Gramicidin S-synthetase. Electrophoretic characterization of the multienzyme.

1. A method characterizing the fully active gramicidin S-synthetase (EC. 6.3.2.-) multienzyme in protein mixtures by a combination of sedimentation and polyacrylamide gel electrophoretic mobility data has been described. 2. The molecular weight of 280000 has been reevaluated by gradient centrifugation, gel filtration, and polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The size of the multienzyme is not changed by sodium dodecyl sulfate treatment. 3. In polyacrylamide gel electrophoresis dimerisation occurs in Tris, while two bands, which may represent monomer and dimer, are observed in phosphate. 4. Reliability of molecular weight determinations of sodium dodecyl sulfate-protein complexes of sizes up to 300000 daltons has been determined, correlating either mobilities or retardation coefficients.     

Substrate-induced changes in sulfhydryl reactivity of bacterial D-amino acid transaminase

D-Amino acid transaminase from Bacillus sphaericus strain ATCC 14577 is a dimer with eight cysteinyl residues per molecule (T.S. Soper, W.M. Jones, and J.M. Manning (1979) J. Biol. Chem. 254, 10,901-10,905). The reaction of the cysteinyl residues with a variety of sulfhydryl reagents has been explored to gain insight into the physical environments around these cysteinyl residues in the absence or the presence of substrates. The native enzyme, in the pyridoxal-P conformation, appears to be a symmetrical dimer, whose SH groups react in pairs with anionic reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) or the halo acids. Two SH groups react with either reagent without altering enzymatic activity. Two additional SH groups react with DTNB with loss of catalytic activity. Positively charged reagents such as beta-bromoethylamine are much more effective in inactivating the pyridoxal-P conformation of the enzyme with almost five of the eight SH groups reacting and this results in a significant loss in catalytic activity. The neutral reagent dithiodipyridine is able to detect some asymmetry in the pyridoxal-P conformation. Upon addition of a D-amino acid substrate, the enzyme is transformed into the pyridoxamine-P conformation. This conformation is much more reactive with anionic reagents and much less reactive with cationic reagents, suggesting that there is a significant change in the net charge around one of the SH groups in the pyridoxamine-P conformation. Also, titration with DTNB indicates that the enzyme is a much more asymmetric dimmer in the pyridoxamine-P conformation than in the pyridoxal-P conformation. Thus, upon binding of a D-amino acid substrate, D-amino acid transaminase is transformed into the pyridoxamine-P conformation. This results in a significant change in the environment of four of the sulfhydryl groups of the enzyme. We conclude that the enzyme is transformed from a symmetrical dimer into an asymmetrical dimer and that the net charge of one of the pairs of cysteinyl groups is changed from a net negative charge into a net positive charge. These results suggest that there is a significant conformational change that occurs during the transition from the pyridoxal-P into the pyridoxamine-P form of this transaminase.     

Cross-linking of hemoglobin, haptoglobin, and hemoglobin-haptoglobin complex with bifunctional imidoesters.

Dimethyl adipimidate was used to cross-link the polypeptides within hemoglobin, haptoglobin, and hemoglobin-haptoglobin complex. Cross-linked hemoglobin retained considerable ability to bind haptoglobin, although the amounts bound were reduced and the haptoglobin reaction could be used to fractionate the modified hemoglobin. With cross-links limited to intramolecular sites, hemoglobin showed four bands on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, identified, with reference to the subunit polypeptides, as monomer, dimer, trimer, and tetramer. The dimer region consisted of at least two separable species. When hemoglobin-haptoglobin complex was cross-linked, a band of hemoglobin dimer was present, which demonstrates that at least two hemoglobin subunits have a close spatial relation when bound to haptoglobin. Some comparisons with adipimidate-reacted hemoglobin were made using malonimidate and suberimidate and some marked differences were noted.     

Exploitation of hormone-induced conformational changes to label selectively a component of rat liver plasma membranes.

