Characterization of unusual families of ATG8-like proteins and ATG12 in the protozoan parasite Leishmania major.
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.
Williams
Roderick A M
RA
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, UK.
Woods
Kerry L
KL
Juliano
Luiz
L
Mottram
Jeremy C
JC
Coombs
Graham H
GH
D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000378:metabolism; D022083:Cytoplasmic Structures / Q000187:drug effects / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D005998:Glycine / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D018320:Leishmania major / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D050356:Lipid Metabolism / Q000187:drug effects; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010271:Parasites / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000187:drug effects
D009842:Oligopeptides; D011480:Protease Inhibitors; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases; D005998:Glycine