Caenorhabditis elegans Gelsolin-like Protein 1 Is a Novel Actin Filament-severing Protein with Four Gelsolin-like Repeats

Abstract

The gelsolin family of proteins is a major class of actin regulatory proteins that sever, cap, and nucleate actin filaments in a calcium-dependent manner and are involved in various cellular processes. Typically, gelsolin-related proteins have three or six repeats of gelsolin-like (G) domain, and each domain plays a distinct role in severing, capping, and nucleation. The Caenorhabditis elegans gelsolin-like protein-1 (gsnl-1) gene encodes an unconventional gelsolin-related protein with four G domains. Sequence alignment suggests that GSNL-1 lacks two G domains that are equivalent to fourth and fifth G domains of gelsolin. In vitro, GSNL-1 severed actin filaments and capped the barbed end in a calcium-dependent manner. However, unlike gelsolin, GSNL-1 remained bound to the side of F-actin with a submicromolar affinity and did not nucleate actin polymerization, although it bound to G-actin with high affinity. These results indicate that GSNL-1 is a novel member of the gelsolin family of actin regulatory proteins and provide new insight into functional diversity and evolution of gelsolin-related proteins.

Calcium-sensitive Activity and Conformation of Caenorhabditis elegans Gelsolin-like Protein 1 Are Altered by Mutations in the First Gelsolin-like Domain

“…However, unlike gelsolin, GSNL-1 remains bound to the side of actin filaments and does not nucleate actin polymerization (14). Analysis of the domainfunction relationship of GSNL-1 shows that G1 and the linker between G1 and G2 are sufficient for actin filament severing in a similar manner to the equivalent part of vertebrate gelsolin (15).…”

“…The critical sequence in G1 is "LDDY" at the C-terminal end of the long helix, whereas the two acidic residues are not conserved in CapG (20). GSNL-1 has "IDDS" at the equivalent position (14). Therefore, we reasoned that the two aspartic acid residues are important for actin-severing activity of GSNL-1.…”

“…Pyrene-labeled actin was prepared as described (22). Bacterially expressed full-length GSNL-1 was purified as described previously (14). Phosphatidylinositol 4,5-bisphosphate (PIP2) (P-4516, Echelon Biosciences) was suspended in water at 1 mg/ml with brief sonication and stored at Ϫ20°C.…”

“…The sequences of protein coding regions were verified by DNA sequencing. Both wild-type and mutant full-length GSNL-1 and the G1b fragment were expressed as fusion proteins with glutathione S-transferase (GST) in Escherichia coli strain BL21(DE3) and purified as described previously for wild-type proteins (14,15). Briefly, E. coli lysates were applied to a glutathione Uniflow (Clontech) column to adsorb the GST fusion proteins, and then the GSNL-1 variants were cleaved by thrombin (Roche Applied Science) on beads and eluted from the column.…”

“…We have previously reported that Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1), which has only four G domains (G1-G4), severs actin filaments and caps barbed ends in a similar manner to gelsolin (14). However, unlike gelsolin, GSNL-1 remains bound to the side of actin filaments and does not nucleate actin polymerization (14).…”