Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system

Kanno, Takeshi; Kitano, Michiko; Kato, Ryuichi; Omori, Akira; Endo, Yaeta; Tozawa, Yuzuru

doi:10.1016/j.pep.2006.09.007

Cited by 15 publications

(13 citation statements)

References 37 publications

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“…We found that the translation products of AGG1 mutants containing Pro3 retained [ 35 S]Met, indicating that cleavage of the initiating Met by MAP did not occur. This result is thus consistent with our previous data obtained from analysis of the sequence specificity and efficiency of endogenous MAP activity in the wheat germ cell-free translation system [35]. The AGG1 mutant proteins containing Pro3 were not modified by N-myristoylation.…”

Section: Discussionsupporting

confidence: 93%

“…3B,C), for which Met residues are present at both positions 1 and 3, indicating that the initiating Met is retained in Myr-AGG1-3P6A and Myr-AGG1-3P6S. This result is in good agreement with our previous finding of an inhibitory effect of Pro at position 3 of full-length translation products on MAP function in the wheat germ cell-free translation system [35]. We also detected low levels of [ 35 S]Met incorporation in the translation products with Gly, Thr, Asp or Glu at position 3 of the myristoylation motif in Myr-AGG1-3X6A or Myr-AGG1-3X6S ( Fig.…”

Section: Relationship Between Initiator Met Elimination and N-myristosupporting

confidence: 91%

“…Given that myristic acid is attached to a Gly at the N‐terminus of a protein, the initiating Met must be cleaved by MAP prior to N‐myristoylation. We have previously shown that a Pro at position 3 markedly inhibits cleavage of the initiator Met by MAP, even if the penultimate amino acid is Ala, Cys, Gly, Pro, Ser, or Thr, all of which generally allow efficient Met removal from a translated polypeptide [35]. Before this finding, the antepenultimate amino acid had not been thought to affect the substrate selectivity of MAP.…”

Section: Discussionmentioning

confidence: 99%

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The consensus motif for N‐myristoylation of plant proteins in a wheat germ cell‐free translation system

Yamauchi¹,

Fusada²,

Hayashi³

et al. 2010

The FEBS Journal

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Protein N‐myristoylation plays key roles in various cellular functions in eukaryotic organisms. To clarify the relationship between the efficiency of protein N‐myristoylation and the amino acid sequence of the substrate in plants, we have applied a wheat germ cell‐free translation system with high protein productivity to examine the N‐myristoylation of various wild‐type and mutant forms of Arabidopsis thaliana proteins. Evaluation of the relationship between removal of the initiating Met and subsequent N‐myristoylation revealed that constructs containing Pro at position 3 do not undergo N‐myristoylation, primarily because of an inhibitory effect of this amino acid on elimination of the initiating Met by methionyl aminopeptidase. Our analysis of the consensus sequence for N‐myristoylation in plants focused on the variability of amino acids at positions 3, 6 and 7 of the motif. We found that not only Ser at position 6 but also Lys at position 7 affects the selectivity for the amino acid at position 3. The results of our analyses allowed us to identify several A. thaliana proteins as substrates for N‐myristoylation that had previously been predicted not to be candidates for such modification with a prediction program. We have thus shown that a wheat germ cell‐free system is a useful tool for plant N‐myristoylome analysis. This in vitro approach will facilitate comprehensive determination of N‐myristoylated proteins in plants.

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Section: Discussionsupporting

confidence: 93%

Section: Relationship Between Initiator Met Elimination and N-myristosupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

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The consensus motif for N‐myristoylation of plant proteins in a wheat germ cell‐free translation system

Yamauchi¹,

Fusada²,

Hayashi³

et al. 2010

The FEBS Journal

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“…This chapter describes the use of wheat germ extract, a highly stable and productive eukaryotic system for cell-free translation (Kanno et al , 2007; Madin et al , 2000). Since wheat germ extract is prepared from a eukaryote, it provides differences in the mechanisms for translation and folding of nascent proteins as compared to bacterial translation (Endo and Sawasaki, 2006).…”

Section: Overview Of Cell-free Translationmentioning

confidence: 99%

“…Some examples of added reagents include detergents to solubilize proteins (Klammt et al , 2007), liposomes to capture functional forms of membrane proteins (Goren and Fox, 2008; Nozawa et al , 2007), cofactors, coenzymes, and metal ions to reconstitute catalytic function (Abe et al , 2004; Boyer et al , 2008; Goren and Fox, 2008), multiple mRNAs to yield cotranslation of heteromeric proteins and protein–protein complexes (Matsumoto et al , 2008), or other enzymes to effect posttranslational modifications (Goerke and Swartz, 2008; Kanno et al , 2007). Moreover, many different types of amino acid (AA) labeling strategies can be accomplished in cell-free translation by a simple substitution of unlabeled AAs with AAs containing isotopic substitutions such as 2 H, 13 C, or 15 N for NMR or selenomethionine (SeMet) for determination of X-ray diffraction phasing (Klammt et al , 2004; Matsuda et al , 2007; Vinarov and Markley, 2005).…”

Section: Introductionmentioning

confidence: 99%

Chapter 37 Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes

Goren

Nozawa

Makino

et al. 2009

Methods in Enzymology

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Wheat germ cell-free translation is shown to be an effective method to produce integral membrane proteins in the presence of unilamelar liposomes. In this chapter, we describe the expression vectors, preparation of mRNA, two types of cell-free translation reactions performed in the presence of liposomes, a simple and highly efficient purification of intact proteoliposomes using density gradient ultracentrifugation, and some of the types of characterization studies that are facilitated by this facile preparative approach. The in vitro transfer of newly translated, membrane proteins into liposomes compatible with direct measurements of their catalytic function is contrasted with existing approaches to extract membrane proteins from biological membranes using detergents and subsequently transfer them back to liposomes for functional studies.

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Tools for analyzing and predicting N‐terminal protein modifications

Meinnel

Giglione

2008

Proteomics

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The vast majority of the proteins encoded in any genome naturally undergo a large number of different N-terminal modifications, hindering their characterization by routine proteomic approaches. These modifications are often irreversible, usually cotranslational and are crucial, as their occurrence may reflect or affect the status, fate and function of the protein. For example, large signal peptide cleavages and N-blocking mechanisms reflect targeting to various cell compartments, whereas N-ligation events tend to be related to protein half-life. N-terminal positional proteomic strategies hold promise as a new generation of approaches to the fine analysis of such modifications. However, further biological investigation is required to resolve problems associated with particular low-abundance or challenging N-terminal modifications. Recent progress in genomics and bioinformatics has provided us with a means of assessing the impact of these modifications in proteomes. This review focuses on methods for characterizing the occurrence and diversity of N-terminal modifications and for assessing their contribution to function in complete proteomes. Progress is being made towards the annotation of databases containing information for complete proteomes, and should facilitate research into all areas of proteomics.

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Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system

Cited by 15 publications

References 37 publications

The consensus motif for N‐myristoylation of plant proteins in a wheat germ cell‐free translation system

The consensus motif for N‐myristoylation of plant proteins in a wheat germ cell‐free translation system

Chapter 37 Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes

Tools for analyzing and predicting N‐terminal protein modifications

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