Tonoplast subcellular localization of maize cytochrome b5 reductases

Plant cytochrome b(5) reductases (b(5)R) are assumed to be part of an ER-associated redox chain that oxidizes NADH to provide electrons via cytochrome b(5) (cyt b(5)) to ER-associated fatty acyl desaturase and related hydroxylases, as in mammalian cells. Here we report on cDNA cloning of a novel maize b5R, NFR II, strongly related to a previously cloned cDNA, NFR I (Bagnaresi ef al., 1999, Biochem. J. 338, 499-505). Maize b(5)R isoforms are produced by a small multi-gene family. The NFR cDNAs were shown to encode active b(5)Rs by heterologous expression in yeast. Both reductases. in addition to Fe3+-chelates, efficiently reduced Cu2+-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(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(5)R. This indicates that alternative ER-associated electron donors for desaturases need to be sought, and that plant b(5)Rs may have previously unexpected functions.