Proteolytic Processing of TgIMC1 during Maturation of the Membrane Skeleton of Toxoplasma gondii

Abstract

Membrane skeletons play an important role in the maintenance of cell shape and integrity in many cell types. In the protozoan parasite Toxoplasma gondii this function is performed by the subpellicular network, a resilient structure composed of tightly interwoven 10-nm filaments. We report here that this network is assembled at an early stage in the development of daughter parasites. The networks of immature and mature parasites differ dramatically with respect to their stability. Although in immature parasites the network is completely solubilized by detergent, the network in mature parasites is entirely detergent-resistant. Conversion of the detergent-labile to the detergent-resistant network occurs late in daughter cell development and appears to be coupled to proteolytic processing of the carboxyl terminus of TgIMC1, the major subunit of the network filaments. A single cysteine residue in the TgIMC1 carboxyl terminus was found to be essential for this processing event. The dramatic change in resistance to detergent extraction probably reflects an overall change in structural stability of the subpellicular network that accompanies maturation of daughter parasites and allows a switch from an assembly-competent but loose structure to one that is rigid and offers mechanical strength to the mature parasite.Membrane skeletons, composed of the plasma membrane and its organized underlying coat, are found in many cell types and are essential for mechanical strength and the maintenance of cell shape. Two widely divergent examples are the spectrinbased membrane skeletal system of the erythrocyte and the articulin-based system in the unicellular Euglena. In the erythrocyte, spectrin along with its associated proteins such as ankyrin form a meshwork underlying the plasma membrane (1). In the Euglena, two articulin proteins interact stoichiometrically to form a membrane skeletal system consisting of 40 interdigitating strips (2-4). Along with the articulins of the Euglena, a number of unique cytoskeletal systems have been described in protists, including the giardins (5, 6), assemblins (7), and tetrins (8) as well as the components of the subpellicular network, a novel cytoskeletal structure recently identified in the protozoan parasite Toxoplasma gondii (9).T. gondii is an obligate intracellular parasite that infects a wide range of nucleated cells. Human infection with the para-

A MORN-repeat protein is a dynamic component of theToxoplasma gondiicell division apparatus

“…We therefore hypothesized that MORN1 might interact with elements of the parasite's cytoskeleton. The T. gondii cytoskeleton is resistant to extraction with Triton X-100 and deoxycholate (Mann et al, 2002;Morrissette et al, 1997) and co-fractionation in extraction assays has been a hallmark of proteins associated with the cytoskeleton (Gaskins et al, 2004). MORN1 showed marked resistance to Triton X-100…”

“…The IMC is structurally organized by a meshwork formed by a group of proteins weakly related to intermediate filaments (Gubbels et al, 2004;Hu et al, 2002a;Mann and Beckers, 2001). Initially the IMC is flexible allowing for growth but proteolytic processing of IMC components is then thought to stabilize the structure in the mature cell (Hu et al, 2002a;Mann et al, 2002). In addition to providing a pellicle, the IMC also plays a critical role as an anchor for the gliding apparatus of the parasite (Gaskins et al, 2004).…”

Immobilization of the Type XIV Myosin Complex inToxoplasma gondii

“…The generation of monospecific antisera to Toxoplasma IMC1 has been described previously (Mann et al, 2002). Monospecific antisera to yellow fluorescent protein (YFP) were generated by injecting rabbits with purified recombinant YFP (Cocalico Biologicals, Reamstown, PA).…”

Toxoplasma gondii: further studies on the subpellicular network

“…Apparently, the subpellicular network provides the shape of the parasite. In daughter parasites, however, this structure is loose and yet susceptible to detergent extraction, to be expanded and remodelled during growth of the parasites (Mann et al 2002). This unstable structure changes into the rigid and stable structure in mature parasites, as observed in our detergent extraction preparations.…”