Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria.

Duronio, Robert J.; Jackson-Machelski, Emily; Heuckeroth, Robert O.; Olins, Peter O.; Devine, Catherine S.; Yonemoto, Wes; Slice, Lee W.; Taylor, Susan S.; Gordon, Jeffrey I.

doi:10.1073/pnas.87.4.1506

Cited by 241 publications

(148 citation statements)

References 36 publications

(34 reference statements)

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“…is unstructured when the N-terminal Gly is not myristyThe crystal structure explains how this is accomplished. lated (Duronio et al, 1990). When the C-subunit and Instead of associating with a membrane, the acyl group NMT are coexpressed in E. coli, the recombinant enzyme folds into the protein, making important contacts with is fully myristylated.…”

Section: )mentioning

confidence: 99%

Crystal structures of the myristylated catalytic subunit of cAMP‐dependent protein kinase reveal open and closed conformations

et al. 1993

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Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of CAMP-dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32,2154-2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 A resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C-terminal30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surrounding the glycine-rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N-terminal40 residues that lie outside the conserved catalytic core are anchored by the N-terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.

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Section: )mentioning

confidence: 99%

Crystal structures of the myristylated catalytic subunit of cAMP‐dependent protein kinase reveal open and closed conformations

et al. 1993

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“…This approach allowed a high expression of the protein in Escherichia coli (86 mg/liter of culture medium). For the myristoylated protein, a pBB131 vector containing yeast N-myristoyltransferase cDNA was co-transformed (21). Both proteins were purified from the heat-stable protein fractions by successive column chromatography on Phenyl-Sepharose and Resource RPC (Amersham Pharmacia Biotech).…”

Section: Methodsmentioning

confidence: 99%

Identification of the Calmodulin-binding Domain of Neuron-specific Protein Kinase C Substrate Protein CAP-22/NAP-22

Takasaki

Hayashi

Matsubara

et al. 1999

Journal of Biological Chemistry

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Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated. Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain. In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain. Phosphorylation of a single serine residue in the N-terminal domain (Ser 5 ) by protein kinase C abolished the binding. Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.

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“…However, ARF3 expressed as a fusion protein with a GAL4-binding domain cannot be modified, because N-myristoyltransferase can only act at the N terminus (21). Therefore, it is presumed that the interaction of ARF3⅐Q71L with clone 1 or clone 2 in yeast is independent of myristoylation.…”

Section: Identification Of An Arf3-interacting Protein By Yeast Two-mentioning

confidence: 99%

“…21) and selected for both ampicillin and kanamycin resistance. Transformed cells were grown at 37°C to A 600 ϭ 0.6, and protein expression was induced with 1 mM isopropyl-1-thio-␤-D-galactopyranoside in the presence of 200 M sodium myristate for 3 h at 27°C.…”

Section: The Nucleotide Sequence(s) Reported In This Paper Has Been Smentioning

confidence: 99%

Arfaptin 1, a Putative Cytosolic Target Protein of ADP-ribosylation Factor, Is Recruited to Golgi Membranes

Kanoh

Williger

Exton

1997

Journal of Biological Chemistry

111

101

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ADP-ribosylation factors (ARFs) have been implicated in vesicle transport in the Golgi complex. Employing yeast two-hybrid screening of an HL60 cDNA library using a constitutively active mutant of ARF3 (ARF3⅐Q71L), as a probe, we have identified a cDNA encoding a novel protein with a calculated molecular mass of 38.6 kDa, which we have named arfaptin 1. The mRNA of arfaptin 1 was ubiquitously expressed, and recombinant arfaptin 1 bound preferentially to class I ARFs, especially ARF1, but only in the GTP-bound form. The interactions were independent of myristoylation of ARF. Arfaptin 1 in cytosol was recruited to Golgi membranes by ARF in a guanosine 5 -O-(3-thiotriphosphate)-dependent and brefeldin A-sensitive manner. When expressed in COS cells, arfaptin 1 was localized to the Golgi complex. The yeast two-hybrid system yielded another clone, which encoded a putative protein, which we have named arfaptin 2. This consisted of the same number of amino acids as arfaptin 1 and was 60% identical to it. Arfaptin 2 was also ubiquitously expressed and bound to the GTP-, but not GDP-liganded form of class I ARFs, especially ARF1. These results suggest that arfaptins 1 and 2 may be direct target proteins of class 1 ARFs. Arfaptin 1 may be involved in Golgi function along with ARF1.

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Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria.

Cited by 241 publications

References 36 publications

Crystal structures of the myristylated catalytic subunit of cAMP‐dependent protein kinase reveal open and closed conformations

Crystal structures of the myristylated catalytic subunit of cAMP‐dependent protein kinase reveal open and closed conformations

Identification of the Calmodulin-binding Domain of Neuron-specific Protein Kinase C Substrate Protein CAP-22/NAP-22

Arfaptin 1, a Putative Cytosolic Target Protein of ADP-ribosylation Factor, Is Recruited to Golgi Membranes

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