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N-myristoylation determines dual targeting of mammalian NADH-cytochrome b5 reductase to ER and mitochondrial outer membranes by a mechanism of kinetic partitioning.

Colombo S, Longhi R, Alcaro S, Ortuso F, Sprocati T, Flora A, Borgese N - J. Cell Biol. (2005)

Bottom Line: Mammalian NADH-cytochrome b5 reductase (b5R) is an N-myristoylated protein that is dually targeted to ER and mitochondrial outer membranes.We find that myristoylation interferes with interaction of the nascent chain with signal recognition particle, so that a portion of the nascent chains escapes from cotranslational integration into the ER and can be post-translationally targeted to the mitochondrial outer membrane.Thus, competition between two cotranslational events, binding of signal recognition particle and modification by N-myristoylation, determines the site of translation and the localization of b5R.

View Article: PubMed Central - PubMed

Affiliation: Consiglio Nazionale delle Ricerche Institute of Neuroscience, Cellular and Molecular Pharmacology Section and Department of Medical Pharmacology, University of Milan, 20129 Milan, Italy.

ABSTRACT
Mammalian NADH-cytochrome b5 reductase (b5R) is an N-myristoylated protein that is dually targeted to ER and mitochondrial outer membranes. The N-linked myristate is not required for anchorage to membranes because a stretch of hydrophobic amino acids close to the NH2 terminus guarantees a tight interaction of the protein with the phospholipid bilayer. Instead, the fatty acid is required for targeting of b5R to mitochondria because a nonmyristoylated mutant is exclusively localized to the ER. Here, we have investigated the mechanism by which N-linked myristate affects b5R targeting. We find that myristoylation interferes with interaction of the nascent chain with signal recognition particle, so that a portion of the nascent chains escapes from cotranslational integration into the ER and can be post-translationally targeted to the mitochondrial outer membrane. Thus, competition between two cotranslational events, binding of signal recognition particle and modification by N-myristoylation, determines the site of translation and the localization of b5R.

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Myristate stabilizes the α-helical conformation of b5R's NH2-terminal peptide. (A) CD spectra of b5R NH2-terminal peptide (G2-A33). Spectra were taken for the unmodified (blue), the N-myristoylated (red), or the N-acetylated (green) peptide, diluted in water (a), 50% TFE (b), or 25 mM SDS (c). In aqueous solution the N-myristoylated peptide has a spectrum indicative of a higher α-helical content than the other two peptides. (B) Result of GRID analysis with the methyl group probe. The b5R G2-A33 peptide in α-helical conformation is represented in wireframe. Contour surfaces with favorable interaction energies of the methyl probe (threshold = −10 kJ/mol) are shown in light blue.
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fig8: Myristate stabilizes the α-helical conformation of b5R's NH2-terminal peptide. (A) CD spectra of b5R NH2-terminal peptide (G2-A33). Spectra were taken for the unmodified (blue), the N-myristoylated (red), or the N-acetylated (green) peptide, diluted in water (a), 50% TFE (b), or 25 mM SDS (c). In aqueous solution the N-myristoylated peptide has a spectrum indicative of a higher α-helical content than the other two peptides. (B) Result of GRID analysis with the methyl group probe. The b5R G2-A33 peptide in α-helical conformation is represented in wireframe. Contour surfaces with favorable interaction energies of the methyl probe (threshold = −10 kJ/mol) are shown in light blue.

Mentions: The above results indicate that myristoylation of b5R nascent chain interferes with SRP binding, resulting in the escape of a portion of RNCs from targeting to the ER. We hypothesized that myristoylation might affect interaction with SRP by a conformational effect on b5R's NH2-terminal region. To test this idea, we investigated the effect of myristoylation on the secondary structure of the NH2-terminal peptide of b5R (from G2 to A33) by circular dichroism (CD). In aqueous solution (Fig. 8 A, a), the nonmyristoylated peptide (blue trace) had a largely unordered structure (∼10% α-helix, calculated according to the equation in Chen et al., 1974), whereas the same peptide carrying N-linked myristate (red trace) showed a higher proportion (∼30%) of α-helix, as indicated by the increase in the negative peaks at 208 and 222 nm. The stabilizing effect of the attached myristate was not due simply to blockade of the NH2 terminus because the N-acetylated G2-A33 peptide (green trace) had a spectrum similar to that of the unmodified peptide (14% helical content). Inclusion of a nonpolar solvent (trifluoroethanol [TFE]; Fig. 8 A, b) or detergent (SDS; Fig. 8 A, c) increased the α-helicity of both the unmodified and acetylated peptide, so that under these conditions all three peptides had very similar spectra.


N-myristoylation determines dual targeting of mammalian NADH-cytochrome b5 reductase to ER and mitochondrial outer membranes by a mechanism of kinetic partitioning.

Colombo S, Longhi R, Alcaro S, Ortuso F, Sprocati T, Flora A, Borgese N - J. Cell Biol. (2005)

Myristate stabilizes the α-helical conformation of b5R's NH2-terminal peptide. (A) CD spectra of b5R NH2-terminal peptide (G2-A33). Spectra were taken for the unmodified (blue), the N-myristoylated (red), or the N-acetylated (green) peptide, diluted in water (a), 50% TFE (b), or 25 mM SDS (c). In aqueous solution the N-myristoylated peptide has a spectrum indicative of a higher α-helical content than the other two peptides. (B) Result of GRID analysis with the methyl group probe. The b5R G2-A33 peptide in α-helical conformation is represented in wireframe. Contour surfaces with favorable interaction energies of the methyl probe (threshold = −10 kJ/mol) are shown in light blue.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171821&req=5

fig8: Myristate stabilizes the α-helical conformation of b5R's NH2-terminal peptide. (A) CD spectra of b5R NH2-terminal peptide (G2-A33). Spectra were taken for the unmodified (blue), the N-myristoylated (red), or the N-acetylated (green) peptide, diluted in water (a), 50% TFE (b), or 25 mM SDS (c). In aqueous solution the N-myristoylated peptide has a spectrum indicative of a higher α-helical content than the other two peptides. (B) Result of GRID analysis with the methyl group probe. The b5R G2-A33 peptide in α-helical conformation is represented in wireframe. Contour surfaces with favorable interaction energies of the methyl probe (threshold = −10 kJ/mol) are shown in light blue.
Mentions: The above results indicate that myristoylation of b5R nascent chain interferes with SRP binding, resulting in the escape of a portion of RNCs from targeting to the ER. We hypothesized that myristoylation might affect interaction with SRP by a conformational effect on b5R's NH2-terminal region. To test this idea, we investigated the effect of myristoylation on the secondary structure of the NH2-terminal peptide of b5R (from G2 to A33) by circular dichroism (CD). In aqueous solution (Fig. 8 A, a), the nonmyristoylated peptide (blue trace) had a largely unordered structure (∼10% α-helix, calculated according to the equation in Chen et al., 1974), whereas the same peptide carrying N-linked myristate (red trace) showed a higher proportion (∼30%) of α-helix, as indicated by the increase in the negative peaks at 208 and 222 nm. The stabilizing effect of the attached myristate was not due simply to blockade of the NH2 terminus because the N-acetylated G2-A33 peptide (green trace) had a spectrum similar to that of the unmodified peptide (14% helical content). Inclusion of a nonpolar solvent (trifluoroethanol [TFE]; Fig. 8 A, b) or detergent (SDS; Fig. 8 A, c) increased the α-helicity of both the unmodified and acetylated peptide, so that under these conditions all three peptides had very similar spectra.

Bottom Line: Mammalian NADH-cytochrome b5 reductase (b5R) is an N-myristoylated protein that is dually targeted to ER and mitochondrial outer membranes.We find that myristoylation interferes with interaction of the nascent chain with signal recognition particle, so that a portion of the nascent chains escapes from cotranslational integration into the ER and can be post-translationally targeted to the mitochondrial outer membrane.Thus, competition between two cotranslational events, binding of signal recognition particle and modification by N-myristoylation, determines the site of translation and the localization of b5R.

View Article: PubMed Central - PubMed

Affiliation: Consiglio Nazionale delle Ricerche Institute of Neuroscience, Cellular and Molecular Pharmacology Section and Department of Medical Pharmacology, University of Milan, 20129 Milan, Italy.

ABSTRACT
Mammalian NADH-cytochrome b5 reductase (b5R) is an N-myristoylated protein that is dually targeted to ER and mitochondrial outer membranes. The N-linked myristate is not required for anchorage to membranes because a stretch of hydrophobic amino acids close to the NH2 terminus guarantees a tight interaction of the protein with the phospholipid bilayer. Instead, the fatty acid is required for targeting of b5R to mitochondria because a nonmyristoylated mutant is exclusively localized to the ER. Here, we have investigated the mechanism by which N-linked myristate affects b5R targeting. We find that myristoylation interferes with interaction of the nascent chain with signal recognition particle, so that a portion of the nascent chains escapes from cotranslational integration into the ER and can be post-translationally targeted to the mitochondrial outer membrane. Thus, competition between two cotranslational events, binding of signal recognition particle and modification by N-myristoylation, determines the site of translation and the localization of b5R.

Show MeSH
Related in: MedlinePlus