Abstract
Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8) employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64) and aspartic (pepstatin) protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine), indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in apicoplast biogenesis. Collectively, our results reveal several atypical features of autophagy in malaria parasites, which may be largely associated with non-degradative processes.
Highlights
Eukaryotic organisms principally rely on two degradation machineries for turnover of dispensable and damaged cellular contents: the ubiquitin proteasome system (UPS) and the lysosomal system
The predicted Plasmodium autophagy repertoire includes proteins involved in induction (Atg1 and Atg17), cargo selection (Atg11), nucleation and assembly of phagophore (Vps34, Vps15, Atg18, and Atg23), expansion of phagophore into autophagosome (Atg3, Atg4, Atg5, Atg7, Atg8, and Atg12), processing and retrieval of Atg8 from the autophagosome (Atg4), degradation of the autophagic body membrane (Atg15), and efflux of the end products (Atg22)
Malaria parasites, including P. falciparum, seem to have limited autophagy machinery, as they contain single homologs for 11 and multiple homologs for 4 of the 33 proteins involved in autophagy in S. cerevisiae
Summary
Eukaryotic organisms principally rely on two degradation machineries for turnover of dispensable and damaged cellular contents: the ubiquitin proteasome system (UPS) and the lysosomal system. If the autophagy cargo contains random cellular contents, it is known as macroautophagy or autophagy; it is called microautophagy (such as mitophagy) if the cargo is selective; it is known as cytoplasm to vacuolar targeting (Cvt) if the cargo is selective and delivered to the lysosome for non-degradative purpose [1, 4, 5]. Autophagy has both housekeeping and regulatory functions in eukaryotic organisms [6]. As autophagy is involved in both degradative and biosynthetic turnover of cellular contents, it is likely to have key roles in malaria parasite development
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