scholarly journals Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides

1993 ◽  
Vol 295 (3) ◽  
pp. 645-648 ◽  
Author(s):  
M M Altamirano ◽  
J A Plumbridge ◽  
H A Barba ◽  
M L Calcagno
Keyword(s):  
Thermal Denaturation ◽  
Cysteine Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Urea Denaturation ◽  
Disulphide Bonds ◽  
Oligomeric Protein ◽  
Sds Page ◽  
Mutant Forms ◽  
Allosteric Properties

Glucosamine-6-phosphate deaminase is an oligomeric protein composed of six identical 29.7 kDa subunits. Each subunit has four cysteine residues located at positions 118, 219, 228 and 239. We have previously shown that Cys-118 and Cys-239 form a pair of vicinal thiols, the reactivity of which changes with the allosteric transition. The site-directed mutations Cys-->Ser corresponding to the other two cysteine residues have been constructed, as well as some selected multiple mutations involving the four cysteines. Thiol and disulphide measurements on the wild-type and mutant enzymes indicate that thiols from Cys-219 are oxidized and form interchain disulphide bonds. The disulphide-linked dimer was demonstrated by SDS/PAGE. This result is consistent with preliminary crystallographic data and thermal denaturation studies, and strongly suggests that glucosamine-6-phosphate deaminase is a trimer of disulphide-linked dimers. The mutant forms of the deaminase lacking the interchain disulphide bond or the thiol at Cys-228 are both stable hexamers showing the same sensitivity to urea denaturation as the wild-type protein. Furthermore, these Cys-->Ser mutants display the same kinetics and allosteric properties as those already described for the wild-type enzyme.

scholarly journals Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

2019 ◽  
Vol 20 (18) ◽  
pp. 4412
Author(s):  
Denis L. Atroshenko ◽  
Mikhail D. Shelomov ◽  
Sophia A. Zarubina ◽  
Nikita Y. Negru ◽  
Igor V. Golubev ◽  
...  
Keyword(s):  
Amino Acid ◽  
Thermal Denaturation ◽  
Catalytic Properties ◽  
Acid Oxidase ◽  
Cephalosporin C ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Amino Acid Oxidase ◽  
Catalytic Constant ◽  
Biotechnological Processes

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.

Lymphoma-Associated Mutations of EZH2 Result In a Change-of-Function

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 707-707
Author(s):  
Victoria M Richon ◽  
Christopher J Sneeringer ◽  
Margaret Porter Scott ◽  
Kevin W Kuntz ◽  
Sarah K Knutson ◽  
...  
Keyword(s):  
Cell Lymphoma ◽  
Catalytic Efficiency ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Malignant Phenotype ◽  
H3k27 Methylation ◽  
Large B Cell Lymphoma ◽  
Mutant Forms ◽  
Large B Cell

