A Membrane Complex Involved in the Synthesis, Transport and Succinylation of Cyclic Beta-1,2-Glucans

GUIDOLIN L. S.; CIOCCHINI A. E.; IÑON DE IANNINO N.; COMERCI D. J

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular SAIB; 2010

Sociedad Argentina de Investigaciones Bioquímica y Biología Molecular

Cyclic a-1,2-glucans (CaG) are periplasmic homopolysaccharides that have been shown to play an important role in several symbiotic and pathogenic relationships. B. abortus CaG synthase (Cgs) is an integral inner membrane (IIM) protein which catalyzes the four different activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of CaG. Once synthesized in the cytoplasm, CaG are transported to the periplasm by the CaG transporter (Cgt) and succinylated by the CaG modifier enzyme (Cgm). Cgt and Cgm, as well as Cgs, are IIM proteins. In this work, we used a bacterial two-hybrid system (BACTH) to study the interaction network between these three IIM proteins. Our results indicate that Cgs interacts with Cgt and Cgm, and that Cgt interacts with Cgm. We have also observed that each one of these proteins form homotypic complexes. Furthermore, an analysis carried out with Cgs truncated or in-frame pentapeptide insertion mutants reveal that Cgs-dimerization as well as Cgs interactions with Cgt and Cgm are mediated by the NH-terminal domain and may be modulated by the C-terminal phosphorylase domain of the protein. We propose that Cgs, Cgt and Cgm form an inner membrane protein complex where the interactions between these proteins may provide a mechanism to coordinate the synthesis, transport to periplasm and succinylation of CaG.B. abortus CaG synthase (Cgs) is an integral inner membrane (IIM) protein which catalyzes the four different activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of CaG. Once synthesized in the cytoplasm, CaG are transported to the periplasm by the CaG transporter (Cgt) and succinylated by the CaG modifier enzyme (Cgm). Cgt and Cgm, as well as Cgs, are IIM proteins. In this work, we used a bacterial two-hybrid system (BACTH) to study the interaction network between these three IIM proteins. Our results indicate that Cgs interacts with Cgt and Cgm, and that Cgt interacts with Cgm. We have also observed that each one of these proteins form homotypic complexes. Furthermore, an analysis carried out with Cgs truncated or in-frame pentapeptide insertion mutants reveal that Cgs-dimerization as well as Cgs interactions with Cgt and Cgm are mediated by the NH-terminal domain and may be modulated by the C-terminal phosphorylase domain of the protein. We propose that Cgs, Cgt and Cgm form an inner membrane protein complex where the interactions between these proteins may provide a mechanism to coordinate the synthesis, transport to periplasm and succinylation of CaG.