Characterization of unusual families of ATG8-like proteins and ATG12 in the protozoan parasiteLeishmania major

Abstract

Leishmania major possesses, apparently uniquely, four families of ATG8-like genes, designated ATG8, ATG8A, ATG8B and ATG8C, and 25 genes in total. L. major ATG8 and examples from the ATG8A, ATG8B and ATG8C families are able to complement a Saccharomyces cerevisiae ATG8-deficient strain, indicating functional conservation. Whereas ATG8 has been shown to form putative autophagosomes during differentiation and starvation of L. major, ATG8A primarily form puncta in response to starvation-suggesting a role for ATG8A in starvation-induced autophagy. Recombinant ATG8A was processed at the scissile glycine by recombinant ATG4.2 but not ATG4.1 cysteine peptidases of L. major and, consistent with this, ATG4.2-deficient L. major mutants were unable to process ATG8A and were less able to withstand starvation than wild-type cells. GFP-ATG8-containing puncta were less abundant in ATG4.2 overexpression lines, in which unlipidated ATG8 predominated, which is consistent with ATG4.2 being an ATG8-deconjugating enzyme as well as an ATG8A-processing enzyme. In contrast, recombinant ATG8, ATG8B and ATG8C were all processed by ATG4.1, but not by ATG4.2. ATG8B and ATG8C both have a distinct subcellular location close to the flagellar pocket, but the occurrence of the GFP-labeled puncta suggest that they do not have a role in autophagy. L. major genes encoding possible ATG5, ATG10 and ATG12 homologues were found to complement their respective S. cerevisiae mutants, and ATG12 localized in part to ATG8-containing puncta, suggestive of a functional ATG5-ATG12 conjugation pathway in the parasite. L. major ATG12 is unusual as it requires C-terminal processing by an as yet unidentified peptidase.

Autophagy in protists

“…For example, it has been initially suggested that the second Atg12-based conjugation system was absent in trypanosomatids. 18 However, later reports 14,15,23 identify Atg5 and Atg10 orthologues, recently experimentally characterized. 23 Interestingly, despite the identification…”

“…Using bioinformatics to map the distribution of known autophagy-related proteins in extant eukaryotes, however, indicates that among the protists canonical autophagy has been subjected to elaboration, moderation and even loss on multiple occasions (Fig. 2). 14 As exemplified by the experimental characterization of autophagy in malarial parasites (discussed under the heading 'Autophagy in Apicomplexa'), or the molecular characterization of ATG8 paralogues in Leishmania major 23 the cellular consequences of likely moderation to canonical autophagy, or the paralogue expansion of core ATGs are already under investigation in some species. The availability of reverse genetic tools for many of the other protists in which core ATGs have either been lost or subject to paralogue expansion, means that other variations in autophagy that are apparent at the genomic level should be amenable to experimental investigation, too.…”

“…For instance, there is a species-specific expansion of ATG8 paralogues into four distinct families totaling 25 genes in Leishmania, but not Trypanosoma species as was noted previously. 23 Preliminary localization of some of these paralogues using GFP-tagging approaches suggests that some ATG8-family members have cellular functions in addition to or other than autophagy (reviewed in ref. 23; see also below, ' Autophagy in Trypanosomatidae as inferred from bioinformatics studies of genome sequences').…”

“…14,[16][17][18][19][20][21][22][23] For some of the species examined so far, genomic signatures are complemented by experimental demonstrations of autophagy and/or analyses of candidate protein function. Significantly, however, bioinformatics surveys of protist genomes reveal some taxa in which autophagy, at least as defined by current paradigms, does not occur and others in which the canonical autophagy pathway has been subject to lineage-specific elaboration or moderation.…”

Characterization of the ATG8-conjugation system in 2Plasmodiumspecies with special focus on the liver stage

“…17,34 We examined whether PbATG8 could likewise functionally complement an atg8Δ yeast strain. To assay complementation of autophagy, PbATG8 was expressed under the control of the PGK (phosphoglycerate kinase) promoter and processing of the precursor form of aminopeptidase I (prApe1) was monitored by western blotting.…”