Abstract Abstract 707 EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2), catalyzes the mono- through tri-methylation of lysine 27 on histone H3 (H3K27). While overexpression of EZH2 and increased H3K27 methylation have generally been associated with both hematologic malignancies and solid tumors, inactivating somatic mutations of Tyr641 (Y641F, Y641N, Y641S and Y641H) of EZH2 were recently reported to be associated with follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) (Morin, Nat Genet 2010; 42: 181). In all cases, occurrence of the mutant EZH2 gene was heterozygous, and expression of both wild type and mutant alleles was detected in the mutant samples profiled by transcriptome sequencing. Further, the mutant forms of EZH2 could be incorporated into the multi-protein PRC2 complex, but the resulting complexes lacked the ability to catalyze trimethylation of an unmethylated H3K27 peptide substrate. To explore further the role of EZH2 in lymphomagenesis, we have evaluated the catalytic activity of the mutant EZH2 proteins in greater detail. Recombinant PRC2 complexes were prepared with wild type and Tyr641 mutant EZH2 forms. As previously reported, the wild type enzyme demonstrated robust activity but none of the mutant enzymes displayed significant methyltransferase activity on an unmodified H3K27 peptide. We next evaluated the activity of the enzymes using native avian erythrocyte olignucleosomes as the substrate in the reaction. In contrast to the peptide result, we found that the wild type and all of the mutant enzymes were active methyltransferases against the native nucleosome substrate. Since native nucleosome represents an admixture of the unmodified and mono-, di- and tri-methylated H3K27 we next evaluated the activity of the wild type and mutant enzymes on unmodified, and mono- and di-methylated H3K27 peptide. We demonstrate that the wild type enzyme displays greatest catalytic efficiency (kcat/K) for the zero to mono-methylation reaction of H3K27, and diminished efficiency for subsequent (mono- to di- and di- to tri-methylation) reactions. In stark contrast, the disease-associated Y641 mutants display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to WT-enzyme. Catalytic coupling between the mutant EZH2 species and PRC2 complexes containing either wild type EZH2 or wild type EZH1 are predicted to augment H3K27 trimethylation and thus produce the malignant phenotype associated with mutant heterozygosity. To test this prediction, the level of H3K27 methylation was evaluated in lymphoma cell lines harboring only wild type EZH2 (OCI-LY-19) or heterozygous for EZH2 Y641N (DB, KARPAS and SU-DHL-6) or EZH2 Y641F (WSU-DLCL2) by immunoblotting. As predicted by simulations, the level of H3K27 trimethylation was elevated in all of the lymphoma cell lines harboring the mutant EZH2 relative the wild type. Additionally, we observe decreased H3K27 dimethylation and monomethylation in the cells harboring the mutated EZH2 relative to wild type enzyme; these reductions in di- and monomethylation are likewise consistent with expectations based on steady state kinetic simulations. The present results imply that the malignant phenotype of follicular lymphoma and diffuse large B cell lymphoma of the GCB subtype, associated with expression of mutant forms of EZH2, results from of an overall gain-of-function with respect to formation of the trimethylated form of H3K27. These data suggest that selective, small molecule inhibitors of EZH2 enzymatic function may form a rational underpinning for molecularly targeted therapeutics against mutant-harboring lymphomas and other malignancies in which EZH2 gain-of-function is pathogenic. Disclosures: Richon: Epizyme, Inc: Employment. Sneeringer:Epizyme: Employment. Porter Scott:Epizyme, Inc: Employment. Kuntz:Epizyme, Inc: Employment. Knutson:Epizyme, Inc.: Employment. Pollock:Epizyme, Inc: Employment. Copeland:Epizyme, Inc: Employment.

scholarly journals Properties of a cysteine-free proton-pumping nicotinamide nucleotide transhydrogenase

1997 ◽  
Vol 324 (2) ◽  
pp. 681-687 ◽  
Author(s):  
Johan MEULLER ◽  
Junwei ZHANG ◽  
Cynthia HOU ◽  
Philip D. BRAGG ◽  
Jan RYDSTRÖM
Keyword(s):  
Proton Pumping ◽  
Free Enzyme ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Β Subunit ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Α Subunit ◽  
State Reduction ◽  
Nicotinamide Nucleotide Transhydrogenase

Nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated with respect to the roles of its cysteine residues. This enzyme contains seven cysteines, of which five are located in the α subunit and two are in the β subunit. All cysteines were replaced by site-directed mutagenesis. The final construct (αC292T, αC339T, αC395S, αC397T, αC435S, βC147S, βC260S) was inserted normally in the membrane and underwent the normal NADPH-dependent conformational change of the β subunit to a trypsin-sensitive state. Reduction of NADP+ by NADH driven by ATP hydrolysis or respiration was between 32% and 65% of the corresponding wild-type activities. Likewise, the catalytic and proton pumping activities of the purified cysteine-free enzyme were at least 30% of the purified wild-type enzyme activities. The H+/H- ratio for both enzymes was 0.5, although the cysteine-free enzyme appeared to be more stable than the wild-type enzyme in proteoliposomes. No bound NADP(H) was detected in the enzymes. Modification of transhydrogenase by diethyl pyrocarbonate and the subsequent inhibition of the enzyme were unaffected by removal of the cysteines, indicating a lack of involvement of cysteines in this process. Replacement of cysteine residues in the α subunit resulted in no or little change in activity, suggesting that the basis for the decreased activity was probably the modification of the conserved β-subunit residue Cys-260 or (less likely) the non-conserved β-subunit residue Cys-147. It is concluded that the cysteine-free transhydrogenase is structurally and mechanistically very similar to the wild-type enzyme, with minor modifications of the properties of the NADP(H) site, possibly mediated by the βC260S mutation. The cysteine-free construct will be a valuable tool for studying structure–function relationships of transhydrogenases.

scholarly journals Formation of one or more intrachain disulphide bonds is required for the intracellular processing and transport of CD36

1997 ◽  
Vol 328 (2) ◽  
pp. 635-642 ◽  
Author(s):  
Paola GRUARIN ◽  
Roberto SITIA ◽  
Massimo ALESSIO
Keyword(s):  
Scavenger Receptor ◽  
Extracellular Domain ◽  
Low Density Lipoproteins ◽  
Cysteine Residues ◽  
Thiol Groups ◽  
Wild Type ◽  
Mobility Shift ◽  
Disulphide Bonds ◽  
Gel Mobility Shift ◽  
Triton X

In monocytes/macrophages, CD36 is thought to have a role as a scavenger receptor, mediating the phagocytosis of apoptotic cells and the endocytic uptake of oxidized low-density lipoproteins and fatty acids. The proposed topology of CD36 predicts that, of ten cysteine residues, six lie in the extracellular domain, whereas four are equally distributed in the two short terminal tails flanking the N-terminal and C-terminal hydrophobic stretches. Here we investigate the formation of intrachain disulphide bonds, on the basis of the assumption that the cysteine residues present in the luminal domains are generally oxidized, whereas those in the cytosol are reduced. As revealed by gel mobility-shift assays, disulphide bonds are present in the extracellular domain of the CD36 molecule. The formation of these bonds is required for the transport of CD36 from endoplasmic reticulum to Golgi. Furthermore reactive thiol groups are present in the CD36 sequence, which upon lysis form an intrachain extra loop as an artifact. This disulphide bond is not formed in either (1) truncated CD36 lacking the two C-terminal cysteine residues or (2) Triton X-100-insoluble wild-type CD36 molecules, suggesting that, in this fraction, the C-terminal thiol groups are modified.

