A ROLE FOR N-MYRISTOYLATION IN PROTEIN TARGETING - NADH-CYTOCHROME B(5) REDUCTASE REQUIRES MYRISTIC ACID FOR ASSOCIATION WITH OUTER MITOCHONDRIAL BUT NOT ER MEMBRANES

N-myristoylation is a cotranslational modification involved in protein -protein interactions as well as in anchoring polypeptides to phosphol ipid bilayers; however, its role in targeting proteins to specific sub cellular compartments has not been clearly defined. The mammalian myri stoylated flavoenzyme NADH-cytochrome bg reductase is integrated into ER and mitochondrial outer membranes via an anchor containing a stretc h of 14 uncharged amino acids downstream to the NH2-terminal myristoyl ated glycine, Since previous studies suggested that the anchoring func tion 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 i nvestigated by immunofluorescence, immuno-EM, and cell fractionation. By all three techniques, the wt protein localized to ER and mitochondr ia, while the nonmyristoylated mutant was found only on ER membranes, Pulse-chase experiments indicated that this altered steady state distr ibution was due to the mutant's inability to target to mitochondria, a nd not to its enhanced instability in that location. Both wt and mutan t reductase were resistant to Na2CO3 extraction and partitioned into t he detergent phase after treatment of a membrane fraction with Triton X-114, demonstrating that myristic acid is not required for tight anch oring of reductase to membranes. Our results indicate that myristoylat ed reductase localizes to ER and mitochondria by different mechanisms, and reveal a novel role for myristic acid in protein targeting.