Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation.

Johnson, D R; Cok, Steven J.; Feldmann, Horst; Gordon, Jeffrey I.

doi:10.1073/pnas.91.21.10158

Cited by 21 publications

(20 citation statements)

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“…Defining the levels of N-myristoylation of cellular Arfs in 5. cerevisiae strains with wild-type and mutant NMTl alleles Temperature-sensitive mutants of NMTZ have proven to be valuable tools for studying the regulation of protein N-myristoylation in S. cerevisiae (Duronio et al, 1991;Johnson et al, 1993Johnson et al, , 1994b. As in C. albicans, substitution of an Asp for the conserved Glyg51 located five residues from the C-terminus of the enzyme causes a temperature-sensitive reduction in the affinity of nmt451Dp for myristoyl-CoA.…”

Section: Resultsmentioning

confidence: 99%

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N-Myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA:protein N-myristoyltransferase

Lodge

Jackson-Machelski

Devadas³

et al. 1997

Microbiology

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Myristoyl-CoA: protein N-myristoyltransf erase (Nmt) catalyses the covalent attachment of myristate to the N-terminal glycine of a small subset of cellular proteins produced during vegetative growth of Candida albicans. nmt447D is a mutant NMT allele encoding an enzyme with a G l F 7 4 Asp substitution and reduced affinity for myristoyl-CoA. Among isogenic NMTINMT, NMTlhnmt and nmthlnmt447D strains, only nmthlnmt447D cells require myristate for growth on yeast/peptone/dextrose media (YPD) at 24 or 37 "C. When switched from YPD/myristate to YPD alone, 60% of the organisms die within 4 h.Antibodies raised against the C-terminal eight residues of Saccharomyces cerevisiae A r f l p were used to probe Western blots of total cellular proteins prepared from these isogenic Candida strains. N-Myristoylation of C. albicans ADP-ribosylation factor (Arf) produced a change in its electrophoretic mobility during SDS-PAGE : the myristoylated species migrated more rapidly than the nonmyristoylated species. In an NMrhmtA strain, 100% of the Arf is Nmyristoylated based on this mobility shift assay. When exponentially growing nmtMnmt447D cells were incubated at 24 "C in YPDfmyristate, < 25% cellular Arf was nonmyristoylated. In contrast, 2 or 4 h after withdrawal of myristate, 2 50 YO of total cellular Arf was nonmyristoylated. This finding suggests that 2 5 0 % reduction in Arf N-myristoylation is a biochemical marker of a growtharrested cell. A similar conclusion was made after assaying isogenic 5. cerevisiae strains containing various combinations of NMT7, nmt1=451D, ARF1, arflA, ARF2 and ad2A alleles and grown at 24-37 "C on YPD or YPD/myristate.Peptidomimetic inhibitors of C. albicans Nmt were synthesized based on the Nterminal sequence of an 5. cerevisiae Arf. SC-59383 has an I C, , of 145 k0.08 pM for purified C. albicans Nmt and is 560-fold selective for the fungal compared to human N-myristoyltransferase. It had an EC, , of 51 f 17 and 67+6 pM, 24 and 48 h after a single administration of the drug to cultures of C. albicans. The Atf gel mobility shift assay indicated that a single dose of 200 pM produced a < 50% reduction in Arf N-myristoylation after 4 h, which is consistent with the fungistatic, but not fungicidal, activity. The effect on Nmt was specific: an enantiomer, SC-59840, had no inhibitory effect on purified C. albicans Nmt (I C, > 1000 pM), and 200 pM of the compound produced no detectable reduction in Arf N-myristoylation in vivo. SC-58272, which is related to SC-59383, was a more potent inhibitor in vitro (IC,, 0056 &0*01 pM), but had no growth inhibitory activity and did not produce any detectable reduction in Arf J. K. LODGE a n d OTHERS N-myristoylation. These findings highlight the utility of the Arf protein gel mobility shift assay for demonstrating the mechanism-based antifungal activity of SC-59383, a selective inhibitor of C. albicans Nmt.

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Section: Resultsmentioning

confidence: 99%

“…Growth arrest occurs within 1 h after shifting to 30-37 "C. Cells remain viable at 37 "C for 8 h. Death begins within 12 h, and no viable cells are detectable after 24 h (Johnson et al, 1994b). Steady state levels of Nmtlp and nmt451Dp are comparable in isogenic strains 1 and 4 h after shifting to 37 "C (Johnson et al, 199413).…”

Section: Resultsmentioning

confidence: 99%

N-Myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA:protein N-myristoyltransferase

Lodge

Jackson-Machelski

Devadas³

et al. 1997

Microbiology

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“…Protein N-myristoylation has been shown to be crucial for the survival of yeast, Drosophila, and murine embryos (14,31,32). Myristoylated proteins play an important role in many cellular processes and signaling pathways, including the TCR cascade (10).…”

