Tonoplast subcellular localization of maize cytochrome b sub(5) reductases

Plant cytochrome b sub(5) reductases (b sub(5)R) are assumed to be part of an ER-associated redox chain that oxidizes NADH to provide electrons via cytochrome b sub(5) (cyt b sub(5)) to ER-associated fatty acyl desaturase and related hydroxylases, as in mammalian cells. Here we report on cDNA cloning of a novel maize b sub(5)R, NFR II, strongly related to a previously cloned cDNA, NFR I (Bagnaresi et al., 1999, Biochem. J. 338, 499-505). Maize b sub(5)R isoforms are produced by a small multi-gene family. The NFR cDNAs were shown to encode active b sub(5)Rs by heterologous expression in yeast. Both reductases, in addition to Fe super(3+)-chelates, efficiently reduced Cu super(2+)-chelates. Using a polyclonal antibody able to recognize both NFR I and NFR II isoforms, no ER or mitochondrial localization could be detected in maize roots. Unexpectedly, maize b sub(5)Rs were found to be targeted to the tonoplast. Using the most specific assay to measure NFR activity, we confirmed that the highest NFR specific activity is associated with tonoplast-enriched maize root fractions. Tonoplast targeting is not consistent with a role in desaturase reactions or with the other functions ascribed to date to plant b sub(5)R. This indicates that alternative ER-associated electron donors for desaturases need to be sought, and that plant b sub(5)Rs may have previously unexpected functions.