Purification, Functional Reconstitution, and Characterization of the Saccharomyces cerevisiae Isoprenylcysteine Carboxylmethyltransferase Ste14p

Anderson, Jessica; Frase, Hilary; Michaelis, Susan; Hrycyna, Christine A.

doi:10.1074/jbc.m410292200

Cited by 45 publications

(75 citation statements)

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“…The diffusion rate obtained for C 20 BAS was slower by a factor of five relative to POPC under the same conditions. When 30 mol% Chol was incorporated in the C 20 BAS membrane, the observed lateral diffusion rate decreased by an order of magnitude, giving a value similar to that obtained for the longer chain C 32 phytBAS derivative.…”

Section: Discussionmentioning

confidence: 76%

“…The impact of bolalipid structure and membrane composition on Ste14p activity, monitored by an in vitro vapor diffusion methyltransferase assay (20), was then determined using vesicles of differing lipid composition, gel to liquid crystalline phase transition temperatures, and lipid mobility. Confocal immunofluorescence microscopy and protein assays were used to determine the extent and distribution of Ste14p in reconstituted vesicle membranes.…”

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confidence: 99%

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Functional Reconstitution of the Integral Membrane Enzyme, Isoprenylcysteine Carboxyl Methyltransferase, in Synthetic Bolalipid Membrane Vesicles

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Three bipolar archaeal-type diglycerophosphocholine tetraether lipids (a.k.a., bolalipids) have been prepared to determine 1) the influence of molecular structure on the physical properties of bolalipid membranes and 2) their impact on the functional reconstitution of Ste14p, a membrane-associated isoprenylcysteine carboxyl methyltransferase from Saccharomyces cerevisiae. The three bolalipids synthesized were: C 20 BAS, C 32 BAS, and C 32 phytBAS. These bolalipid structures differ in that the C 20 BAS derivative has a short sn-1 glyceryl diether C 20 H 40 transmembrane alkyl chain and two ether-linked sn-2 n-decyl chains, whereas the C 32 BAS and C 32 phytBAS derivatives have a longer sn-1 diether C 32 H 64 membrane-spanning chain and two ether-linked sn-2 n-hexadecyl or phytanyl chains, respectively. Differential scanning calorimetry and temperature-dependent 31 P NMR was used to determine the gel-to-liquid crystalline phase transition temperatures of the bolalipids (C 32 BAS T m > 85 °C; C 32 phytBAS T m = 14 °C; C 20 BAS T m = 17°C). The bolalipid lateral diffusion coefficients, determined by fluorescence recovery after photobleaching at 20 °C, were 1.5 × 10 −8 and 1.8 × 10 −9 cm 2 /s for C 20 BAS and C 32 phytBAS, respectively. The mobility of C 32 BAS could not be measured at this temperature. Ste14p activity was monitored by an in vitro methyltransferase assay in reconstituted vesicle dispersions composed of DMPC, C 20 BAS:E. coli polar lipid, C 20 BAS:POPC, C 32 phytBAS:E. coli polar lipid, and C 32 phytBAS:POPC. Ste14p activity was lost in vesicles composed of 75-100 mol% C 20 BAS and 0-100 mol% C 32 BAS, but retained in vesicles with 0-50 mol% C 20 BAS and 0-100 mol% C 32 phytBAS. Confocal immunofluorescence microscopy confirmed the presence of Ste14p in 100 mol% C 20 BAS and 100 mol% C 32 phytBAS vesicle dispersions, even though the lamellar liquid crystalline phase thickness of C 20 BAS is only 32 Å. Since Ste14p activity was not affected by either the gel to liquid-crystal phase transition temperature of the lipid or the temperature of the assay, the low activity observed in 75-100 mol% C 20 BAS membranes can be attributed to hydrophobic mismatch between this bolalipid and the hydrophobic surface of Ste14p.Bolalipids, also called bolaamphiphiles, are a class of bipolar lipids found in the cell membranes of some Archaea. In many cases, these organisms can survive in extreme environments due to the presence of isoprenoid-based tetraether bolalipids in their membranes (1-5). The membrane-spanning bipolar structural motif confers increased stability to these membranes by effectively cross-linking the apposed leaflets of a bilayer membrane, thereby producing a monolayer membrane that functionally mimics a conventional membrane bilayer. † This work was supported by NIH CA112427, the NIH "Diversity in Biomedical Science Program" at Purdue University, and the Indiana 21 st Century Fund.Correspondence to: Christine A. Hrycyna; David H. Thompson. NIH Public Access Author ManuscriptBiochemistry. Author ...