The kinetics for inactivation of rat liver plasma membrane adenylate cyclase by iodoacetic acid and iodoacetamide has been measured in the presence and absence of glucagon. Glucagon stimulated the rate of iodoacetic acid inhibition by a factor 9f 2.3-fold and iodoacetamide inhibition by 10-fold. These results suggest that interaction of glucagon with its receptor in the membrane resulted in conformational changes which increased either the exposure or nucleophilicity of one or more sulfhydryl groups crucial for adenylate cyclase activity. Membranes were treated with radioactively labeled iodoacetamide or iodoacetic acid in the presence or absence of glucagon and run on 5 and 7.5% sodium dodecylsulfate polyacrylamide gels. These labeling experiments revealed that two membrane components were more extensively labeled in the presence of glucagon. The first component had an apparent molecular weight of 240,000 on sodium dodecyl sulfate gels and stained positive with Coomassie blue and periodate Schiff reagent. This polypeptide accounted for approximately 1.3% of the total membrane protein. The second component had an apparent molecular weight less than 10,000 and could not be correlated directly with a well defined protein band on sodium dodecyl sulfate polyacrylamide gels. The enhancement in labeling of the 240,000 molecular weight component seen in the presence of glucagon agreed very well with that predicted from the kinetics for inhibition of adenylate cyclase activity in the presence and absence of glucagon. This correlation suggests that the component selectively labeled by this technique may be an integral component of the adenylate cyclase system and that hormone-induced conformational changes may be used to selectively label components of the adenylate cyclase system in mammalian membranes.     

Wheat germ phosphoglycerate mutase: purification, polymorphism, and inhibition

An improved method for purifying the bisphosphoglycerate-independent phosphoglycerate mutase from wheat germ has been devised. The method yields enzyme with a specific activity of 2,300 units/mg in 0.1 M Tris-C1 at pH 8.7 and 30 degrees C. Electrophoresis on electrofocusing and analytical polyacrylamide gels reveals only one protein band (pI = 7.3); however, under denaturing conditions (sodium dodecyl sulfate polyacrylamide gel electrophoresis), two prominent enzyme forms, with molecular masses of 63 and 74 kDa, manifest themselves along with several minor, high molecular mass components (126-141 kDa). Non-denaturing exclusion chromatography shows that both major species are catalytically active, and suggests that each species is capable of participating in reversible monomer/dimer association. Wheat germ mutase is inhibited by time-dependent reactions involving either polydentate chelators or sulfhydryl reagents.     

Saturating Irradiance-Induced Photoinhibition without Monomerisation of Photosystem 2 Dimer in Soybean Leaves

The oligomeric state of photosystem 2 (PS2) complex in soybean leaves treated with saturating irradiance was studied by non-denaturing polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography. PS2 dimers resolved by non-denaturing PAGE accounted for about 75 % of total PS2 complex and there was no significant difference in the ratio of PS2 dimer to monomer between samples from saturating irradiance-treated and fully dark-adapted leaves. Furthermore, BBY particles were resolved into four chlorophyll-enriched fractions by gel filtration chromatography. From their molecular masses and protein components, these fractions were deduced to be PS2 dimer, PS2 monomer, oligomeric light-harvesting complex 2 (LHC2), and monomeric LHC2. Also, no change in the proportion of PS2 dimer in total PS2 was observed in the granal region of thylakoid membranes from soybean leaves after saturating irradiation. Hence the dimer is the predominant natural form of PS2 in vivo and no monomerisation of PS2 dimer occurs during saturating irradiance-induced photoinhibition in soybean leaves.     

