Limits...
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.

Show MeSH

Related in: MedlinePlus

Myristoylation of b5R forms in the wheat germ extract and effect of myristoylation on interaction with SRP. (A) wt b5R (lane 1), G2A (lane 2), and b5Rext (lane 3) were translated in the presence of unlabeled Met and 3H-MyrCoA (see Materials and methods for details). Immunoprecipitates were run on 11% SDS–polyacrylamide gels, followed by blotting and phosphorimaging analysis. wt b5R and its extended mutant are myristoylated, whereas G2A is not. (B) Stoichiometry of in vitro myristoylation of wt b5R determined by double labeling. See Materials and methods for details on the experimental procedure. Calculation of the molar ratio of myristate to protein is based on the known specific radioactivities of the added compounds, and on the number of Met residues in the b5R sequence (8, not considering Met1). Any dilution of the specific radioactivity of 3H-MyrCoA or 35S-Met by the endogenous compounds is not considered. (C) Comparison of stoichiometry of in vitro myristoylation of wt b5R and b5Rext shows equal efficiency for the two proteins. wt b5R and b5Rext transcripts were translated in the wheat germ extract in the presence of 35S-Met and 3H-MyrCoA (21 μM). Calculation of the molar ratio of myristate to translated protein was as in B. Bars indicate the SEM (n = 5). (D) MyrCoA blunts the effect of SRP on the translation of wt b5R, but not of G2A and b5Rext. Transcripts coding for each of the three b5R forms were translated in wheat germ extract together with luciferase mRNA, 50 nM SRP, 35S-Met, and the indicated concentrations of unlabeled MyrCoA. For each concentration of MyrCoA, translation efficiency is the ratio of b5R band intensity to that of luciferase in the presence of SRP normalized to the same ratio in the absence of SRP, which was set at 100. Bars indicate the SEM (n = 5). (E) Inhibition by MyrCoA of the association of wt b5R nascent chains with SRP, assessed by cross-linking. Truncated mRNAs coding for the first 108 amino acids of wt b5R (lanes 1 and 2) and G2A (lanes 6 and 7), the first 113 amino acids of b5Rext (lanes 3–5), and the first 125 residues of cytochrome b(5) (lane 8) were translated for 30 min in reticulocyte lysate with or without 75 μM MyrCoA, as indicated. RNCs were recovered by centrifugation through a high salt sucrose cushion as detailed in the Materials and methods section. For each construct, equal amounts of TCA precipitable radioactivity were cross-linked with DSS and then immunoprecipitated with an anti-SRP54 antibody, with the exception of lane 5, in which precipitation was performed with a nonimmune serum. Adducts corresponding to cross-linked products of SRP and the nascent polypeptide chains of the three forms of reductase, but not of cytochrome b(5), were detected when anti-SRP54 was used for the immunoprecipitation. Note the strong inhibitory effect of added MyrCoA on wt b5R nascent chain cross-linking (lane 2), but not on the mutant b5R forms. Exposure times were 3 d for lanes 1–5 and 12 d for lanes 6–8.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171821&req=5

fig6: Myristoylation of b5R forms in the wheat germ extract and effect of myristoylation on interaction with SRP. (A) wt b5R (lane 1), G2A (lane 2), and b5Rext (lane 3) were translated in the presence of unlabeled Met and 3H-MyrCoA (see Materials and methods for details). Immunoprecipitates were run on 11% SDS–polyacrylamide gels, followed by blotting and phosphorimaging analysis. wt b5R and its extended mutant are myristoylated, whereas G2A is not. (B) Stoichiometry of in vitro myristoylation of wt b5R determined by double labeling. See Materials and methods for details on the experimental procedure. Calculation of the molar ratio of myristate to protein is based on the known specific radioactivities of the added compounds, and on the number of Met residues in the b5R sequence (8, not considering Met1). Any dilution of the specific radioactivity of 3H-MyrCoA or 35S-Met by the endogenous compounds is not considered. (C) Comparison of stoichiometry of in vitro myristoylation of wt b5R and b5Rext shows equal efficiency for the two proteins. wt b5R and b5Rext transcripts were translated in the wheat germ extract in the presence of 35S-Met and 3H-MyrCoA (21 μM). Calculation of the molar ratio of myristate to translated protein was as in B. Bars indicate the SEM (n = 5). (D) MyrCoA blunts the effect of SRP on the translation of wt b5R, but not of G2A and b5Rext. Transcripts coding for each of the three b5R forms were translated in wheat germ extract together with luciferase mRNA, 50 nM SRP, 35S-Met, and the indicated concentrations of unlabeled MyrCoA. For each concentration of MyrCoA, translation efficiency is the ratio of b5R band intensity to that of luciferase in the presence of SRP normalized to the same ratio in the absence of SRP, which was set at 100. Bars indicate the SEM (n = 5). (E) Inhibition by MyrCoA of the association of wt b5R nascent chains with SRP, assessed by cross-linking. Truncated mRNAs coding for the first 108 amino acids of wt b5R (lanes 1 and 2) and G2A (lanes 6 and 7), the first 113 amino acids of b5Rext (lanes 3–5), and the first 125 residues of cytochrome b(5) (lane 8) were translated for 30 min in reticulocyte lysate with or without 75 μM MyrCoA, as indicated. RNCs were recovered by centrifugation through a high salt sucrose cushion as detailed in the Materials and methods section. For each construct, equal amounts of TCA precipitable radioactivity were cross-linked with DSS and then immunoprecipitated with an anti-SRP54 antibody, with the exception of lane 5, in which precipitation was performed with a nonimmune serum. Adducts corresponding to cross-linked products of SRP and the nascent polypeptide chains of the three forms of reductase, but not of cytochrome b(5), were detected when anti-SRP54 was used for the immunoprecipitation. Note the strong inhibitory effect of added MyrCoA on wt b5R nascent chain cross-linking (lane 2), but not on the mutant b5R forms. Exposure times were 3 d for lanes 1–5 and 12 d for lanes 6–8.

Mentions: The comparable interaction of wt and G2A b5R nascent chains with SRP could have been due either to a lack of effect of N-myristoylation on this interaction or to low or no myristoylation occurring in vitro. Although the wheat germ extract is equipped with the enzymes required for N-myristoylation (Deichaite et al., 1988), the concentration of endogenous Myristoyl-CoA (MyrCoA) in the extract provided by Promega could be insufficient to support the reaction. When 3H-MyrCoA was included in the translation mixes (prepared with unlabeled Met), wt b5R and the extended mutant were myristoylated, whereas G2A b5R was not (Fig. 6 A). To obtain a quantitative estimate of the degree of in vitro myristoylation of wt b5R, 35S-Met and 3H-MyrCoA of known specific activities were included together in the same incubation. After SDS-PAGE and blotting onto nitrocellulose, the radioactive b5R band was excised, solubilized, and counted in double label mode. From the 3H/35S ratio and the known methionine content of b5R, we were able to calculate the amount of added myristic acid attached to the in vitro–synthesized protein. As seen in Fig. 6 B, the calculated ratio increased with increasing concentration of MyrCoA, reaching a value of ∼1 at 120 μM MyrCoA. We could not test the effect of higher concentrations of MyrCoA because they interfered with in vitro translation. However, because a 1:1 stoichiometry of myristic acid to protein is the maximum value attainable, the result indicates that MyrCoA endogenous to the wheat germ extract does not significantly contribute to the reaction. Thus, the MyrCoA concentration in these extracts appears to be low, and limiting for the myristoylation reaction.


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)

Myristoylation of b5R forms in the wheat germ extract and effect of myristoylation on interaction with SRP. (A) wt b5R (lane 1), G2A (lane 2), and b5Rext (lane 3) were translated in the presence of unlabeled Met and 3H-MyrCoA (see Materials and methods for details). Immunoprecipitates were run on 11% SDS–polyacrylamide gels, followed by blotting and phosphorimaging analysis. wt b5R and its extended mutant are myristoylated, whereas G2A is not. (B) Stoichiometry of in vitro myristoylation of wt b5R determined by double labeling. See Materials and methods for details on the experimental procedure. Calculation of the molar ratio of myristate to protein is based on the known specific radioactivities of the added compounds, and on the number of Met residues in the b5R sequence (8, not considering Met1). Any dilution of the specific radioactivity of 3H-MyrCoA or 35S-Met by the endogenous compounds is not considered. (C) Comparison of stoichiometry of in vitro myristoylation of wt b5R and b5Rext shows equal efficiency for the two proteins. wt b5R and b5Rext transcripts were translated in the wheat germ extract in the presence of 35S-Met and 3H-MyrCoA (21 μM). Calculation of the molar ratio of myristate to translated protein was as in B. Bars indicate the SEM (n = 5). (D) MyrCoA blunts the effect of SRP on the translation of wt b5R, but not of G2A and b5Rext. Transcripts coding for each of the three b5R forms were translated in wheat germ extract together with luciferase mRNA, 50 nM SRP, 35S-Met, and the indicated concentrations of unlabeled MyrCoA. For each concentration of MyrCoA, translation efficiency is the ratio of b5R band intensity to that of luciferase in the presence of SRP normalized to the same ratio in the absence of SRP, which was set at 100. Bars indicate the SEM (n = 5). (E) Inhibition by MyrCoA of the association of wt b5R nascent chains with SRP, assessed by cross-linking. Truncated mRNAs coding for the first 108 amino acids of wt b5R (lanes 1 and 2) and G2A (lanes 6 and 7), the first 113 amino acids of b5Rext (lanes 3–5), and the first 125 residues of cytochrome b(5) (lane 8) were translated for 30 min in reticulocyte lysate with or without 75 μM MyrCoA, as indicated. RNCs were recovered by centrifugation through a high salt sucrose cushion as detailed in the Materials and methods section. For each construct, equal amounts of TCA precipitable radioactivity were cross-linked with DSS and then immunoprecipitated with an anti-SRP54 antibody, with the exception of lane 5, in which precipitation was performed with a nonimmune serum. Adducts corresponding to cross-linked products of SRP and the nascent polypeptide chains of the three forms of reductase, but not of cytochrome b(5), were detected when anti-SRP54 was used for the immunoprecipitation. Note the strong inhibitory effect of added MyrCoA on wt b5R nascent chain cross-linking (lane 2), but not on the mutant b5R forms. Exposure times were 3 d for lanes 1–5 and 12 d for lanes 6–8.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Myristoylation of b5R forms in the wheat germ extract and effect of myristoylation on interaction with SRP. (A) wt b5R (lane 1), G2A (lane 2), and b5Rext (lane 3) were translated in the presence of unlabeled Met and 3H-MyrCoA (see Materials and methods for details). Immunoprecipitates were run on 11% SDS–polyacrylamide gels, followed by blotting and phosphorimaging analysis. wt b5R and its extended mutant are myristoylated, whereas G2A is not. (B) Stoichiometry of in vitro myristoylation of wt b5R determined by double labeling. See Materials and methods for details on the experimental procedure. Calculation of the molar ratio of myristate to protein is based on the known specific radioactivities of the added compounds, and on the number of Met residues in the b5R sequence (8, not considering Met1). Any dilution of the specific radioactivity of 3H-MyrCoA or 35S-Met by the endogenous compounds is not considered. (C) Comparison of stoichiometry of in vitro myristoylation of wt b5R and b5Rext shows equal efficiency for the two proteins. wt b5R and b5Rext transcripts were translated in the wheat germ extract in the presence of 35S-Met and 3H-MyrCoA (21 μM). Calculation of the molar ratio of myristate to translated protein was as in B. Bars indicate the SEM (n = 5). (D) MyrCoA blunts the effect of SRP on the translation of wt b5R, but not of G2A and b5Rext. Transcripts coding for each of the three b5R forms were translated in wheat germ extract together with luciferase mRNA, 50 nM SRP, 35S-Met, and the indicated concentrations of unlabeled MyrCoA. For each concentration of MyrCoA, translation efficiency is the ratio of b5R band intensity to that of luciferase in the presence of SRP normalized to the same ratio in the absence of SRP, which was set at 100. Bars indicate the SEM (n = 5). (E) Inhibition by MyrCoA of the association of wt b5R nascent chains with SRP, assessed by cross-linking. Truncated mRNAs coding for the first 108 amino acids of wt b5R (lanes 1 and 2) and G2A (lanes 6 and 7), the first 113 amino acids of b5Rext (lanes 3–5), and the first 125 residues of cytochrome b(5) (lane 8) were translated for 30 min in reticulocyte lysate with or without 75 μM MyrCoA, as indicated. RNCs were recovered by centrifugation through a high salt sucrose cushion as detailed in the Materials and methods section. For each construct, equal amounts of TCA precipitable radioactivity were cross-linked with DSS and then immunoprecipitated with an anti-SRP54 antibody, with the exception of lane 5, in which precipitation was performed with a nonimmune serum. Adducts corresponding to cross-linked products of SRP and the nascent polypeptide chains of the three forms of reductase, but not of cytochrome b(5), were detected when anti-SRP54 was used for the immunoprecipitation. Note the strong inhibitory effect of added MyrCoA on wt b5R nascent chain cross-linking (lane 2), but not on the mutant b5R forms. Exposure times were 3 d for lanes 1–5 and 12 d for lanes 6–8.
Mentions: The comparable interaction of wt and G2A b5R nascent chains with SRP could have been due either to a lack of effect of N-myristoylation on this interaction or to low or no myristoylation occurring in vitro. Although the wheat germ extract is equipped with the enzymes required for N-myristoylation (Deichaite et al., 1988), the concentration of endogenous Myristoyl-CoA (MyrCoA) in the extract provided by Promega could be insufficient to support the reaction. When 3H-MyrCoA was included in the translation mixes (prepared with unlabeled Met), wt b5R and the extended mutant were myristoylated, whereas G2A b5R was not (Fig. 6 A). To obtain a quantitative estimate of the degree of in vitro myristoylation of wt b5R, 35S-Met and 3H-MyrCoA of known specific activities were included together in the same incubation. After SDS-PAGE and blotting onto nitrocellulose, the radioactive b5R band was excised, solubilized, and counted in double label mode. From the 3H/35S ratio and the known methionine content of b5R, we were able to calculate the amount of added myristic acid attached to the in vitro–synthesized protein. As seen in Fig. 6 B, the calculated ratio increased with increasing concentration of MyrCoA, reaching a value of ∼1 at 120 μM MyrCoA. We could not test the effect of higher concentrations of MyrCoA because they interfered with in vitro translation. However, because a 1:1 stoichiometry of myristic acid to protein is the maximum value attainable, the result indicates that MyrCoA endogenous to the wheat germ extract does not significantly contribute to the reaction. Thus, the MyrCoA concentration in these extracts appears to be low, and limiting for the myristoylation reaction.

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