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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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A kinetic partitioning model to explain the dual ER/MOM targeting of b5R. The different steps of alternative pathways are numbered and explained in the text. SRP is represented by the brown elongated body. The green segment of the nascent chain represents the myristoylation consensus, the red segment the hydrophobic, SRP-interacting region. The wavy black line represents the myristoyl moiety attached to the NH2 terminus of the nascent chain. The two blue forms in pathway 4 depict unknown chaperones that might be involved in the post-translational targeting of b5R to the MOM. See Discussion for further explanation.
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fig9: A kinetic partitioning model to explain the dual ER/MOM targeting of b5R. The different steps of alternative pathways are numbered and explained in the text. SRP is represented by the brown elongated body. The green segment of the nascent chain represents the myristoylation consensus, the red segment the hydrophobic, SRP-interacting region. The wavy black line represents the myristoyl moiety attached to the NH2 terminus of the nascent chain. The two blue forms in pathway 4 depict unknown chaperones that might be involved in the post-translational targeting of b5R to the MOM. See Discussion for further explanation.

Mentions: The kinetic partitioning model for the dual ER/MOM targeting of b5R that we propose, and that is consistent with the data on the three b5R forms investigated in this paper, is illustrated in Fig. 9. Upon emergence from the ribosome, the NH2-terminal region of wt b5R, because of its hydrophobic stretch (shown in red) and its myristoylation consensus sequence (shown in green), has the opportunity to interact both with SRP and with N-myristoyl-CoA:protein myristoyltransferase (NMT), the latter interaction preceded by removal of the NH2-terminal Met by methionine aminopeptidase (MAP). Based on the results with b5Rext, we postulate that occupation by SRP blocks accessibility to NMT. However, different signal peptides have widely differing binding constants for SRP (Flanagan et al., 2003). The NH2-terminal portion of wt b5R, because of its moderate affinity for SRP, will spend enough time in the unbound state to allow for its quantitative myristoylation before SRP-mediated targeting to the membrane can occur (Fig. 9, pathway 1). Once myristoylated, the affinity of the nascent peptide for SRP is further reduced so that only a fraction of the translating ribosomes manage to be targeted to the ER via the SRP pathway (Fig. 9, pathway 2). Nonetheless, because targeting to the translocation complex of a single translating ribosome will result in the association of the entire polysome with the ER, it is plausible that ribosomes translating such a membrane-associated mRNA are able to directly deliver myristoylated nascent peptide to nearby vacant translocons in an SRP-independent pathway (not depicted), resulting in the insertion of a sizeable portion of wt b5R molecules into the ER membrane.


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)

A kinetic partitioning model to explain the dual ER/MOM targeting of b5R. The different steps of alternative pathways are numbered and explained in the text. SRP is represented by the brown elongated body. The green segment of the nascent chain represents the myristoylation consensus, the red segment the hydrophobic, SRP-interacting region. The wavy black line represents the myristoyl moiety attached to the NH2 terminus of the nascent chain. The two blue forms in pathway 4 depict unknown chaperones that might be involved in the post-translational targeting of b5R to the MOM. See Discussion for further explanation.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: A kinetic partitioning model to explain the dual ER/MOM targeting of b5R. The different steps of alternative pathways are numbered and explained in the text. SRP is represented by the brown elongated body. The green segment of the nascent chain represents the myristoylation consensus, the red segment the hydrophobic, SRP-interacting region. The wavy black line represents the myristoyl moiety attached to the NH2 terminus of the nascent chain. The two blue forms in pathway 4 depict unknown chaperones that might be involved in the post-translational targeting of b5R to the MOM. See Discussion for further explanation.
Mentions: The kinetic partitioning model for the dual ER/MOM targeting of b5R that we propose, and that is consistent with the data on the three b5R forms investigated in this paper, is illustrated in Fig. 9. Upon emergence from the ribosome, the NH2-terminal region of wt b5R, because of its hydrophobic stretch (shown in red) and its myristoylation consensus sequence (shown in green), has the opportunity to interact both with SRP and with N-myristoyl-CoA:protein myristoyltransferase (NMT), the latter interaction preceded by removal of the NH2-terminal Met by methionine aminopeptidase (MAP). Based on the results with b5Rext, we postulate that occupation by SRP blocks accessibility to NMT. However, different signal peptides have widely differing binding constants for SRP (Flanagan et al., 2003). The NH2-terminal portion of wt b5R, because of its moderate affinity for SRP, will spend enough time in the unbound state to allow for its quantitative myristoylation before SRP-mediated targeting to the membrane can occur (Fig. 9, pathway 1). Once myristoylated, the affinity of the nascent peptide for SRP is further reduced so that only a fraction of the translating ribosomes manage to be targeted to the ER via the SRP pathway (Fig. 9, pathway 2). Nonetheless, because targeting to the translocation complex of a single translating ribosome will result in the association of the entire polysome with the ER, it is plausible that ribosomes translating such a membrane-associated mRNA are able to directly deliver myristoylated nascent peptide to nearby vacant translocons in an SRP-independent pathway (not depicted), resulting in the insertion of a sizeable portion of wt b5R molecules into the ER membrane.

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