Abstract
Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized “orphan” Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1–65Arp7A·LIM2–3Tes·EVH1Mena complex reveals that residues 28–49 of Arp7A contact the LIM2–3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2–3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.
Highlights
Actin, one of the major components of the eukaryotic cytoskeleton, is an evolutionarily conserved protein that has structural and functional homologues in prokaryotes [1,2,3,4]
We have extended these earlier studies and shown that Arp7A is localized in the subacrosomal layer, which is known as the acroplaxome [40]
The acroplaxome is a morphologically distinct junctional complex that is thought to play an important role in anchoring the acrosome to the nuclear envelope of round spermatids [41]
Summary
Antibodies and Immunofluorescence Analysis—The Arp7A antibody was produced by immunization of rabbits with a peptide corresponding to residues 1– 65 of human Arp7A. Monoclonal antibodies against actin (AC74 and AC40 Sigma; MAB1501R Chemicon International); ␣-actinin (MAB1682 Chemicon International); GFP (3E1) and Tes (1A9, 7F6, and 5E6) (Cancer Research UK); GM130 (2C10, Abcam); Mena [21] and paxillin (m349) (BD Biosciences), and zyxin (164D4) (SySy) were used. Mammalian Expression Vectors, Pulldown, and Immunoprecipitation Assays—Arp7A (GenBankTM: NM_006687) was amplified by PCR from a human testis Marathon-Ready cDNA library (Clontech) and cloned into the NotI-EcoRI sites of CB6N-GFP [30] to generate GFP-Arp7A for expression in mammalian cells. All CB6-N-GFP Tes expression vectors have been previously described [27]. Mammalian cell extracts containing the different GFP-tagged proteins were prepared and incubated with His- or GST-tagged protein resins as described previously [27, 28]. The resulting supernatant was incubated for 2 h at 4 °C with Tes antibody for immunoprecipitation and processed as described previously [28]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
