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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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Schematic representation of the NH2-terminal regions of the three constructs used in this study. (A) The residues that constitute the myristoylation consensus are in boldface, the Gly that accepts the myristoyl moiety is boxed. This residue is mutated to Ala in G2A b5R. The rectangle downstream to Arg10 represents the hydrophobic region in all three constructs; this region is lengthened by five residues (filled part of the rectangle) in b5Rext. (B) Amino acid sequence (one-letter code) of the hydrophobic region (flanked by a basic residue on both sides) of the three constructs. The residues inserted in b5Rext are shown in boldface. The average hydrophobicity of the 12 residues after Arg10 was calculated according to the STA PRIFT scale (Cornette et al., 1987), and is displayed in italics to the right of the sequences.
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fig1: Schematic representation of the NH2-terminal regions of the three constructs used in this study. (A) The residues that constitute the myristoylation consensus are in boldface, the Gly that accepts the myristoyl moiety is boxed. This residue is mutated to Ala in G2A b5R. The rectangle downstream to Arg10 represents the hydrophobic region in all three constructs; this region is lengthened by five residues (filled part of the rectangle) in b5Rext. (B) Amino acid sequence (one-letter code) of the hydrophobic region (flanked by a basic residue on both sides) of the three constructs. The residues inserted in b5Rext are shown in boldface. The average hydrophobicity of the 12 residues after Arg10 was calculated according to the STA PRIFT scale (Cornette et al., 1987), and is displayed in italics to the right of the sequences.

Mentions: Like members of the bcl-2 family, most of the mass of b5R, comprising its FAD and NADH binding domains, is exposed to the cytosol. However, whereas bcl-2 proteins are C-tail anchored, b5R is bound to the bilayer by a short anchor at its NH2 terminus. This region consists of a moderately hydrophobic stretch of 14 amino acids preceded by a myristoylation consensus sequence (see Fig. 1). The NH2-terminal glycine of the membrane-bound form of b5R is indeed myristoylated (Ozols et al., 1984), and this modification is present in a 1:1 molar ratio to protein in both the MOM- and the ER-associated forms (Borgese and Longhi, 1990). N-myristoylation is a cotranslational, generally irreversible modification that is often necessary, although not sufficient, for membrane binding of the modified protein (for review see Resh, 1999). In the case of b5R, however, myristoylation does not detectably alter the strength of the enzyme's association with phospholipid bilayers because the 14-residue-long hydrophobic stretch is sufficient to tightly anchor the nonmyristoylated form to both artificial (Strittmatter et al., 1993) and ER membranes (Borgese et al., 1996). Myristoylation also does not affect the catalytic activity of the enzyme (Strittmatter et al., 1993; Borgese et al., 1996). Instead, this modification has a pronounced effect on targeting of b5R because the nonmyristoylated mutant (in which the acceptor Gly is mutated to Ala) is no longer delivered to mitochondria and localizes exclusively to the ER in cultured mammalian cells (Borgese et al., 1996). Thus, in the case of b5R, N-linked myristate appears to have a specific role in targeting rather than functioning simply as a membrane anchor.


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)

Schematic representation of the NH2-terminal regions of the three constructs used in this study. (A) The residues that constitute the myristoylation consensus are in boldface, the Gly that accepts the myristoyl moiety is boxed. This residue is mutated to Ala in G2A b5R. The rectangle downstream to Arg10 represents the hydrophobic region in all three constructs; this region is lengthened by five residues (filled part of the rectangle) in b5Rext. (B) Amino acid sequence (one-letter code) of the hydrophobic region (flanked by a basic residue on both sides) of the three constructs. The residues inserted in b5Rext are shown in boldface. The average hydrophobicity of the 12 residues after Arg10 was calculated according to the STA PRIFT scale (Cornette et al., 1987), and is displayed in italics to the right of the sequences.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Schematic representation of the NH2-terminal regions of the three constructs used in this study. (A) The residues that constitute the myristoylation consensus are in boldface, the Gly that accepts the myristoyl moiety is boxed. This residue is mutated to Ala in G2A b5R. The rectangle downstream to Arg10 represents the hydrophobic region in all three constructs; this region is lengthened by five residues (filled part of the rectangle) in b5Rext. (B) Amino acid sequence (one-letter code) of the hydrophobic region (flanked by a basic residue on both sides) of the three constructs. The residues inserted in b5Rext are shown in boldface. The average hydrophobicity of the 12 residues after Arg10 was calculated according to the STA PRIFT scale (Cornette et al., 1987), and is displayed in italics to the right of the sequences.
Mentions: Like members of the bcl-2 family, most of the mass of b5R, comprising its FAD and NADH binding domains, is exposed to the cytosol. However, whereas bcl-2 proteins are C-tail anchored, b5R is bound to the bilayer by a short anchor at its NH2 terminus. This region consists of a moderately hydrophobic stretch of 14 amino acids preceded by a myristoylation consensus sequence (see Fig. 1). The NH2-terminal glycine of the membrane-bound form of b5R is indeed myristoylated (Ozols et al., 1984), and this modification is present in a 1:1 molar ratio to protein in both the MOM- and the ER-associated forms (Borgese and Longhi, 1990). N-myristoylation is a cotranslational, generally irreversible modification that is often necessary, although not sufficient, for membrane binding of the modified protein (for review see Resh, 1999). In the case of b5R, however, myristoylation does not detectably alter the strength of the enzyme's association with phospholipid bilayers because the 14-residue-long hydrophobic stretch is sufficient to tightly anchor the nonmyristoylated form to both artificial (Strittmatter et al., 1993) and ER membranes (Borgese et al., 1996). Myristoylation also does not affect the catalytic activity of the enzyme (Strittmatter et al., 1993; Borgese et al., 1996). Instead, this modification has a pronounced effect on targeting of b5R because the nonmyristoylated mutant (in which the acceptor Gly is mutated to Ala) is no longer delivered to mitochondria and localizes exclusively to the ER in cultured mammalian cells (Borgese et al., 1996). Thus, in the case of b5R, N-linked myristate appears to have a specific role in targeting rather than functioning simply as a membrane anchor.

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