Section: Discussionmentioning

confidence: 99%

Immunosuppression and Aberrant T Cell Development in the Absence of N-Myristoylation

Rampoldi

Bonrouhi

Boehm

et al. 2015

The Journal of Immunology

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N-myristoylation refers to the attachment of myristic acid to the N-terminal glycine of proteins and substantially affects their intracellular targeting and functions. The thymus represents an organ with a prominent N-myristoylation activity. To elucidate the role of protein N-myristoylation for thymocyte development, we generated mice with a T cell lineage–specific deficiency in N-myristoyl transferase (Nmt)1 and 2. Depletion of Nmt activity in T cells led to a defective transmission of TCR signals, a developmental blockage of thymocytes at the transition from double-negative 3 to 4 stages, and a reduction of all the following stages. We could demonstrate that Lck and myristoylated alanine-rich C kinase substrate, two main myristoylated kinases in T cells, were mislocalized in the absence of Nmt activity. N-myristoylation was also indispensable for early and distal TCR signaling events such as CD3ζ, Zap70, and Erk activation and for release of cytokines such as IFN-γ and IL-2. As a consequence, the initiation and propagation of the TCR signaling cascade was severely impaired. Furthermore, we showed that the absence of myristoylation had an immunosuppressive effect on T cells in vivo after treatment with CpG and stimulation of the TCR with the staphylococcal enterotoxin B superantigen. Therefore, protein myristoylation is indispensable in T cell development and activation and its inhibition might offer a novel strategy to achieve immunosuppression.

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“…Pheromone-induced gene expression was assayed using a plasmid (pRS424-FUS1-lacZ [Johnson et al, 1994]) that contains the pheromone-inducible reporter FUS1-lacZ. Cells in early logarithmic phase (A 600 , 0.35-0.5) were treated with various concentrations of ␣-factor for 2 h. ␤-Galactosidase activity in permeabilized cells was determined (McCaffrey et al, 1987) Gpa1p protein levels were determined by immunoblot of 100 g of yeast total cell lysate from CMY23 and CMY24.…”

Section: Phenotypic Analysis Of Mutantsmentioning

confidence: 99%

Dual Lipid Modification Motifs in G_αand G_γSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

Manahan

Patnana

Blumer

et al. 2000

MBoC

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To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide-binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein ␥ subunit (Ste18p) is unusual among G ␥ subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation-or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the G ␥ subunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of G ␤␥ after receptor-stimulated release from G ␣ . The G protein ␣ subunit (Gpa1p) is tandemly modified at its N terminus with amide-and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway. INTRODUCTIONLipid modifications anchor heterotrimeric guanine nucleotide-binding proteins (G proteins) to the inner leaflet of the plasma membrane. G protein ␣ subunits are fatty acylated with amide-linked myristate, thioester-linked palmitate, or both. G protein ␥ subunits are prenylated with either farnesyl or geranylgeranyl moieties through stable thioether linkages. Prenylation of ␥ subunits and myristoylation of ␣ subunits are essential for the function of G proteins. These modifications promote plasma membrane association and facilitate high-affinity protein-protein interactions (reviewed in Wedegaertner et al., 1995). The functional consequences of thioacylation are less well understood. Thioacylation does not appear to be a major determinant of membrane avidity, at least in the presence of G ␤␥ subunits, but may play a role in targeting G ␣ specifically to the plasma membrane (Dunphy et al., 1996;Morales et al., 1998;Fishburn et al., 1999;Huang et al., 1999). In vitro studies have demonstrated the importance of thioester-linked lipid in mediating protein-protein interactions of G ␣ subunits. Thioacylation increases the affinity of G s␣ for G ␤␥ approximately fivefold (Iiri et al., 1996) and negatively regulates the interaction between regulators of G protein signaling and G ␣ subunits (Tu et al., 1997). Thus, thioacylation may impact protein-protein interactions, as well as the subcellular distribution of modified proteins.We have investigated thioacylation of G proteins in the genetically tractable organism Saccharomyces cerevisiae. The pheromone res...

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Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation.

Cited by 21 publications

References 28 publications

N-Myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA:protein N-myristoyltransferase

N-Myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA:protein N-myristoyltransferase

Immunosuppression and Aberrant T Cell Development in the Absence of N-Myristoylation

Dual Lipid Modification Motifs in G_αand G_γSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

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Suppressors of nmtl-181, a conditional lethal allele of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene, reveal proteins involved in regulating protein N-myristoylation.

Cited by 21 publications

References 28 publications

N-Myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA:protein N-myristoyltransferase

N-Myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA:protein N-myristoyltransferase

Immunosuppression and Aberrant T Cell Development in the Absence of N-Myristoylation

Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

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Dual Lipid Modification Motifs in G_αand G_γSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae