In vitro studies on the assembly properties of the lens proteins CP49, CP115: coassembly with alpha-crystallin but not with vimentin.
A rapid one-step purification procedure for CP49, an intermediate filament protein found in the lens, is described using reverse-phase HPLC. This protein is one of the major intermediate filament proteins of the lens fibre cells and is found in both the water insoluble fraction (WIF) and the water soluble fraction (WSF) of the lens. In order to better understand the physiological role of CP49 in lens transparency we have purified CP49 from both compartments and compared the in vitro assembly characteristics of both by electron microscopy and sedimentation assays. Our studies showed that CP49, when mixed with another lens intermediate filament protein, CP115, forms 10 nm intermediate filaments. Vimentin, another intermediate filament protein found in the lens, was unable to coassemble with CP115, thus demonstrating the specificity of the interaction of CP49 with CP115. CP49 isolated from either the WIF or the WSF formed 10-nm filaments with CP115 and indicated that CP49 from both these lens cell compartments had similar in vitro assembly characteristics. This also suggested that the post-translational modifications observed for CP49 from the different compartments was of little apparent consequence to filament formation. The inability to reconstitute beaded filaments from CP49 and CP115 suggested that other lens proteins may be needed in the reconstitution assay before these lens specific cytoskeletal elements could be repolymerised from their purified protein components. CP49 and CP115 were therefore assembled in the presence of alpha-crystallins and a beaded filament structure was observed as has been seen with type III intermediate filament proteins assembled with alpha-crystallins.
Carter
J M
JM
Department of Biochemistry, The University, Dundee, U.K.
Hutcheson
A M
AM
Quinlan
R A
RA
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000737:chemistry* / Q000648:ultrastructure; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D007381:Intermediate Filament Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D007908:Lens, Crystalline / Q000737:chemistry*; D008854:Microscopy, Electron; D014746:Vimentin / Q000737:chemistry / Q000648:ultrastructure
D003459:Crystallins; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D014746:Vimentin; C079098:filensin; C084808:phakinin