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Section: Discussionmentioning

confidence: 76%

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Functional Reconstitution of the Integral Membrane Enzyme, Isoprenylcysteine Carboxyl Methyltransferase, in Synthetic Bolalipid Membrane Vesicles

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“…Because of its membrane spans and because it lacks a classical methyltransferase consensus motif, Icmt is an atypical member of the methyltransferase family of enzymes 40 . However, the recent purification of Ste14p from yeast, and its reconstitution in liposomes in an enzymatically active form, have provided conclusive evidence that it is the sole component comprising Icmt activity 40,41 . Using the purified enzyme, researchers showed that yeast Ste14p recognizes the farnesylated and geranylgeranyl substrates N-acetyl-S-farnesylcysteine and N-acetyl-S-geranylgeranylcysteine equivalently.…”

Section: Enzymatic Processing Of Prenylated Proteinsmentioning

confidence: 99%

“…Given the importance of carboxyl methylation for Ras-induced onco-genesis shown by the Icmt mouse studies mentioned above, the development of Icmt inhibitors as anticancer drugs is of obvious interest 19,41,47 . The well-known chemotherapeutic compound methotrexate (an antifolate that blocks thymidine synthesis, which is necessary for tumor growth) causes accumulation of S-adenosylhomocysteine, a potent inhibitor of nearly all cellular methyltransferases; it has been suggested that at least some of methotrexate's antitumor activity may stem from its ability to inhibit Icmt, though specificity for Icmt is lacking 48 .…”

Section: Enzymatic Processing Of Prenylated Proteinsmentioning

confidence: 99%

“…Proposed roles for the carboxyl methylation of prenylated proteins, for which there is evidence in particular cases, include influencing intracellular protein localization, membrane attachment, metabolic stability and interactions with other proteins 19,20,45,46 . Although carboxyl methylation of prenylated proteins has been suggested to be reversible, and therefore could represent a regulatory event, evidence for this reversibility in cells has not been forthcoming.Given the importance of carboxyl methylation for Ras-induced onco-genesis shown by the Icmt mouse studies mentioned above, the development of Icmt inhibitors as anticancer drugs is of obvious interest 19,41,47 . The well-known chemotherapeutic compound methotrexate (an antifolate that blocks thymidine synthesis, which is necessary for tumor growth) causes accumulation of S-adenosylhomocysteine, a potent inhibitor of nearly all cellular methyltransferases; it has been suggested that at least some of methotrexate's antitumor activity may stem from its ability to inhibit Icmt, though specificity for Icmt is lacking 48 .…”

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Therapeutic intervention based on protein prenylation and associated modifications

