Research Article: Characterization of the Autophagy Marker Protein Atg8 Reveals Atypical Features of Autophagy in Plasmodium falciparum

Date Published: November 26, 2014

Publisher: Public Library of Science

Author(s): Rahul Navale, Aparna Devi Allanki, Puran Singh Sijwali, Volker Theo Heussler.

http://doi.org/10.1371/journal.pone.0113220

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.

Partial Text

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. Autophagy is a lysosome-dependent process that delivers a variety of cellular contents, including organelles, to the lysosome, primarily for degradation purpose, and this type of process is regarded as conventional autophagy [1], [2]. However, a number of cellular contents are also selectively delivered to the lysosome for degradative or non-degradative purpose, which is broadly considered selective or unconventional autophagy [3]. 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]. Thus, autophagy has both housekeeping and regulatory functions in eukaryotic organisms [6]. While autophagy has also been shown to have roles in defence against pathogens, several pathogens also exploit the host autophagy machinery for their advantage [7]. Malaria parasites develop through multiple stages in diverse environments, and this multi-stage development is accompanied by acquisition and disposal of several stage-specific cellular contents, including organelles. As autophagy is involved in both degradative and biosynthetic turnover of cellular contents, it is likely to have key roles in malaria parasite development.

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. The Plasmodium autophagy repertoire may have more than 15 proteins, as we excluded proteins that showed marginal homology with yeast Atg proteins (e-value<0.001) or lacked known conserved domains. Additionally, malaria parasites must have an ortholog of ScAtg15 for lysis of the autophagic body membrane, one or more proteases to degrade the cargo, and Atg22 to efflux degradation products. P. falciparum has 20 putative lipases; 11 of these are present in P. falciparum only and the remaining 9 are conserved. A conserved lipase (PF3D7_1427100), which shares an N-terminus hydrophobic region and a C-terminus lipase domain with ScAtg15, may function as Atg15. Cellular localization studies will be required to determine which of these are present in the food vacuole and function as Atg15. Multiple classes of proteases (falcipains, papain-like cysteine proteases; plasmepsins, aspartic proteases; falcilysin, metallo protease; dipeptidylaminopeptidase, cathepsin C-like cysteine protease; and aminopeptidases) have been shown to be present in the food vacuole wherein they have been shown or implicated in haemoglobin degradation, and these proteases may also degrade the autophagy cargo [48], [49], [51], [53], [69], [70], [71]. Several proteins are annotated as transporters on the PlasmoDB, including 5 putative amino acid transporters; cellular localization studies are necessary to determine which of these are present in the food vacuole.   Source: http://doi.org/10.1371/journal.pone.0113220