The Effect of Unpaired Cysteine Residues and the C Domains On the Expression of Von Willebrand Factor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1078-1078
Author(s):  
Thomas A J Mckinnon ◽  
Susie Shaperio ◽  
Agata Anna Nowak ◽  
M. Laffan
Keyword(s):  
Von Willebrand Factor ◽  
Point Mutations ◽  
Full Length ◽  
Cysteine Residues ◽  
Wild Type ◽  
Free Thiol ◽  
Disulphide Bonds ◽  
Self Association ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 1078 Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein that mediates platelet adhesion under high shear stress and is the carrier molecule for FVIII. VWF contains a large number of cysteine residues that were previously thought to all participate in either intra- or inter-molecular disulphide bonds. However, recently it has been shown that VWF contains a proportion of unpaired cysteine residues, or free thiols and these may be involved in lateral self association of VWF. Initially Choi et al concluded that two free thiols are present in the D3 domain C889 and C898) and seven in the C domains (C2448, C2451, C2453, C2490, C2491, C2528 and C2533). Moreover, Ganderton et al recently showed that expression of the isolated VWF C2 domain resulted in the formation of disulphide linked oligomers and suggested that lateral self-association of VWF involved the C2431-C2453 bond located in the VWF C2 domain. When they mutated C2453 to Alanine the extent of oligomerisaton was enhanced. However it is not clear how this relates to the full length VWF molecule. In the present study we investigated the effect of mutating the predicted unpaired cysteine residues on the expression of full length VWF. Initially we used the binding of MPB to VWF to compare the relative amount of free thiol on plasma derived and recombinant VWF (rVWF). Interestingly, rVWF presented an increased free thiol content compared to purified plasma derived VWF indicating that free thiols, at least in rVWF are formed without exposure to the circulation. Next we created nine individual point mutations, based the observations of Choi et al, changing the predicted unpaired cysteine residues to alanine in full length VWF and analysed their expression in HEK293T cells. Interestingly, all of the point mutations failed to secrete from HEK293T cells, with the protein being retained within the cell lysate. A double point mutant, C2431A-C2453A, similarly failed to secrete. Analysis of the pro-VWF:mature-VWF ratio and Endo-H digestion of intracellular VWF demonstrated that all the mutants were retained within the endoplasmic reticulum (ER). Co-expression experiments with wild type VWF partially restored expression of some mutants, however co-expression with a deletion A1/A3 construct, demonstrated that the molecules containing the cysteine point mutations were retained predominately in the ER. Together these data suggest that in full length VWF, correct disulphide bonding within the ER is required for protein secretion. Since the point mutations did not express we created a series of deletion mutants to remove portions of the C-terminus of VWF. While VWF with either its A1, A2, A3 or D4 domain was expressed at comparable levels to wild type, all of the created C-terminal deletion variants: ΔC1C6(2255–2720), ΔC1C2(2255–2428), ΔC3(2431–2494), ΔC3C4-(2400–2515) and ΔC3C6-(2400–2662) also failed to secrete at significant levels. This data suggest that in the full length VWF molecule an intact sequence of C-domains is required for proper expression. To establish if the cysteine mutants could be expressed in smaller VWF constructs we introduced the same mutations into VWF molecules spanning the A2-CK, A2C6 and C3-CK domains. Interestingly, the mutants failed to express in VWF-A2CK and A2C6, again being retained in the ER, but were secreted in VWF-C3CK although to a significantly less extent than wild type C3CK. Furthermore, the A2C6 construct only expressed as monomers with very few dimers. Together these data demonstrate that correct disulphide bonding and an intact series of C domains are required for passage through the ER into the Golgi and efficient VWF secretion. The location and mechanism by which certain disulphide bonds break, forming free thiols remains to be established. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Altering Catalytic Properties of 3-Chlorocatechol-Oxidizing Extradiol Dioxygenase fromSphingomonas xenophaga BN6 by Random Mutagenesis

2001 ◽  
Vol 183 (7) ◽  
pp. 2322-2330 ◽  
Author(s):  
Ulrich Riegert ◽  
Sibylle Bürger ◽  
Andreas Stolz
Keyword(s):  
Amino Acid ◽  
Random Mutagenesis ◽  
Ring Cleavage ◽  
Amino Acid Substitutions ◽  
The Novel ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Extradiol Dioxygenase ◽  
Mutant Forms ◽  
Sphingomonas Xenophaga

ABSTRACT The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.

scholarly journals Cancer-associated variants of human NQO1: impacts on inhibitor binding and cooperativity

2019 ◽  
Vol 39 (9) ◽  
Author(s):  
Clare F. Megarity ◽  
David J. Timson
Keyword(s):  
Thermal Denaturation ◽  
Hill Coefficient ◽  
Detectable Effect ◽  
Negative Cooperativity ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Quinone Oxidoreductase ◽  
Inhibitor Binding ◽  
Dt Diaphorase ◽  
The Hill

