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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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Analysis of myristoylation of b5R forms by metabolic labeling. MDCK cells expressing the indicated b5R forms were incubated with either 0.1 mCi/ml of 35S-Promix for 3 h (top) or 0.1 mCi/ml of 3H-myristic acid for 6 h. b5R was immunoprecipitated from the detergent lysates and analyzed by 11% SDS-PAGE fluorography. Both wt b5R (lane 1) and its extended mutant (lane 3) are myristoylated, whereas G2A (lane 2) is not. The position of the 31-kD size marker is shown. (B) b5Rext is less efficiently myristoylated than the wt protein. The band intensities of the gels of B were quantified by scanning. The 3H/35S ratio for wt b5R was arbitrarily set to 1.
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fig3: Analysis of myristoylation of b5R forms by metabolic labeling. MDCK cells expressing the indicated b5R forms were incubated with either 0.1 mCi/ml of 35S-Promix for 3 h (top) or 0.1 mCi/ml of 3H-myristic acid for 6 h. b5R was immunoprecipitated from the detergent lysates and analyzed by 11% SDS-PAGE fluorography. Both wt b5R (lane 1) and its extended mutant (lane 3) are myristoylated, whereas G2A (lane 2) is not. The position of the 31-kD size marker is shown. (B) b5Rext is less efficiently myristoylated than the wt protein. The band intensities of the gels of B were quantified by scanning. The 3H/35S ratio for wt b5R was arbitrarily set to 1.

Mentions: To further characterize the constructs, we investigated their myristoylation status by metabolic labeling with 3H-myristic acid or 35S-Met/cys in parallel dishes, followed by immunoprecipitation. As expected, the G2A mutant, although present, was not myristoylated (Fig. 3 A, lane 2 in top and bottom), whereas both the wt and extended mutant of b5R incorporated the labeled fatty acid (Fig. 3 A, lanes 1 and 3). However, when the degree of myristoylation of these two constructs was evaluated by the ratio of 3H to 35S radioactivity, we consistently found that b5Rext was myristoylated with <40% the efficiency of the wt protein (Fig. 3 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)

Analysis of myristoylation of b5R forms by metabolic labeling. MDCK cells expressing the indicated b5R forms were incubated with either 0.1 mCi/ml of 35S-Promix for 3 h (top) or 0.1 mCi/ml of 3H-myristic acid for 6 h. b5R was immunoprecipitated from the detergent lysates and analyzed by 11% SDS-PAGE fluorography. Both wt b5R (lane 1) and its extended mutant (lane 3) are myristoylated, whereas G2A (lane 2) is not. The position of the 31-kD size marker is shown. (B) b5Rext is less efficiently myristoylated than the wt protein. The band intensities of the gels of B were quantified by scanning. The 3H/35S ratio for wt b5R was arbitrarily set to 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171821&req=5

fig3: Analysis of myristoylation of b5R forms by metabolic labeling. MDCK cells expressing the indicated b5R forms were incubated with either 0.1 mCi/ml of 35S-Promix for 3 h (top) or 0.1 mCi/ml of 3H-myristic acid for 6 h. b5R was immunoprecipitated from the detergent lysates and analyzed by 11% SDS-PAGE fluorography. Both wt b5R (lane 1) and its extended mutant (lane 3) are myristoylated, whereas G2A (lane 2) is not. The position of the 31-kD size marker is shown. (B) b5Rext is less efficiently myristoylated than the wt protein. The band intensities of the gels of B were quantified by scanning. The 3H/35S ratio for wt b5R was arbitrarily set to 1.
Mentions: To further characterize the constructs, we investigated their myristoylation status by metabolic labeling with 3H-myristic acid or 35S-Met/cys in parallel dishes, followed by immunoprecipitation. As expected, the G2A mutant, although present, was not myristoylated (Fig. 3 A, lane 2 in top and bottom), whereas both the wt and extended mutant of b5R incorporated the labeled fatty acid (Fig. 3 A, lanes 1 and 3). However, when the degree of myristoylation of these two constructs was evaluated by the ratio of 3H to 35S radioactivity, we consistently found that b5Rext was myristoylated with <40% the efficiency of the wt protein (Fig. 3 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