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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 mRNA is distributed on both free and bound polysomes, whereas mutant transcripts are translated exclusively on bound polysomes. (A) Blot analysis of RNA extracted from free (F) and bound (B) polysome fractions prepared from nontransfected MDCK (lanes 1 and 2) or from cells stably transfected with wt b5R (lanes 3 and 4), G2A (lanes 5 and 6), and b5Rext (lanes 7 and 8). 3.6 μg RNA from free and 1.8 μg from the bound polysomes were loaded. The blot was stained with methylene blue and then sequentially hybridized with probes for ribophorin I, GAPDH, and b5R as indicated. The bottom panel shows the methylene blue–stained 18S ribosomal RNA. The position of the 18S RNA in all panels is indicated. (B) Quantification by phosphorimaging of the signals of the experiment illustrated in A. The ratios of the intensities of the signal in the bound versus the free polysome fraction are shown after correction for the different RNA loads.
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fig4: b5R mRNA is distributed on both free and bound polysomes, whereas mutant transcripts are translated exclusively on bound polysomes. (A) Blot analysis of RNA extracted from free (F) and bound (B) polysome fractions prepared from nontransfected MDCK (lanes 1 and 2) or from cells stably transfected with wt b5R (lanes 3 and 4), G2A (lanes 5 and 6), and b5Rext (lanes 7 and 8). 3.6 μg RNA from free and 1.8 μg from the bound polysomes were loaded. The blot was stained with methylene blue and then sequentially hybridized with probes for ribophorin I, GAPDH, and b5R as indicated. The bottom panel shows the methylene blue–stained 18S ribosomal RNA. The position of the 18S RNA in all panels is indicated. (B) Quantification by phosphorimaging of the signals of the experiment illustrated in A. The ratios of the intensities of the signal in the bound versus the free polysome fraction are shown after correction for the different RNA loads.

Mentions: ER membrane proteins with b5R's topology are generally inserted cotranslationally because their NH2-terminal hydrophobic domain interacts with SRP (Goder and Spiess, 2001). On the other hand, MOM proteins are targeted post-translationally (Shore et al., 1995), and early studies (Borgese and Gaetani, 1980; Okada et al., 1982) reported that in liver, b5R is synthesized on free polyribosomes, suggesting that its insertion into both the ER and the MOM is a post-translational event. As a first step to elucidate the in vivo–targeting pathways of our constructs, we analyzed by Northern blotting the distribution of the corresponding mRNAs between free and bound polysome fractions prepared from the transfected MDCK cell lines. In three separate experiments, we observed that all three transcripts were recovered in the bound polysome fraction, but that the mRNA specifying wt b5R was present also in the free fraction, at a concentration equal to 20–30% of that in bound polysomes; for the transcripts specifying the two mutant b5Rs the corresponding percentage was ∼5 (Fig. 4 A, third panel from top). The distribution of the endogenous b5R mRNA in nontransfected cells (visible after longer exposure times not depicted in Fig. 4) was similar to that of the transfected wt 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)

b5R mRNA is distributed on both free and bound polysomes, whereas mutant transcripts are translated exclusively on bound polysomes. (A) Blot analysis of RNA extracted from free (F) and bound (B) polysome fractions prepared from nontransfected MDCK (lanes 1 and 2) or from cells stably transfected with wt b5R (lanes 3 and 4), G2A (lanes 5 and 6), and b5Rext (lanes 7 and 8). 3.6 μg RNA from free and 1.8 μg from the bound polysomes were loaded. The blot was stained with methylene blue and then sequentially hybridized with probes for ribophorin I, GAPDH, and b5R as indicated. The bottom panel shows the methylene blue–stained 18S ribosomal RNA. The position of the 18S RNA in all panels is indicated. (B) Quantification by phosphorimaging of the signals of the experiment illustrated in A. The ratios of the intensities of the signal in the bound versus the free polysome fraction are shown after correction for the different RNA loads.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171821&req=5

fig4: b5R mRNA is distributed on both free and bound polysomes, whereas mutant transcripts are translated exclusively on bound polysomes. (A) Blot analysis of RNA extracted from free (F) and bound (B) polysome fractions prepared from nontransfected MDCK (lanes 1 and 2) or from cells stably transfected with wt b5R (lanes 3 and 4), G2A (lanes 5 and 6), and b5Rext (lanes 7 and 8). 3.6 μg RNA from free and 1.8 μg from the bound polysomes were loaded. The blot was stained with methylene blue and then sequentially hybridized with probes for ribophorin I, GAPDH, and b5R as indicated. The bottom panel shows the methylene blue–stained 18S ribosomal RNA. The position of the 18S RNA in all panels is indicated. (B) Quantification by phosphorimaging of the signals of the experiment illustrated in A. The ratios of the intensities of the signal in the bound versus the free polysome fraction are shown after correction for the different RNA loads.
Mentions: ER membrane proteins with b5R's topology are generally inserted cotranslationally because their NH2-terminal hydrophobic domain interacts with SRP (Goder and Spiess, 2001). On the other hand, MOM proteins are targeted post-translationally (Shore et al., 1995), and early studies (Borgese and Gaetani, 1980; Okada et al., 1982) reported that in liver, b5R is synthesized on free polyribosomes, suggesting that its insertion into both the ER and the MOM is a post-translational event. As a first step to elucidate the in vivo–targeting pathways of our constructs, we analyzed by Northern blotting the distribution of the corresponding mRNAs between free and bound polysome fractions prepared from the transfected MDCK cell lines. In three separate experiments, we observed that all three transcripts were recovered in the bound polysome fraction, but that the mRNA specifying wt b5R was present also in the free fraction, at a concentration equal to 20–30% of that in bound polysomes; for the transcripts specifying the two mutant b5Rs the corresponding percentage was ∼5 (Fig. 4 A, third panel from top). The distribution of the endogenous b5R mRNA in nontransfected cells (visible after longer exposure times not depicted in Fig. 4) was similar to that of the transfected wt 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