Abstract Human NAD(P)H quinone oxidoreductase (DT-diaphorase, NQO1) exhibits negative cooperativity towards its potent inhibitor, dicoumarol. Here, we addressed the hypothesis that the effects of the two cancer-associated polymorphisms (p.R139W and p.P187S) may be partly mediated by their effects on inhibitor binding and negative cooperativity. Dicoumarol stabilized both variants and bound with much higher affinity for p.R139W than p.P187S. Both variants exhibited negative cooperativity towards dicoumarol; in both cases, the Hill coefficient (h) was approximately 0.5 and similar to that observed with the wild-type protein. NQO1 was also inhibited by resveratrol and by nicotinamide. Inhibition of NQO1 by resveratrol was approximately 10,000-fold less strong than that observed with the structurally similar enzyme, NRH quinine oxidoreductase 2 (NQO2). The enzyme exhibited non-cooperative behaviour towards nicotinamide, whereas resveratrol induced modest negative cooperativity (h = 0.85). Nicotinamide stabilized wild-type NQO1 and p.R139W towards thermal denaturation but had no detectable effect on p.P187S. Resveratrol destabilized the wild-type enzyme and both cancer-associated variants. Our data suggest that neither polymorphism exerts its effect by changing the enzyme’s ability to exhibit negative cooperativity towards inhibitors. However, it does demonstrate that resveratrol can inhibit NQO1 in addition to this compound’s well-documented effects on NQO2. The implications of these findings for molecular pathology are discussed.

scholarly journals Identification of essential histidine residues in UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T1

1997 ◽  
Vol 328 (1) ◽  
pp. 193-197 ◽  
Author(s):  
Stephanie WRAGG ◽  
K. Fred HAGEN ◽  
A. Lawrence TABAK
Keyword(s):  
Recombinant Proteins ◽  
Initial Rate ◽  
Initial Step ◽  
Enzymic Activity ◽  
Histidine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Histidine Residues ◽  
Sds Page ◽  
Enzyme Biosynthesis

UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) catalyse the initial step of mucin-type O-glycosylation. The activity of bovine ppGaNTase-T1 isoenzyme was inhibited by diethyl pyrocarbonate (DEPC) modification. Activity was partially restored by hydroxylamine treatment, indicating that one of the reactive residues was a histidine. The transferase was protected against DEPC inactivation when UDP-GalNAc and EPO-G, a peptide pseudo-substrate PPDAAGAAPLR, were simultaneously present, while presence of EPO-G alone did not alter DEPC inactivation. However, inclusion of UDP-GalNAc alone potentiated DEPC-inhibition of the enzyme, suggesting that UDP-GalNAc binding changes the accessibility or reactivity of an essential histidine residue. Deletion of the first 56 amino acids (including one hisitidine residue) yielded a fully active secreted form of the bovine ppGaNTase-T1 enzyme. Each of the 14 remaining histidines in the enzyme were mutated to alanine, and the recombinant mutants were recovered from COS7 cells. The mutation of histidine residues His211 → Ala and His344 → Ala resulted in recombinant proteins with no detectable enzymic activity. A significant decrease in the initial rate of GalNAc transfer to the substrate was observed with mutants His125 → Ala and His341 → Ala (1% and 6% of wild-type activity respectively). Mutation of the remaining ten histidine residues yielded mutants that were indistinguishable from the wild-type enzyme. Mutagenesis and SDS/PAGE analysis of all N-glycosylation sequons revealed that positions N-95 and N-552 are occupied by N-linked sugars in COS7 cells. Ablation of either site did not perturb enzyme biosynthesis or enzyme activity.

scholarly journals A subunit interface mutant of yeast pyruvate kinase requires the allosteric activator fructose 1,6-bisphosphate for activity

1995 ◽  
Vol 310 (1) ◽  
pp. 117-123 ◽  
Author(s):  
R A Collins ◽  
T McNally ◽  
L A Fothergill-Gilmore ◽  
H Muirhead
Keyword(s):  
Pyruvate Kinase ◽  
Mutant Enzyme ◽  
Hill Coefficient ◽  
Kinetic Properties ◽  
Variant Form ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Kinase Gene ◽  
Fructose 1,6 Bisphosphate ◽  
Allosteric Properties

