Assessment of phosphorylation in T oxoplasma glideosome assembly and function

Summary Members of the phylum Apicomplexa possess a highly conserved molecular motor complex anchored in the parasite pellicle and associated with gliding motility, invasion and egress from infected cells. This machinery, called the glideosome, is structured around the acylated gliding‐associated protein GAP 45 that recruits the motor complex composed of myosin A and two associated myosin light chains ( TgMLC 1 and TgELC 1). This motor is presumably firmly anchored to the inner membrane complex underneath the plasma membrane via an interaction with two integral membrane proteins, GAP 50 and GAP 40. To determine if the previously mapped phosphorylation sites on TgGAP 45 and TgMLC 1 have a direct significance for glideosome assembly and function, a series of phospho‐mimetic and phospho‐null mutants were generated. Neither the overexpression nor the allelic replacement of TgMLC 1 with phospho‐mutants impacted on glideosome assembly and parasite motility. TgGAP 45 phosphorylation mutants were functionally investigated using a complementation strategy in a TgGAP 45 inducible knockout background. The loss of interaction with TgGAP 50 by one previously reported GAP 45‐mutant appeared to depend only on the presence of a remaining competing wild type copy of TgGAP 45. Accordingly, this mutant displayed no phenotype in complementation experiments. Unexpectedly, GAP 45 lacking the region encompassing the cluster of twelve phosphorylation sites did not impact on its dual function in motor recruitment and pellicle integrity. Despite the extensive phosphorylation of TgMLC 1 and TgGAP 45, this post‐translational modification does not appear to be critical for the assembly and function of the glideosome.