Altered N----F transition in bovine serum albumin dimer and tetramer

The monomer, dimer and tetramer of bovine serum albumin were separated by gel filtration on a Sephacryl S-300 column. Their purity was established by gel filtration, polyacrylamide gel electrophoresis and SDS polyacrylamide gel electrophoresis. These preparations were identical in bilirubin binding, cross-reactivity against anti-bovine serum albumin antiserum and UV spectral features. However, the three preparations differed in their N----F transition. The mid points of the N----F transitions for monomer, dimer and tetramer were found to be 3.75, 3.60 and 3.40 respectively. We presume that intermolecular interactions and/or alterations in the pK values of carboxyl groups are responsible for increased stability in the case of dimer and tetramer.     

Metabolism of leukotriene E4 to 5-hydroxy-6-mercapto7,9-trans-11,14-cis-eicosatetraenoic acid by microfloral cysteine-conjugate beta-lyase and rat cecum contents

Leukotriene E4 was incubated with cysteine-conjugate beta-lyase isolated from the intestinal bacterium Eubacterium limosum. The reaction was terminated by addition of iodoacetic acid or dimethyl sulfate, and the products formed were isolated by reverse-phase high-performance liquid chromatography. The structures of two adducts of a metabolite were determined by uv spectroscopy, by gas-liquid radiochromatography, and by comparisons with chemically synthesized reference compounds. They were 5-hydroxy-6-S-carboxymethylthio-7,9-trans-11,14-cis-eicosatetraeno ic acid (iodoacetic acid adduct) and 5-hydroxy-6-S-methylthio-7,9-trans-11,14-cis-eicosatetraenoic acid (dimethyl sulfate adduct) indicating that the structure of the underivatized metabolite was 5-hydroxy-6-mercapto-7,9,11,14-eicosatetraenoic acid (5,6-HMETE). The latter product is formed by beta-lyase-catalyzed cleavage of the cysteine C-S bond in leukotriene E4. Leukotriene E4 was also metabolized to 5,6-HMETE by rat cecal contents. A product formed was trapped as the iodoacetic acid derivative and identified as 5-hydroxy-6-S-carboxy-methylthio-7,9,11,14-eicosatetraenoic acid. It is concluded that intestinal leukotriene E4, originating from biliary excretion of systemic cysteinyl leukotrienes or produced in the intestine, is converted by microfloral cysteine-conjugate beta-lyase to 5,6-HMETE.     

Plastocyanin: possible significance of quaternary structure.

Some properties of the blue copper protein plastocyanin from the green alga Scenedesmus have been investigated and compared with that from spinach, including amino acid composition, isoelectric point and copper content. The protein from Scenedesmus contains two, that from spinach four copper atoms per molecular weight of 40000. A combination of sodium dodecylsulfate/polyacrylamide gel electrophoresis and quantification of sulfhydryl groups indicates a strong preference for a species composed of 4 polypeptide chains of identical amino acid composition representing the enzymically active entity. Due to various treatments the subunits of both plastocyanins are detected as either monomer species alone or as monomer and dimer in a molar ratio of 2 : 1 on sodium dodecylsulfate gels. The four -SH groups per molecule are of different reactivity: two -SH groups can be detected after destruction of the chromophore; two more (forming an S-S bridge in the dimer) become evident after appropriate reduction. A KCN treatment for production of apoprotein is reported and the use of electrodialysis to improve incomplete apoprotein formation. These studies lend support to the proposal of a quaternary structure. Apoproteins were subjected to dodecylsulfate gel analysis, which proved to be an effective means of estimating both the extent of apoprotein formation and its reconstitution to the haloprotein.     

