly changed the protein's apparent binding capacity for either GDP or GTP and altered its intrinsic GTPase activity. These mutations were without effect on cellular growth. The YPTgly17,thr65 mutant protein displayed a significantly altered relative capacity for guanine nucleotide binding but a GTPase activity comparable to the wild-type protein. In contrast to the Ala65----Thr substitution, the double mutant displayed a significantly reduced capacity for autophosphorylation and allowed cells to grow only poorly. Cellular growth was improved when this mutant protein was overproduced.