Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA

Protein N-myristoyltransferase by co-expressing it with mammalian G protein α subunits in Escherichia coli

Robert J. Duronio, David A. Rudnick, Steven P. Adams, Dwight A. Towler, Jeffrey I. Gordon

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

A dual plasmid system was used to examine the protein and acyl-CoA specificities of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by co-expressing it in Escherichia coli with each of four homologous a subunits of the signal-transducing, heterotrimeric G proteins. Exogenous [3H]myristate was incorporated into rat Giα1 and rat G but not into bovine G or human G. Oxygen for methylene group substitutions in myristate result in analogs with comparable chain length and stereochemistry but marked reductions in hydrophobicity. Metabolic labeling studies with 6-, 11-, or 13-[3H]oxatetradecanoic acid indicated that they were incorporated into rat Giα1 and G with an efficiency that could be correlated with their accumulation into E. coli and their interactions with purified NMT in vitro. Octapeptides derived from the NH2-terminal sequences of these four Gα polypeptides were tested as substrates for purified S. cerevisiae NMT. None were bound by the enzyme. Acidic residues at positions 7 and 8 appear to contribute to this effect; deletion of these two amino acids or addition of the next 9 residues of rat G produced active substrates. These results imply that productive interactions between NMT and Gα protein substrates in vivo require structural features that are not fully represented within their NH2-terminal 8 residues.

Original languageEnglish (US)
Pages (from-to)10498-10504
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number16
StatePublished - Dec 1 1991

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Protein Subunits
Substrate Specificity
GTP-Binding Proteins
Yeast
Escherichia coli
Saccharomyces cerevisiae
Rats
Myristic Acid
Substrates
Heterotrimeric GTP-Binding Proteins
Acyl Coenzyme A
Stereochemistry
Hydrophobicity
Hydrophobic and Hydrophilic Interactions
Chain length
Labeling
Plasmids
Substitution reactions
Oxygen
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA : Protein N-myristoyltransferase by co-expressing it with mammalian G protein α subunits in Escherichia coli. / Duronio, Robert J.; Rudnick, David A.; Adams, Steven P.; Towler, Dwight A.; Gordon, Jeffrey I.

In: Journal of Biological Chemistry, Vol. 266, No. 16, 01.12.1991, p. 10498-10504.

Research output: Contribution to journalArticle

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