The sidedness of the COOH terminus of the acetylcholine receptor delta subunit

The nicotinic acetylcholine receptor from Torpedo sp. occurs as a dimer, disulfide-cross-linked between delta subunits. We determined the sidedness of the COOH terminus of the acetylcholine receptor delta subunit by locating the delta-delta disulfide relative to the membrane and by identifying the Cys residue forming the disulfide. We used receptor-rich native membrane vesicles isolated from Torpedo californica electric tissue and characterized as to orientation and intactness. These vesicles had not been extracted and retained v ("43-kDa protein") as a marker of the cytoplasmic surface. Using the reduction of v as an assay of permeability, we showed that two reductants, 2-mercaptoethanesulfonate and reduced glutathione, were relatively impermeant. Both of these reductants reduced the delta-delta disulfide in sealed right-side-out vesicles equally in the presence and absence of saponin, and 2-mercaptoethanesulfonate reduced this disulfide equally in the presence and absence of Triton X-100. By contrast, surfactants enhanced the reduction of dimer in inside-out and sequestered vesicles. We conclude that the disulfide is extracellular. To identify the Cys residue forming the disulfide, we labeled the sulfhydryls both in receptor dimer and in monomer generated by mild reduction of dimer. By high performance liquid chromatography and NH2-terminal sequencing of cyanogen bromide fragments of labeled delta-delta dimer and delta monomer, we found that the penultimate residue, delta-Cys-500, uniquely formed an intersubunit disulfide and that this disulfide was uniquely reduced when receptor dimer was reduced to monomer. Therefore, the delta COOH terminus is extracellular.     

Demonstration of heavy and light protomers of human testosterone-estradiol-binding globulin

Human testosterone-estradiol-binding globulin (hTeBG) was purified from pregnancy serum by sequential ammonium sulfate precipitation, affinity chromatography, and hydroxylapatite chromatography. An overall purification of 2800-fold was achieved with a 27% total yield. Apparent homogeneity of the final product was shown by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate (SDS). The equilibrium dissociation constant (Kd) at 4 degrees C for 5 alpha-dihydrotestosterone (DHT) was estimated to be 1.94 +/- 0.95 X 10(-9) M. Analysis of the purified protein revealed microheterogeneity with regard to size on polyacrylamide gel in the presence of SDS and to charge on isoelectric focusing gels. The apparent molecular weight of native hTeBG determined by gradient gel electrophoresis was 115,000. SDS-polyacrylamide gel electrophoresis indicated that hTeBG is comprised of two molecular weight components of 53,000 and 46,000, which are designated as heavy (hTeBGH) and light (hTeBGL) protomers, respectively. Photolysis of purified hTeBG with [1,2-3H]17 beta-hydroxy-4,6-androstadien-3-one [( 3H]delta 6-testosterone) resulted in specific labeling of its binding sites. Analysis of photolabeled products by SDS-polyacrylamide gel electrophoresis revealed two radioactive products with electrophoretic mobilities identical to those of the hTeBGH and hTeBGL. The ratio of hTeBGH to hTeBGL was about 10:1. The H and the L protomers were separated and examined by peptide mapping using protease V8 and chymotrypsin. Comparison of the fragmentation patterns produced by these proteases revealed that hTeBGH and hTeBGL components were nearly identical. Removal of sialic acid or carbohydrate residues from hTeBG did not affect the presence of two molecular components. Isoelectric focusing of native hTeBG demonstrated three isoelectric variants with pIs at 4.75, 4.85 and 4.90. After treatment with neuraminidase and other glycosidases, only two isoelectric species were observed with more alkaline pIs. Although purified hTeBG appeared heterogeneous with regard to size and charge, it was remarkably homogeneous in its ability to absorb to Concanavalin A-Sepharose. We conclude that hTeBg, like the androgen binding proteins of the rabbit and rat, is a dimer whose monomer exhibits two protomeric forms.     

Sequence of the cysteinyl-containing peptides of 4-aminobutyrate aminotransferase. Identification of sulfhydryl residues involved in intersubunit linkage

Three cysteine-containing tryptic peptides were isolated and sequenced from mitochondrial 4-aminobutyrate aminotransferase using DABIA (4-dimethylaminoazobenzene-4-iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species. To identify the cysteinyl residues undergoing reversible oxidation/reduction, the S-DABIA-labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl-containing peptides (peptides II, III) have been oxidized. The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4-aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds.     

