The disks of the vertebrate retinal rod Outer Segment (OS), devoid of mitochondria, are the site ofvisual transduction, a very energy demanding process. In a previous proteomic study we reported theexpression of the respiratory chain complexes I–IV and the oxidative phosphorylation Complex V (F1F0-ATP synthase) in disks. In the present study, the functional localization of these proteins in disks wasinvestigated by biochemical analyses, oxymetry, membrane potential measurements, and confocal laserscanning microscopy. Disk preparations, isolated by Ficoll flotation, were characterized for purity. Anoxygen consumption, stimulated by NADH and Succinate and reverted by rotenone, antimycin A andKCN was measured in disks, either in coupled or uncoupled conditions. Rhodamine-123 fluorescencequenching kinetics showed the existence of a proton potential difference across the disk membranes.Citrate synthase activity was assayed and found enriched in disks with respect to ROS. ATP synthesisby disks (0.7mol ATP/min/mg), sensitive to the common mitochondrial ATP synthase inhibitors, wouldlargely account for the rod ATP need in the light.Overall, data indicate that an oxidative phosphorylation occurs in rod OS, which do not contain mitochondria,thank to the presence of ectopically located mitochondrial proteins. These findings may provideimportant new insight into energy production in outer segments via aerobic metabolism and additionalinformation about protein components in OS disk membranes.
Evidence for aerobic metabolism in retinal rod outer segment disks
TACCHETTI, CARLO;
2009-01-01
Abstract
The disks of the vertebrate retinal rod Outer Segment (OS), devoid of mitochondria, are the site ofvisual transduction, a very energy demanding process. In a previous proteomic study we reported theexpression of the respiratory chain complexes I–IV and the oxidative phosphorylation Complex V (F1F0-ATP synthase) in disks. In the present study, the functional localization of these proteins in disks wasinvestigated by biochemical analyses, oxymetry, membrane potential measurements, and confocal laserscanning microscopy. Disk preparations, isolated by Ficoll flotation, were characterized for purity. Anoxygen consumption, stimulated by NADH and Succinate and reverted by rotenone, antimycin A andKCN was measured in disks, either in coupled or uncoupled conditions. Rhodamine-123 fluorescencequenching kinetics showed the existence of a proton potential difference across the disk membranes.Citrate synthase activity was assayed and found enriched in disks with respect to ROS. ATP synthesisby disks (0.7mol ATP/min/mg), sensitive to the common mitochondrial ATP synthase inhibitors, wouldlargely account for the rod ATP need in the light.Overall, data indicate that an oxidative phosphorylation occurs in rod OS, which do not contain mitochondria,thank to the presence of ectopically located mitochondrial proteins. These findings may provideimportant new insight into energy production in outer segments via aerobic metabolism and additionalinformation about protein components in OS disk membranes.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