A variant form of yeast pyruvate kinase (EC 2.7.1.40) with Ser-384 mutated to proline has been engineered in order to study the allosteric properties of this enzyme. Both the mutant and wild-type enzymes were overexpressed in a strain of yeast in which the genomic copy of the pyruvate kinase gene had been disrupted by an insertion of the Ura3 gene. Both enzymes were purified to homogeneity and their kinetic properties characterized. The wild-type enzyme displays sigmoid kinetics with respect to phosphoenolpyruvate (PEP) concentration, and is activated by the allosteric effect fructose 1,6-bisphosphate with concomitant reduction in co-operativity. In contrast, the mutant was found to be dependent on the presence of the effector for catalytic activity and was inactive in its absence. The fully activated mutant enzyme had a kcat. 1.6 times greater than that of the wild-type enzyme. The mutation introduced into the enzyme is in an intersubunit contact which is known to be critical for the allosteric properties of the enzyme, and is far removed from the active site. The major effect of the mutation seems to be to stabilize the low-affinity T state of the apoenzyme, although kcat. is also affected. The S0.5 for PEP and S0.5 for ADP of the wild-type enzyme were 0.22 +/- 0.004 and 0.15 +/- 0.01 mM respectively (means +/- S.E.M.). In the activated mutant enzyme, these kinetic parameters increased to 0.67 +/- 0.03 and 0.43 +/- 0.03 mM respectively. The cooperativity between ADP-binding sites was altered in the mutant enzyme, with the Hill coefficient (h) for ADP increasing to 1.65 +/- 0.07 in the presence of the effector, compared with a value of 0.01 +/- 0.07 for the wild-type enzyme under the same conditions. CD spectroscopy revealed the secondary structure of the mutant enzyme to be little different from that of the wild-type enzyme, indicating that the two enzymes have similar secondary structures in solution. Precise tertiary and quaternary structures such as intersubunit and interdomain interactions may be modified. An improved purification procedure has been devised that allows large quantities of enzyme to be rapidly prepared.

scholarly journals Dual Lipid Modification of the Yeast Gγ Subunit Ste18p Determines Membrane Localization of Gβγ

1999 ◽  
Vol 19 (11) ◽  
pp. 7705-7711 ◽  
Author(s):  
Jodi E. Hirschman ◽  
Duane D. Jenness
Keyword(s):  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cysteine Residue ◽  
Wild Type ◽  
Molecular Weights ◽  
Sds Page ◽  
Membrane Attachment ◽  
The Stability ◽  
Mutant Forms

ABSTRACT The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein. The Gβγ subunit (a complex of Ste4p and Ste18p) is associated with both internal and plasma membranes, and a portion is not stably associated with either membrane fraction. Like Ras, Ste18p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmitoylation. Mutant Ste18p containing serine at position 106 (mutation ste18-C106S) migrated more rapidly than wild-type Ste18p during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic mobility of wild-type Ste18p (but not the mutant Ste18p) was sensitive to hydroxylamine treatment, consistent with palmitoyl modification at Cys 106. Furthermore, immunoprecipitation of the Gβγ complex from cells cultured in the presence of [3H]palmitic acid resulted in two radioactive species on nonreducing SDS-PAGE gels, with molecular weights corresponding to Gγ and Gβγ. Substitution of serine for either Cys 107 or Cys 106 resulted in the failure of Gβγ to associate with membranes. The Cys 107 substitution also resulted in reduced steady-state accumulation of Ste18p, suggesting that the stability of Ste18p requires modification at Cys 107. All of the mutant forms of Ste18p formed complexes with Ste4p, as assessed by coimmunoprecipitation. We conclude that tight membrane attachment of the wild-type Gβγ depends on palmitoylation at Cys 106 and prenylation at Cys 107 of Ste18p.

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