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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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Dual localization of wt b5R, and ER-restricted distribution of G2A and b5Rext mutants, in stably transfected MDCK cells. Cells expressing each of the three proteins, as indicated to the left of the panels, were doubly stained with goat anti-b5R antibodies, followed by biotinylated anti–goat IgG and AlexaFluor 488–conjugated streptavidin (a, d, and g in A and B), and polyclonal anti-complex III antibodies or anti-protein disulfide isomerase (PDI) antibodies (b, e, and h in A and B respectively), both followed by Texas red–conjugated anti–rabbit IgG. In the merged images (shown in c, f, and i of both panels), the yellow color indicates colocalization. wt b5R colocalizes with the mitochondrial marker, but in addition shows a more widespread distribution (A), due to its ER localization demonstrated by colocalization with PDI (B). G2A and b5Rext do not show colocalization with the mitochondrial marker (A), but extensively colocalize with PDI (B). Bars, 10 μm. The closed boxes in the merged images indicate the area that is enlarged in the corresponding inset.
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fig2: Dual localization of wt b5R, and ER-restricted distribution of G2A and b5Rext mutants, in stably transfected MDCK cells. Cells expressing each of the three proteins, as indicated to the left of the panels, were doubly stained with goat anti-b5R antibodies, followed by biotinylated anti–goat IgG and AlexaFluor 488–conjugated streptavidin (a, d, and g in A and B), and polyclonal anti-complex III antibodies or anti-protein disulfide isomerase (PDI) antibodies (b, e, and h in A and B respectively), both followed by Texas red–conjugated anti–rabbit IgG. In the merged images (shown in c, f, and i of both panels), the yellow color indicates colocalization. wt b5R colocalizes with the mitochondrial marker, but in addition shows a more widespread distribution (A), due to its ER localization demonstrated by colocalization with PDI (B). G2A and b5Rext do not show colocalization with the mitochondrial marker (A), but extensively colocalize with PDI (B). Bars, 10 μm. The closed boxes in the merged images indicate the area that is enlarged in the corresponding inset.

Mentions: We compared the intracellular localization of the three constructs in the cell lines by confocal analysis (Fig. 2). b5R was undetectable by immunofluorescence in nontransfected MDCK cells under the conditions used (not depicted). As previously reported, the transfected wt protein colocalized with markers both of mitochondria (Fig. 2 A, a–c) and of the ER (Fig. 2 B, a–c), whereas the G2A mutant localized to the ER (Fig. 2 B, d–f), but not to mitochondria (Fig. 2 A, d–f). A similar distribution, with no antigen detectable on mitochondria, was seen for the b5Rext mutant (Fig. 2, A and B; g–i). We confirmed the lack of mitochondrial localization for b5Rext also by cell fractionation (Fig. S1 B). Thus, the effect of elimination of myristoylation (in G2A) and of increasing the length/hydrophobicity of the membrane anchor (in b5Rext) similarly abolished mitochondrial targeting of b5R.


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)

Dual localization of wt b5R, and ER-restricted distribution of G2A and b5Rext mutants, in stably transfected MDCK cells. Cells expressing each of the three proteins, as indicated to the left of the panels, were doubly stained with goat anti-b5R antibodies, followed by biotinylated anti–goat IgG and AlexaFluor 488–conjugated streptavidin (a, d, and g in A and B), and polyclonal anti-complex III antibodies or anti-protein disulfide isomerase (PDI) antibodies (b, e, and h in A and B respectively), both followed by Texas red–conjugated anti–rabbit IgG. In the merged images (shown in c, f, and i of both panels), the yellow color indicates colocalization. wt b5R colocalizes with the mitochondrial marker, but in addition shows a more widespread distribution (A), due to its ER localization demonstrated by colocalization with PDI (B). G2A and b5Rext do not show colocalization with the mitochondrial marker (A), but extensively colocalize with PDI (B). Bars, 10 μm. The closed boxes in the merged images indicate the area that is enlarged in the corresponding inset.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171821&req=5

fig2: Dual localization of wt b5R, and ER-restricted distribution of G2A and b5Rext mutants, in stably transfected MDCK cells. Cells expressing each of the three proteins, as indicated to the left of the panels, were doubly stained with goat anti-b5R antibodies, followed by biotinylated anti–goat IgG and AlexaFluor 488–conjugated streptavidin (a, d, and g in A and B), and polyclonal anti-complex III antibodies or anti-protein disulfide isomerase (PDI) antibodies (b, e, and h in A and B respectively), both followed by Texas red–conjugated anti–rabbit IgG. In the merged images (shown in c, f, and i of both panels), the yellow color indicates colocalization. wt b5R colocalizes with the mitochondrial marker, but in addition shows a more widespread distribution (A), due to its ER localization demonstrated by colocalization with PDI (B). G2A and b5Rext do not show colocalization with the mitochondrial marker (A), but extensively colocalize with PDI (B). Bars, 10 μm. The closed boxes in the merged images indicate the area that is enlarged in the corresponding inset.
Mentions: We compared the intracellular localization of the three constructs in the cell lines by confocal analysis (Fig. 2). b5R was undetectable by immunofluorescence in nontransfected MDCK cells under the conditions used (not depicted). As previously reported, the transfected wt protein colocalized with markers both of mitochondria (Fig. 2 A, a–c) and of the ER (Fig. 2 B, a–c), whereas the G2A mutant localized to the ER (Fig. 2 B, d–f), but not to mitochondria (Fig. 2 A, d–f). A similar distribution, with no antigen detectable on mitochondria, was seen for the b5Rext mutant (Fig. 2, A and B; g–i). We confirmed the lack of mitochondrial localization for b5Rext also by cell fractionation (Fig. S1 B). Thus, the effect of elimination of myristoylation (in G2A) and of increasing the length/hydrophobicity of the membrane anchor (in b5Rext) similarly abolished mitochondrial targeting of b5R.

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