Characterization of unusual families of ATG8-like proteins and ATG12 in the protozoan parasite <i>Leishmania major</i>

<i>Leishmania major</i> possesses, apparently uniquely, four families of <i>ATG8</i>-like genes, designated <i>ATG8</i>, <i>ATG8A</i>, <i>ATG8B</i> and <i>ATG8C</i>, and 25 genes in total. <i>L. major ATG8</i> and examples from the <i>ATG8A</i>, <i>ATG8B</i> and <i>ATG8C</i> families are able to complement a <i>Saccharomyces cerevisiae ATG8</i>-deficient strain, indicating functional conservation. Whereas ATG8 has been shown to form putative autophagosomes during differentiation and starvation of <i>L. major</i>, 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 <i>L. major</i> and, consistent with this, <i>ATG4.2</i>-deficient <i>L. major</i> 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. <i>L. major</i> genes encoding possible <i>ATG5</i>, <i>ATG10</i> and <i>ATG12</i> homologues were found to complement their respective <i>S. cerevisiae</i> mutants, and ATG12 localized in part to ATG8-containing puncta, suggestive of a functional ATG5-ATG12 conjugation pathway in the parasite. <i>L. major</i> ATG12 is unusual as it requires C-terminal processing by an as yet unidentified peptidase