The 53kDa polypeptide component of the bovine fibre cell cytoskeleton is derived from the 115kDa beaded filament protein: evidence for a fibre cell specific intermediate filament protein.
The 115kDa protein found enriched in the PMCC (plasma membrane-cytoskeleton complex) fraction of the cortex in bovine lens fibre cells is proteolytically processed to a stable 53kDa product. The 115 kDa protein and the 53kDa polypeptide have been purified by a combination of ion exchange and hydroxyapatite chromatography. Tryptic peptide mapping using reverse phase HPLC and subsequent peptide sequencing confirmed that the 53kDa polypeptide is derived from the 115kDa protein. The 53kDa fragment is also a component of the PMCC as well as being a major component of the urea soluble fraction of lens plasma membranes which have been extracted with buffers containing 1M KC1. The 53kDa polypeptide has escaped identification as a breakdown product of the 115kDa protein because it is not recognised by a commonly used monoclonal antibody, R2D2, specific for the bovine 115kDa protein. This result suggests that proteolysis is important in determining the function(s) of the 115kDa protein, and that part of this function is satisfied by the 53kDa protein core. Both the purified 115kDa protein and the 53kDa polypeptide were unable to form either beaded or intermediate filaments on their own but they were able to form short 10nm rods indicative of an intermediate stage in intermediate filament assembly. Comparison ot the assembly properties of the 53 and 115kDa proteins indicate that there are sequences in the 115kDa protein which inhibit in vitro assembly. This is similar to the situation with neurofilament proteins. We suggest that the 115kDa protein is a lens-specific intermediate filament protein.
Quinlan
R A
RA
Department of Biochemistry, Medical Sciences Institute, The University, Dundee, UK.
Carter
J M
JM
Hutcheson
A M
AM
Campbell
D G
DG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003599:Cytoskeleton / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007904:Lens Cortex, Crystalline / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D007381:Intermediate Filament Proteins; D010455:Peptides