The initial events of vision at low light take place in vertebrate retinal rods. The rod outer segmentconsists of a stack of flattened disks surrounded by the plasma membrane. A list of the proteins thatreside in disks has not been achieved yet. We present the first comprehensive proteomic analysis ofpurified rod disks, obtained by combining the results of two-dimensional gel electrophoresis separationof disk proteins to MALDI-TOF or nLC-ESI-MS/MS mass spectrometry techniques. Intact disks wereisolated from bovine retinal rod outer segments by a method that minimizes contamination from innersegment. Out of a total of 187 excised spots, 148 proteins were unambiguously identified. An additionalset of 61 proteins (partially overlapping with the previous ones) was generated by one-dimensional(1D) gel nLC-ESI-MS/MS method. Proteins involved in vision as well as in aerobic metabolism werefound, among which are the five complexes of oxidative phosphorylation. Results from biochemical,Western blot, and confocal laser scanning microscopy immunochemistry experiments suggest thatF1Fo-ATP synthase is located and catalytically active in ROS disk membranes. This study represents astep toward a global physiological characterization of the disk proteome and provides informationnecessary for future studies on energy supply for phototransduction.
Proteomic analysis of the retinal rod outer segment disks
TACCHETTI, CARLO;
2008-01-01
Abstract
The initial events of vision at low light take place in vertebrate retinal rods. The rod outer segmentconsists of a stack of flattened disks surrounded by the plasma membrane. A list of the proteins thatreside in disks has not been achieved yet. We present the first comprehensive proteomic analysis ofpurified rod disks, obtained by combining the results of two-dimensional gel electrophoresis separationof disk proteins to MALDI-TOF or nLC-ESI-MS/MS mass spectrometry techniques. Intact disks wereisolated from bovine retinal rod outer segments by a method that minimizes contamination from innersegment. Out of a total of 187 excised spots, 148 proteins were unambiguously identified. An additionalset of 61 proteins (partially overlapping with the previous ones) was generated by one-dimensional(1D) gel nLC-ESI-MS/MS method. Proteins involved in vision as well as in aerobic metabolism werefound, among which are the five complexes of oxidative phosphorylation. Results from biochemical,Western blot, and confocal laser scanning microscopy immunochemistry experiments suggest thatF1Fo-ATP synthase is located and catalytically active in ROS disk membranes. This study represents astep toward a global physiological characterization of the disk proteome and provides informationnecessary for future studies on energy supply for phototransduction.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