A novel concatenated dimer of recombinant bovine somatotropin

A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized inEscherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.     

Synchrotron radiation based STXM analysis and micro-XRF mapping of differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on Fe and S

The differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on substrates Fe^2 + and S⁰ was investigated by using synchrotron radiation based scanning transmission X-ray microscopy (STXM) imaging and microbeam X-ray fluorescence (μ-XRF) mapping. The extracellular thiol groups (SH) were first alkylated by iodoacetic acid forming Protein-SCH₂COOH and then the P-SCH₂COOH was marked by calcium ions forming P-SCH₂COOCa. The STXM imaging and μ-XRF mapping of SH were based on analysis of SCH₂COO-bonded Ca^2 +. The results indicated that the thiol group content of A. ferrooxidans grown on S⁰ is 3.88 times to that on Fe^2 +. Combined with selective labeling of SH by Ca^2 +, the STXM imaging and μ-XRF mapping provided an in situ and rapid analysis of differential expression of extracellular thiol groups.     

Half-of-the-sites and all-of-the-sites reactivity in human plasma blood coagulation factor XIIIa

The reactivities of human plasma factor XIIIa toward iodoacetic acid and toward alpha-bromo-4-hydroxy-3-nitroacetophenone have been studied under conditions where this dimeric enzyme reacts with the reagents in half-of-the-sites fashion and under conditions where it reacts with the reagents in all-of-the-sites fashion. Direct measurements of alkylation of active site -- SH groups in the apparently identical subunits of the enzyme as functions of remaining catalytic activity are in agreement with the observed reactivities. In addition to extending earlier evidence for half-of-the-sites reactions in factor XIIIa (Chung, S. I., Lewis, M. S., and Folk, J. E. (1974) J. Biol. Chem. 249, 940-950), the present findings suggest that the all-of-the-sites reactivity results from a positively cooperative interaction between enzyme subunits.     

Characterization of rat ovarian lutropin receptor. Role of thiol groups in receptor association

Rat ovarian lutropin receptor occurs predominantly as a monomer of an apparent molecular mass of 70 or 80 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing and reducing conditions, respectively. The receptor contains 0.4% free cysteine and 1.9% cysteine as cystine, determined by amino acid analysis of the S-carboxymethyl receptor prepared before and after reduction. The presence of free thiol groups was further shown by the specific adsorption of the receptor on p-chloromercuribenzoate-agarose and its susceptibility to 3H labeling with [3H]N-ethylmaleimide or [3H]iodoacetic acid. The receptor readily undergoes association into homo-oligomers. Evidence suggests that the association was caused by the intermolecular oxidation of the free -SH groups to form disulfide bonds. The aggregation could be induced by H2O2 or molecular O2 and was inhibited by sulfhydryl protecting agents such as N-ethylmaleimide, iodoacetic acid, dithiothreitol, cysteine, and Zn(II). The oligomers could be dissociated by reduction into a monomer. 125I-Labeling of the S-carboxymethyl- or N-ethylmaleyl receptor gave a single band of molecular mass 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Furthermore, S-alkylation of the receptor did not affect its binding to the ligand. On reduction, however, it lost its ability to bind to the ligand, but the reduced receptor retained its ability to bind to a specific polyclonal rabbit antireceptor antibody indicating the separation of the ligand and antibody binding sites. Endoproteinase Glu-C cleaved the receptor at a single glutamyl residue to give two components, 46 and 36 kDa. The 36-kDa component was extracellularly located since it contained the carbohydrate. On deglycosylation with endoglycosidase F, it yielded two components, 27 and 25 kDa. The deglycosylation of the reduced intact receptor (80 kDa) with endoglycosidase F occurred in two steps giving 73- and 64-kDa polypeptides, indicating the presence of about 20% carbohydrate contained in two or more N-linked chains.     