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In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of 15-carbon farnesyl groups or 20-carbon geranylgeranyl groups that function both as anchors for fixing proteins to membranes and as molecular handles for facilitating binding of these lipidated proteins to other proteins. Additional modification of these prenylated proteins includes C-terminal proteolysis and methylation, and attachment of a 16-carbon palmitoyl group; these modifications augment membrane anchoring and alter the dynamics of movement of proteins between different cellular membrane compartments. The enzymes in the protein prenylation pathway have been isolated and characterized. Blocking protein prenylation is proving to be therapeutically useful for the treatment of certain cancers, infection by protozoan parasites and the rare genetic disease Hutchinson-Gilford progeria syndrome.Isoprenoids are lipids made of five-carbon blocks, and they constitute one of the main classes of natural products. A subset of isoprenoids called prenyl groups are found on a variety of biological substances, including proteins. Prenylated proteins and peptides are found in most (if not all) eukaryotes. They arise from the post-translational attachment of 15-carbon farnesyl or 20-carbon geranylgeranyl groups to the C-terminal segment of proteins. Protein prenyl groups not only are hydrophobic elements that bind proteins to membranes, but, at least in some cases, they also function as molecular handles that bind to hydrophobic grooves on the surface of soluble protein factors; these factors then remove the prenylated protein from the membrane in a regulated manner. NIH Public Access Author ManuscriptNat Chem Biol. Author manuscript; available in PMC 2010 June 28. Published in final edited form as:Nat Chem Biol. 2006 October ; 2(10): 518-528. doi:10.1038/nchembio818. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptProtein prenylation has been vigorously studied over the past ~15 years because it is found on several signaling proteins (including heterotrimeric G proteins) that connect cell surface receptors to intracellular effectors, and also on Ras proteins, one of the most common oncoproteins found in human tumors. Ras proteins serve as molecular switches at cellular membranes to control propagation of growth signals from cell surface receptors to nuclear transcription factors. Because Ras mutations are important in many human cancers, there has been extensive focus on Ras prenylation. Discovery of protein prenyl groups and structural varietiesThe first reports of prenylated proteins and peptides described the secreted pheromone peptides from jelly fungi 1,2 , whose structure resembles that of the well-known a-factor mating pheromone from baker's yeast (Saccharomyces cerevisiae), which contains a cysteine methylester farnesylated at the C terminus 3 . In the 1980s, studies on the timing of cholesterol biosynthesis with respect to the cell cycle in human cells led to the discovery that a compound deriv...

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Liver prenylated methylated protein methyl esterase is an organophosphate-sensitive enzyme

Lamango

2005

J. Biochem. Mol. Toxicol.

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Prenylation and subsequent methylation are essential modifications on a significant proportion of eucaryotic proteins. Proteins such as the G-gamma subunits of G-protein coupled receptors, nuclear lamins, and guanine nucleotide-binding proteins such as Ras are prenylated and undergo methylation. Prenylated methylated protein methyl esterase (PMPMEase) readily hydrolyses the prenylated protein methyl esters, thus making this step reversible and possibly regulatory. Benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM) was developed as a specific PMPMEase substrate and characterized by electron spray ionization mass spectrometry (ESI-MS) to be of the calculated molecular mass. Rat liver and brain PMPMEase hydrolyzed BzGFCM, forming benzoyl-glycyl-farnesyl-cysteine (BzGFC) in a time- and concentration-dependent manner. Both enzymes cleaved BzGFCM with K(m) values of 4.58 +/- 0.30 and 25.57 +/- 2.36 microM and V(max) values of 2.21 +/- 0.03 and 0.17 +/- 0.003 nmol/min/mg, respectively. The liver enzyme eluted from a gel-filtration column as a single peak of apparent size, 89 kDa. The brain enzyme eluted as two main peaks of 53 and 890 kDa. Organophosphorus pesticides (OPs), which are suspected to be involved in human disorders such as parkinsonism, neuronal, and retinal degeneration, inhibited the liver enzyme with IC(50) values from 4.77 muM for parathion to 0.04 microM for paraoxon, respectively. Only about 25% of the brain enzyme was inhibited by 0.5-1 mM solutions of mipafox, while 0.1 and 1 mM paraoxon inhibited over 50% and 95% of the enzyme, respectively. Paraoxon is thus about 2,250 times less potent against the brain than the liver PMPMEase. BzGFCM was not hydrolyzed by various cholinesterases, indicating its specificity for PMPMEase. Perturbations in prenylated protein metabolism might play a role in noncholinergic OPs-induced toxicity, since prenylated proteins play such important roles in cell signaling, proliferation, differentiation, and apoptosis.

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Purification, Functional Reconstitution, and Characterization of the Saccharomyces cerevisiae Isoprenylcysteine Carboxylmethyltransferase Ste14p

Cited by 45 publications

References 48 publications

Functional Reconstitution of the Integral Membrane Enzyme, Isoprenylcysteine Carboxyl Methyltransferase, in Synthetic Bolalipid Membrane Vesicles

Functional Reconstitution of the Integral Membrane Enzyme, Isoprenylcysteine Carboxyl Methyltransferase, in Synthetic Bolalipid Membrane Vesicles

Therapeutic intervention based on protein prenylation and associated modifications

Liver prenylated methylated protein methyl esterase is an organophosphate-sensitive enzyme

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