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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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Myristoylation inhibits recruitment of b5R-synthesizing polysomes to ER membranes. (A) The truncated mRNAs coding for the NH2-terminal portion of the three b5R forms (described in the legend to Fig. 6 E) were translated in wheat germ extract without other additions (left column), or with the addition of DPM + SRP and MyrCoA, as indicated (see Materials and methods). The translated samples were brought to 1.8 M sucrose and run on high salt-sucrose flotation gradients. TCA-precipitated fractions from the gradients were analyzed by 14% SDS-PAGE phosphorimaging. Fraction 2 contains the 0.3/1.6 M sucrose interface. Lane 5 contains the bottom fraction plus the pellet. Note the shift of the nascent chains from the bottom of the gradient, containing the free polysomes, to the 0.3/1.6 M interface, when DPM were added. When MyrCoA was present, wt b5R nascent chains were partially shifted back to the free polysome fraction. The arrow on the right indicates the position of the 14-kD size marker. (B) Quantification of three independent experiments like the one of A. The bands indicated by the asterisk in lanes 2 and 5 were quantified. Shown is the percentage recovered in lane 2 with respect to the sum of the intensities in lanes 2 + 5. Bars indicate the SEM. **, highly significant difference between the − and + MyrCoA samples (P = 0.0061 by t test).
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fig7: Myristoylation inhibits recruitment of b5R-synthesizing polysomes to ER membranes. (A) The truncated mRNAs coding for the NH2-terminal portion of the three b5R forms (described in the legend to Fig. 6 E) were translated in wheat germ extract without other additions (left column), or with the addition of DPM + SRP and MyrCoA, as indicated (see Materials and methods). The translated samples were brought to 1.8 M sucrose and run on high salt-sucrose flotation gradients. TCA-precipitated fractions from the gradients were analyzed by 14% SDS-PAGE phosphorimaging. Fraction 2 contains the 0.3/1.6 M sucrose interface. Lane 5 contains the bottom fraction plus the pellet. Note the shift of the nascent chains from the bottom of the gradient, containing the free polysomes, to the 0.3/1.6 M interface, when DPM were added. When MyrCoA was present, wt b5R nascent chains were partially shifted back to the free polysome fraction. The arrow on the right indicates the position of the 14-kD size marker. (B) Quantification of three independent experiments like the one of A. The bands indicated by the asterisk in lanes 2 and 5 were quantified. Shown is the percentage recovered in lane 2 with respect to the sum of the intensities in lanes 2 + 5. Bars indicate the SEM. **, highly significant difference between the − and + MyrCoA samples (P = 0.0061 by t test).

Mentions: The MyrCoA-induced resistance of b5R nascent chains to the action of SRP should result in the escape of the translating polysomes from recruitment to the ER membrane. To test this prediction, we translated the same truncated mRNAs used for the cross-linking experiments in the presence of dog pancreas microsomes (DPM), and investigated the effect of MyrCoA on the distribution of the resulting RNCs between free and bound polysomes by flotation on high salt sucrose gradients. As shown in Fig. 7, translation of the truncated mRNAs resulted in two major species, of which the one with slower mobility (Fig. 7 A, asterisks) presumably corresponds to the full-length truncated nascent chain. When translations were performed in the absence of added DPM, the nascent chains were quantitatively recovered in the bottom fraction and pellet of the gradient (Fig. 7 A, lane 5, left). If the translation was performed in the presence of DPM and SRP (Fig. 7 A, middle) the majority of nascent chains of all three b5R forms floated to the 1.6 M/0.3 M sucrose interface (fraction 2). As shown in the right panel of Fig. 7 A, addition of MyrCoA to the translation mix resulted in a shift of part of the translated truncated wt b5R products from the bound (fraction 2) to the free fraction (fraction 5). This effect was seen only for the wt protein, and not for the two mutants. Quantification of three separate experiments confirmed that MyrCoA decreased the fraction of wt b5R RNCs associated with DPM by ∼30% (P < 0.01 by t test), whereas it had no significant effect on the distribution of G2A and b5Rext nascent peptides (Fig. 7 B).


