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Actin-related proteins in Actinobacillus pleuropneumoniae and their interactions with actin-binding proteins

Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 14-740, México, DF 07000, Mexico¹
Departamento de Ingeniería Genética de Plantas, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 629, Irapuato, Gto. 36500, Mexico²
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas IPN, Prolongación de Carpio y Plan de Ayala s/n, México, DF 11340, Mexico³
División de Investigación Básica, Instituto Nacional de Cancerología, San Fernando No 22, Tlalpan, DF 14000, Mexico⁴

Author for correspondence: Magdalena Segura-Nieto. Tel: +52 46 23 96 00 ext. 665/413. Fax: +52 46 24 58 49. e-mail: msegura{at}irapuato.ira.cinvestav.mx

A group of prokaryotic actin-related proteins (PARP) with an M_r of 43000 was detected in Actinobacillus pleuropneumoniae. These proteins were enriched by a depolymerization/polymerization cycle, under similar conditions to those used to polymerize muscle actin, and purified by affinity chromatography on a DNase I-Sepharose column. Three isoforms of A. pleuropneumoniae PARP (Ap-PARP) with pI values of 5·8, 6·15 and 6·2 were detected. Ap-PARP were recognized by four different anti-actin antibodies (one anti-muscle and three anti-cytoplasmic isoforms). Ap-PARP were also recognized by antibodies against Anabaena variabilis PARP (Av-PARP) and against actin-binding proteins such as -actinin and spectrin, and also by a monoclonal antibody against heat-shock cognate protein 70 (Hsc70). Specific binding of phalloidin to Ap-PARP was detected both in permeabilized cells and in vitro. Purified Ap-PARP can polymerize under similar conditions to those required for skeletal muscle actin polymerization and the filaments formed appear to be decorated with myosin subfragment-1 (S1) as observed by transmission electron microscopy. The amino acid composition of Ap-PARP revealed more similarities to muscle -actin and the cytoplasmic ß-actin isoform than to eukaryotic actin-related proteins.

Keywords: prokaryotic actin-related proteins, actin-related proteins, actin-binding proteins, cytoskeleton-like structure, Actinobacillus pleuropneumoniae

Abbreviations: ABP, actin-binding proteins; ARP, actin-related proteins; CD, cytochalasin D; Hsc, heat-shock cognate protein; Hsp, heat-shock protein; PARP, prokaryotic actin-related proteins; S1, myosin subfragment-1; TRITC, tetramethylrhodamine isothiocyanate; Ap, Actinobacillus pleuropneumoniae; Av, Anabaena variabilis

^a Present address: Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3 CP 62250, Cuernavaca, Mor, Mexico.