Tonoplast subcellular localization of maize cytochrome b(5) reductases

Plant cytochrome b(5) reductases (b(5)R) are assumed to be part of an ER-as sociated redox chain that oxidizes NADH to provide electrons via cytochrome b(5) (cyt b(5)) to ER-associated fatty acyl desaturase and related hydroxy lases, as in mammalian cells. Here we report on cDNA cloning of a novel mai ze b5R, NFR II, strongly related to a previously cloned cDNA, NFR I (Bagnar esi ef al., 1999, Biochem. J. 338, 499-505). Maize b(5)R isoforms are produ ced 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 t o Fe3+-chelates, efficiently reduced Cu2+-chelates. Using a polyclonal anti body able to recognize both NFR I and NFR II isoforms, no ER or mitochondri al localization could be detected in maize roots. Unexpectedly, maize b(5)R s were found to be targeted to the tonoplast. Using the most specific assay to measure NFR activity, we confirmed that the highest NFR specific activi ty is associated with tonoplast-enriched maize root fractions. Tonoplast ta rgeting is not consistent with a role in desaturase reactions or with the o ther functions ascribed to date to plant b(5)R. This indicates that alterna tive ER-associated electron donors for desaturases need to be sought, and t hat plant b(5)Rs may have previously unexpected functions.