A single mRNA, transcribed from an alternative, erythroid-specific, promoter, codes for two non-myristylated forms of NADH-cytochrome b5 reductase

Abstract

Abstract. Two forms of NADH-cytochrome b5 reductase are produced from one gene: a myristylated membrane-bound enzyme, expressed in all tissues, and a soluble, erythrocyte-specific, isoform. The two forms are identical in a large cytoplasmic domain (Mr = 30,000) and differ at the NH2-terminus, which, in the membrane form, is responsible for binding to the bilayer, and which contains the myristylation consensus sequence and an additional 14 uncharged amino acids. To investigate how the two differently targeted forms of the reductase are produced, we cloned a reductase transcript from reticulocytes, and studied its relationship to the previously cloned liver cDNA. The reticulocyte transcript differs from the liver transcript in the 5' non-coding portion and at the beginning of the coding portion, where the seven codons specifying the myristoylatlon consensus are replaced by a reticulocytespecific sequence which codes for 13 non-charged amino acids. Analysis of genomic reductase clones indicated that the ubiquitous transcript is generated from an upstream "housekeeping" type promoter, while the reticulocyte transcript originates from a downstream, erythroid-specific, promoter. In vitro translation of the reticulocyte-specific mRNA generated two products: a minor one originating from the first AUG, and a major one starting from a downstream AUG, as indicated by mutational analysis. Both the AUGs used as initiation codons were in an unfavorable sequence context. The major, lower relative molecular mass product behaved as a soluble protein, while the NH2-terminally extended minor product interacted with microsomes in vitro. The generation of soluble reductase from a downstream AUG was confirmed in vivo, in Xenopus oocytes. Thus, differently localized products, with respect both to tissues and to subcellular compartments, are generated from the same gene by a combination of transcriptional and translational mechanisms.

Determination of Concentration of Cytosolic NADH-Cytochrome b5 Reductase in Erythrocytes from Normal Chinese Adults, Neonates and Patients with Hereditary Methemoglobinemia by Double-Antibody Sandwich ELISA

“…There is evidence suggesting that b5R bound to red cell membrane may be different from that bound to somatic cellular membranes [11]. The controversy over whether erythrocyte cytosolic soluble b5R is a posttranslational derivative of membrane-bound b5R or is derived directly from its own template remains unsolved [12, 13]. …”

Nuclear Localization of NADPH:Cytochromec(P450) Reductase Enhances the Cytotoxicity of Mitomycin C to Chinese Hamster Ovary Cells

“…These findings are consistent with the production of the semiquinone anion radical by NBR and the subsequent redox cycling of this intermediate in the presence of oxygen. NPR resides in the endoplasmic reticulum, whereas NBR is known to localize predominantly in the mitochondrial outer membrane (Pietrini et al, 1992) and NQO1 is located in the cytosol (Seow et al, 2004a). The locations of major MC-activating enzymes suggest that activation of this antineoplastic agent in a cellular region distal to its presumed cytotoxic target, nuclear DNA, may not result in optimal cell killing.…”

The Intracellular Location of NADH:Cytochromeb5 Reductase Modulates the Cytotoxicity of the Mitomycins to Chinese Hamster Ovary Cells

“…The expression of the FpD gene involves a combination of transcriptional and translational mechanisms. Two transcripts encoding the three known forms of the rat FpD enzyme are produced from a single gene (22,23). One transcript produces the myristylated membrane-bound enzyme and is expressed in all tissues.…”