Membrane-binding properties of filensin, a cytoskeletal protein of the lens fiber cells.
Filensin is a 100/110 kDa membrane-associated protein found in lens fiber cells. Previous studies have shown that this protein polymerizes in vitro and binds strongly to vimentin and to another 47 kDa lens membrane protein. Using cosedimentation assays, flotation assays and immunoelectron microscopy, we have examined the properties of purified filensin and measured its binding to lens membranes. Filensin behaves as a ureaextractable, hydrophilic protein which does not partition with Triton X-114 and is not affected by 1 M hydroxylamine at alkaline pH, an agent known to release fatty-acylated proteins from the membrane. Immunoblotting of urea-extracted lens membranes with two different affinity-purified antibodies reveals that, unlike intact filensin, a COOH-terminal filensin degradation product (51 kDa) remains tightly associated with the membranes. Purified filensin binds directly to urea-stripped lens membranes, but not to protein-free vesicles reconstituted from total lens lipids. The binding of filensin is not significantly influenced by the purified 47 kDa protein. Interestingly, the filensin-binding capacity of urea-extracted membranes is increased at least two-fold after trypsin treatment, which removes entirely the 51 kDa peptide from the membranes and presumably unmasks additional filensin-acceptor sites. Consistent with this, filensin binds to trypsinized and non-trypsinized membranes with similar affinities (2 x 10(-7) and 4 x 10(-7) M, respectively). Treatment of the membranes with thrombin, which also eliminates the 51 kDa peptide, does not increase their binding capacity, apparently because filensin-acceptor sites are also destroyed during proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Brunkener
M
M
Program of Cell Biology, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Georgatos
S D
SD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007381:Intermediate Filament Proteins; D007908:Lens, Crystalline / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D013552:Swine
D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; C079098:filensin