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)

Myristoylation inhibits recruitment of b5R-synthesizing polysomes to ER membranes. (A) The truncated mRNAs coding for the NH2-terminal portion of the three b5R forms (described in the legend to Fig. 6 E) were translated in wheat germ extract without other additions (left column), or with the addition of DPM + SRP and MyrCoA, as indicated (see Materials and methods). The translated samples were brought to 1.8 M sucrose and run on high salt-sucrose flotation gradients. TCA-precipitated fractions from the gradients were analyzed by 14% SDS-PAGE phosphorimaging. Fraction 2 contains the 0.3/1.6 M sucrose interface. Lane 5 contains the bottom fraction plus the pellet. Note the shift of the nascent chains from the bottom of the gradient, containing the free polysomes, to the 0.3/1.6 M interface, when DPM were added. When MyrCoA was present, wt b5R nascent chains were partially shifted back to the free polysome fraction. The arrow on the right indicates the position of the 14-kD size marker. (B) Quantification of three independent experiments like the one of A. The bands indicated by the asterisk in lanes 2 and 5 were quantified. Shown is the percentage recovered in lane 2 with respect to the sum of the intensities in lanes 2 + 5. Bars indicate the SEM. **, highly significant difference between the − and + MyrCoA samples (P = 0.0061 by t test).
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Related In: Results  -  Collection

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fig7: Myristoylation inhibits recruitment of b5R-synthesizing polysomes to ER membranes. (A) The truncated mRNAs coding for the NH2-terminal portion of the three b5R forms (described in the legend to Fig. 6 E) were translated in wheat germ extract without other additions (left column), or with the addition of DPM + SRP and MyrCoA, as indicated (see Materials and methods). The translated samples were brought to 1.8 M sucrose and run on high salt-sucrose flotation gradients. TCA-precipitated fractions from the gradients were analyzed by 14% SDS-PAGE phosphorimaging. Fraction 2 contains the 0.3/1.6 M sucrose interface. Lane 5 contains the bottom fraction plus the pellet. Note the shift of the nascent chains from the bottom of the gradient, containing the free polysomes, to the 0.3/1.6 M interface, when DPM were added. When MyrCoA was present, wt b5R nascent chains were partially shifted back to the free polysome fraction. The arrow on the right indicates the position of the 14-kD size marker. (B) Quantification of three independent experiments like the one of A. The bands indicated by the asterisk in lanes 2 and 5 were quantified. Shown is the percentage recovered in lane 2 with respect to the sum of the intensities in lanes 2 + 5. Bars indicate the SEM. **, highly significant difference between the − and + MyrCoA samples (P = 0.0061 by t test).
Mentions: The MyrCoA-induced resistance of b5R nascent chains to the action of SRP should result in the escape of the translating polysomes from recruitment to the ER membrane. To test this prediction, we translated the same truncated mRNAs used for the cross-linking experiments in the presence of dog pancreas microsomes (DPM), and investigated the effect of MyrCoA on the distribution of the resulting RNCs between free and bound polysomes by flotation on high salt sucrose gradients. As shown in Fig. 7, translation of the truncated mRNAs resulted in two major species, of which the one with slower mobility (Fig. 7 A, asterisks) presumably corresponds to the full-length truncated nascent chain. When translations were performed in the absence of added DPM, the nascent chains were quantitatively recovered in the bottom fraction and pellet of the gradient (Fig. 7 A, lane 5, left). If the translation was performed in the presence of DPM and SRP (Fig. 7 A, middle) the majority of nascent chains of all three b5R forms floated to the 1.6 M/0.3 M sucrose interface (fraction 2). As shown in the right panel of Fig. 7 A, addition of MyrCoA to the translation mix resulted in a shift of part of the translated truncated wt b5R products from the bound (fraction 2) to the free fraction (fraction 5). This effect was seen only for the wt protein, and not for the two mutants. Quantification of three separate experiments confirmed that MyrCoA decreased the fraction of wt b5R RNCs associated with DPM by ∼30% (P < 0.01 by t test), whereas it had no significant effect on the distribution of G2A and b5Rext nascent peptides (Fig. 7 B).

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