Overproduction, purification and crystallization of PfTic22, a component of the import apparatus from the apicoplast ofPlasmodium falciparum

Abstract

Tic22 is a component of the protein-import apparatus of the chloroplasts of plants and algae and the apicoplasts of the Apicomplexa, a large group of organisms that includes the parasites that cause malaria. Tic22 is important for protein import into these organelles and for organelle biogenesis. It lies between the two membranes of chloroplasts, making interactions with components of both the TIC and TOC complexes. In the apicoplast, it is predicted to be located between the inner two membranes and to play a similar role in import. Although Tic22 is ubiquitous, its function is as yet uncertain. Tic22 from Plasmodium falciparum was therefore overproduced, purified and crystallized. A data set extending to 2.15 Å resolution has been collected from a crystal containing selenomethionine-labelled protein and structure determination is under way.

Tic22 Is an Essential Chaperone Required for Protein Import into the Apicoplast

“…TgTic20 and TgDer1), loss of apicoplast import is promptly followed by major defects in apicoplast biogenesis (18,20), consistent with the importance of apicoplast-localized proteins for organellar maintenance. The loss of apicoplasts is the most likely reason for the subsequent inhibition of parasite growth in TgTic22-less parasites.…”

“…However, we succeeded in expressing residues 68 -279 of PfTic22, corresponding to the predicted mature form (17), and were able to generate crystals. Microseeding allowed the growth of large, single crystals of seleno-methionine labeled protein that diffracted to 2.15 Å resolution (20). A three wavelength MAD dataset was collected, allowing phase determination and building of a final model containing the majority of the protein (residues 68 -151 and 162-275).…”

Cryo-electron tomography reveals four-membrane architecture of the Plasmodium apicoplast

“…Much has been discussed about how proteins are transported across the apicoplast’s surrounding membranes, with various membranous translocon proteins already described [37-41]. In the acquired cryo-tomograms, structures were observed in merozoites’ apicoplast of both P. berghei and P. falciparum , potentially resembling a translocon (Figures 4C and 5C), although this cannot be resolved with the current images.…”