Abstract
gCap39 is an actin filament end-capping protein which has a threefold repeated domain structure similar to the N-terminal half of gelsolin. However, unlike gelsolin, gCap39 does not sever actin filaments and dissociates completely from filament ends after calcium removal. We have capitalized on these differences to explore the structural basis for actin filament capping, severing, and their regulation. Using truncated gCap39, generated by limited proteolysis or deletion mutagenesis, we found that actin filament capping requires multiple gCap domains, and almost the entire molecule is necessary for optimal activity. gCap39 domain I, like the equivalent domain in gelsolin, contains an actin monomer binding site. gCap39 domains II-III are, however, different from gelsolin in that they do not bind to the side of actin filaments. Since filament side binding is hypothesized to be the first step in severing, lack of side binding may explain why gCap39 does not sever. This is confirmed directly by swapping gCap39 domains II-III for the side-binding gelsolin domains to generate a chimera which severs actin filaments. The chimera is Ca2+ independent in actin filament severing and capping, although gCap39 domain I itself is regulated by Ca2+.
Highlights
In spite of 49% amino acid identity to gelsolin and similar structural organization into repeated domains [1, 5],gCap39 does not sever actin filaments, while all other gelsolin-like proteins sever actin filamentsand cap their ends[6,7,8]. Since this family of proteins is likely to be important in regulating actin polymerization and organization in cells (9, lo), we and others have sought to identify the actin-binding domains required for these functions [11,12,13]. gCap39 is the only nonsevering member of the gelsolin family of proteins and is, suitable for studying capping without interference from severing
C-terminal Truncated Constructs-gCap39 cDNA in pGEM4Z was digested with Sac1, blunted with T4 DNA polymerase and ligated with BamHI linkers
The DNA was digested with BamHI, gel-purified and self-ligated.The plasmid DNA containing the new BamHI site was digested with PstI, PpuMI, or NarI, blunted by Klenow or T4 DNA polymerase, and ligated to BamHI linkers with multiple stop codons (GTAGCTGACTAGGATC and CTAGTCAGCTAC)
Summary
GCap39 is a newly identified Ca2+-and polyphosphoinositide-regulated actin filament “barbed” end-capping protein and a unique member of the extensive gelsolin family [1,2,3,4]. In spite of 49% amino acid identity to gelsolin and similar structural organization into repeated domains [1, 5],gCap39 does not sever actin filaments, while all other gelsolin-like proteins sever actin filamentsand cap their ends[6,7,8].
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