Synthesis and biological properties of antiparallel and parallel dimers of alpha-human atrial natriuretic peptide

To obtain antiparallel and parallel dimers of alpha-human atrial natriuretic peptide (alpha-hANP), two fully protected peptides I and II having the same amino acid sequence as alpha-hANP with different protective groups at the cysteinyl residues were synthesized, the former having Acm and Npys and the latter MeBzl and Acm. Equivalent amounts of peptides I and II were mixed and subjected to HF deprotection. Next, the first disulfide bond was linked between the remaining Npys group in I and the liberated SH group in II to form a monodisulfide dimer. The second disulfide bond was formed within the newly formed dimer between the remaining Acm groups by treatment with iodine, giving an antiparallel dimer. The parallel dimer of alpha-hANP was synthesized similarly starting from the protected peptide II. These dimers could be clearly segregated on HPLC. The retention time on HPLC of the antiparallel dimer was identical with that of natural beta-hANP. Both dimers showed biological activities as high as one third to one sixth of alpha-hANP in smooth muscle spasmolytic activity, and almost the same level of natriuretic activity as alpha-hANP at a high dose (10 nmol/kg) but about one fifth the activity at a low dose (1 nmol/kg). In these assay systems, the antiparallel dimer showed a slower onset and a tendency of longer duration than alpha-hANP.     

Modulation of dimer stability in yeast pyrophosphatase by mutations at the subunit interface and ligand binding to the active site

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 microm, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH > or = 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and p-hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys(83)), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg(2+) cofactor, P(i) product, and the PP(i) analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k(cat) remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys(83) residue in the dimer.     

Enzymatic methylation of 23-29-kDa bovine retinal rod outer segment membrane proteins. Evidence for methyl ester formation at carboxyl-terminal cysteinyl residues

A group of 23-29-kDa polypeptides in the membranes of bovine rod outer segments are substrates for S-adenosylmethionine-dependent methylation reactions. The bulk of the methyl group incorporation is in base-labile ester-like linkages, and does not appear to be due to the widespread D-aspartyl/L-isoaspartyl methyltransferase (EC 2.1.1.77). To determine the site(s) of methylation, 3H-methylated proteins separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate were eluted and digested with papain, leucine aminopeptidase-M, and prolidase. After performic acid oxidation of the digest, a base-labile radioactive material was recovered that coeluted with a synthetic standard of cysteic acid methyl ester upon cation exchange and G-15 gel filtration chromatography, as well as in two thin-layer electrophoresis and two thin-layer chromatography systems. These results provide direct evidence for the methylation of the alpha-carboxyl group of a carboxyl-terminal cysteinyl residue, a modification that has been proposed for the 21-kDa Ha-ras product and other cellular proteins (Clarke, S., Vogel, J. P., Deschenes, R. J., and Stock, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4643-4647).     

Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae

Abplp is a yeast cortical actin-binding protein that contains an SH3 domain similar to those found in signal transduction proteins that function at the membrane/cytoskeleton interface. Although no detectable phenotypes are associated with a disruption allele of ABP1, mutations that create a requirement for this protein have now been isolated in the previously identified gene SAC6 and in two new genes, SLA1 and SLA2. The SAC6 gene encodes yeast fimbrin, an actin filament-bundling protein. Null mutations in SLA1 and SLA2 cause temperature-sensitive growth defects. Sla1p contains three SH3 domains and is essential for the proper formation of the cortical actin cytoskeleton. The COOH terminus of Sla2p contains a 200 amino acid region with homology to the COOH terminus of talin, a membrane cytoskeletal protein which is a component of fibroblast focal adhesions. Sla2p is required for cellular morphogenesis and polarization of the cortical cytoskeleton. In addition, synthetic-lethal interactions were observed for double- mutants containing null alleles of SLA2 and SAC6. In total, the mutant phenotypes, sequences, and genetic interactions indicate that we have identified novel proteins that cooperate to control the dynamic cytoskeletal rearrangements that are required for the development of cell polarity in budding yeast.