A role for N-myristoylation in protein targeting: NADH-cytochrome b5 reductase requires myristic acid for association with outer mitochondrial but not ER membranes

Nica Borgese, Diego Aggujaro, Paola Carrera, Grazia Pietrini, Monique Bassetti

Research output: Contribution to journalArticle

Abstract

N-myristoylation is a cotranslational modification involved in protein- protein interactions as well as in anchoring polypeptides to phospholipid bilayers; however, its role in targeting proteins to specific subcellular compartments has not been clearly defined. The mammalian myristoylated flavoenzyme NADH-cytochrome b5 reductase is integrated into ER and mitochondrial outer membranes via an anchor containing a stretch of 14 uncharged amino acids downstream to the NH2-terminal myristoylated glycine. Since previous studies suggested that the anchoring function could be adequately carried out by the 14 uncharged residues, we investigated a possible role for myristic acid in reductase targeting. The wild type (wt) and a nonmyristoylatable reductase mutant (gly2→ala) were stably expressed in MDCK cells, and their localization was investigated by immunofluorescence, immuno-EM, and cell fractionation. By all three techniques, the wt protein localized to ER and mitochondria, while the nonmyristoylated mutant was found only on ER membranes. Pulse-chase experiments indicated that this altered steady state distribution was due to the mutant's inability to target to mitochondria, and not to its enhanced instability in that location. Both wt and mutant reductase were resistant to Na2CO3 extraction and partitioned into the detergent phase after treatment of a membrane fraction with Triton X-114, demonstrating that myristic acid is not required for tight anchoring of reductase to membranes. Our results indicate that myristoylated reductase localizes to ER and mitochondria by different mechanisms, and reveal a novel role for myristic acid in protein targeting.

Original languageEnglish
Pages (from-to)1501-1513
Number of pages13
JournalJournal of Cell Biology
Volume135
Issue number6
DOIs
Publication statusPublished - 1996

ASJC Scopus subject areas

  • Cell Biology

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