Proteomic analysis of the retinal rod outer segment disks.
The initial events of vision at low light take place in vertebrate retinal rods. The rod outer segment consists of a stack of flattened disks surrounded by the plasma membrane. A list of the proteins that reside in disks has not been achieved yet. We present the first comprehensive proteomic analysis of purified rod disks, obtained by combining the results of two-dimensional gel electrophoresis separation of disk proteins to MALDI-TOF or nLC-ESI-MS/MS mass spectrometry techniques. Intact disks were isolated from bovine retinal rod outer segments by a method that minimizes contamination from inner segment. Out of a total of 187 excised spots, 148 proteins were unambiguously identified. An additional set 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 were found, among which are the five complexes of oxidative phosphorylation. Results from biochemical, Western blot, and confocal laser scanning microscopy immunochemistry experiments suggest that F 1F o-ATP synthase is located and catalytically active in ROS disk membranes. This study represents a step toward a global physiological characterization of the disk proteome and provides information necessary for future studies on energy supply for phototransduction.
Panfoli
Isabella
I
Department of Biology, University of Genoa, V.le Benedetto XV 3, 16132 Genova, Italy. Isabella.Panfoli@unige.it, bachi.angela@hsr.it
Musante
Luca
L
Bachi
Angela
A
Ravera
Silvia
S
Calzia
Daniela
D
Cattaneo
Angela
A
Bruschi
Maurizio
M
Bianchini
Paolo
P
Diaspro
Alberto
A
Morelli
Alessandro
A
Pepe
Isidoro M
IM
Tacchetti
Carlo
C
Candiano
Giovanni
G
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D021122:Protein Subunits / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D021122:Protein Subunits; D020543:Proteome; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases