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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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b5R and its mutant forms interact with SRP. (A) Synthetic transcripts coding for the indicated b5R forms were translated in wheat germ extract for 20 min in the absence or presence of 100 nM SRP, and analyzed by 11% SDS-PAGE autoradiography. SRP inhibits translation of the wt, G2A, and extended b5R (lanes 1–6), while having much less effect on the translation of a soluble b5R form that lacks the hydrophobic anchor (sol, lanes 7 and 8). (B) Dependence of translational slow-down on SRP concentration. wt b5R (top), G2A (middle), and b5Rext (bottom) transcripts were translated together with luciferase mRNA for 40 min in the presence of the indicated concentrations of SRP, and were analyzed by 11% SDS-PAGE phosphorimaging. The arrow and arrowhead indicate b5R and luciferase, respectively. (C) The intensities of the bands in six different experiments like the one shown in B were quantified. For each b5R form and in each experiment, the ratio of intensities of b5R to luciferase in the absence of SRP was set to 100% translation efficiency. Shown are averages with SEM.
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fig5: b5R and its mutant forms interact with SRP. (A) Synthetic transcripts coding for the indicated b5R forms were translated in wheat germ extract for 20 min in the absence or presence of 100 nM SRP, and analyzed by 11% SDS-PAGE autoradiography. SRP inhibits translation of the wt, G2A, and extended b5R (lanes 1–6), while having much less effect on the translation of a soluble b5R form that lacks the hydrophobic anchor (sol, lanes 7 and 8). (B) Dependence of translational slow-down on SRP concentration. wt b5R (top), G2A (middle), and b5Rext (bottom) transcripts were translated together with luciferase mRNA for 40 min in the presence of the indicated concentrations of SRP, and were analyzed by 11% SDS-PAGE phosphorimaging. The arrow and arrowhead indicate b5R and luciferase, respectively. (C) The intensities of the bands in six different experiments like the one shown in B were quantified. For each b5R form and in each experiment, the ratio of intensities of b5R to luciferase in the absence of SRP was set to 100% translation efficiency. Shown are averages with SEM.

Mentions: In the wheat germ extract, addition of SRP in the absence of microsomal membranes slows down elongation of signal peptide bearing nascent chains (Walter and Blobel, 1983b), and this effect can be exploited to probe for signal peptide–SRP interaction. First, we analyzed the effect of high concentrations (100 nM) of SRP on the in vitro translation of b5R mRNAs. As control, we used a transcript coding for a soluble form of the enzyme, which lacks the entire hydrophobic NH2-terminal domain (Pietrini et al., 1992). As seen in Fig. 5 A, translation of all three membrane-binding constructs of b5R was blocked under these conditions, whereas the translation of the soluble form was much less affected (lanes 7 and 8). Thus, the NH2-terminal domain of b5R can indeed be defined as an SRP-interacting signal anchor.


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)

b5R and its mutant forms interact with SRP. (A) Synthetic transcripts coding for the indicated b5R forms were translated in wheat germ extract for 20 min in the absence or presence of 100 nM SRP, and analyzed by 11% SDS-PAGE autoradiography. SRP inhibits translation of the wt, G2A, and extended b5R (lanes 1–6), while having much less effect on the translation of a soluble b5R form that lacks the hydrophobic anchor (sol, lanes 7 and 8). (B) Dependence of translational slow-down on SRP concentration. wt b5R (top), G2A (middle), and b5Rext (bottom) transcripts were translated together with luciferase mRNA for 40 min in the presence of the indicated concentrations of SRP, and were analyzed by 11% SDS-PAGE phosphorimaging. The arrow and arrowhead indicate b5R and luciferase, respectively. (C) The intensities of the bands in six different experiments like the one shown in B were quantified. For each b5R form and in each experiment, the ratio of intensities of b5R to luciferase in the absence of SRP was set to 100% translation efficiency. Shown are averages with SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171821&req=5

fig5: b5R and its mutant forms interact with SRP. (A) Synthetic transcripts coding for the indicated b5R forms were translated in wheat germ extract for 20 min in the absence or presence of 100 nM SRP, and analyzed by 11% SDS-PAGE autoradiography. SRP inhibits translation of the wt, G2A, and extended b5R (lanes 1–6), while having much less effect on the translation of a soluble b5R form that lacks the hydrophobic anchor (sol, lanes 7 and 8). (B) Dependence of translational slow-down on SRP concentration. wt b5R (top), G2A (middle), and b5Rext (bottom) transcripts were translated together with luciferase mRNA for 40 min in the presence of the indicated concentrations of SRP, and were analyzed by 11% SDS-PAGE phosphorimaging. The arrow and arrowhead indicate b5R and luciferase, respectively. (C) The intensities of the bands in six different experiments like the one shown in B were quantified. For each b5R form and in each experiment, the ratio of intensities of b5R to luciferase in the absence of SRP was set to 100% translation efficiency. Shown are averages with SEM.
Mentions: In the wheat germ extract, addition of SRP in the absence of microsomal membranes slows down elongation of signal peptide bearing nascent chains (Walter and Blobel, 1983b), and this effect can be exploited to probe for signal peptide–SRP interaction. First, we analyzed the effect of high concentrations (100 nM) of SRP on the in vitro translation of b5R mRNAs. As control, we used a transcript coding for a soluble form of the enzyme, which lacks the entire hydrophobic NH2-terminal domain (Pietrini et al., 1992). As seen in Fig. 5 A, translation of all three membrane-binding constructs of b5R was blocked under these conditions, whereas the translation of the soluble form was much less affected (lanes 7 and 8). Thus, the NH2-terminal domain of b5R can indeed be defined as an SRP-interacting signal anchor.

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