Contributions of the structural domains of filensin in polymer formation and filament distribution.
Filensin and phakinin constitute the subunits of a heteropolymeric, lens-specific intermediate filament (IF) system known as the beaded-chain filaments (BFs). Since the rod of filensin is four heptads shorter than the rods of all other IF proteins, we decided to examine the specific contribution of this protein in filament assembly. For these purposes, we constructed chimeric proteins in which regions of filensin were exchanged with the equivalent ones of vimentin, a self-polymerizing IF protein. Our in vitro studies show that the filensin rod domain does not allow homopolymeric filament elongation. However, the filensin rod is necessary for co-polymerization of filensin with phakinin and seems to counteract the inherent tendency of the latter protein to homopolymerize into large, laterally associated filament bundles. Apart from the rod domain, the presence of an authentic or substituted tail domain in filensin is also essential for co-assembly with the naturally tail-less phakinin and formation of extended filaments in vitro. Finally, transfection experiments in CHO and MCF-7 cells show that the rod domain of filensin plays an important role in de novo filament formation and distribution. The same type of analysis further suggests that the end-domains of filensin interact with cell-specific, assembly-modulating factors.
Goulielmos
G
G
Program of Cell Biology, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Remington
S
S
Schwesinger
F
F
Georgatos
S D
SD
Gounari
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D017931:DNA Primers; D005136:Eye Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007382:Intermediate Filaments / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D011108:Polymers; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D011108:Polymers; D011993:Recombinant Fusion Proteins; C079098:filensin