Topology and Function of the S. cerevisiae Autophagy Protein Atg15

The putative phospholipase Atg15 is required for the intravacuolar lysis of autophagic bodies and MVB vesicles. Intracellular membrane lysis is a highly sophisticated mechanism that is not fully understood. The amino-terminal transmembrane domain of Atg15 contains the sorting signal for entry into t...

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Bibliographic Details
Published in:Cells (Basel, Switzerland) Switzerland), 2023-08, Vol.12 (16), p.2056
Main Authors: Marquardt, Lisa, Montino, Marco, Mühe, Yvonne, Schlotterhose, Petra, Thumm, Michael
Format: Article
Language:English
Subjects:
Acidification
Amino acids
autophagic body
Autophagy
Chimeras
Cytosol
Enzymatic activity
Glucose
Lysis
macroautophagy
Membrane proteins
Membrane vesicles
microautophagy
Microscopy
Monoclonal antibodies
Nitrogen
phospholipase
Plasmids
Yeast
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Summary:The putative phospholipase Atg15 is required for the intravacuolar lysis of autophagic bodies and MVB vesicles. Intracellular membrane lysis is a highly sophisticated mechanism that is not fully understood. The amino-terminal transmembrane domain of Atg15 contains the sorting signal for entry into the MVB pathway. By replacing this domain, we generated chimeras located in the cytosol, the vacuole membrane, and the lumen. The variants at the vacuole membrane and in the lumen were highly active. Together with the absence of Atg15 from the phagophore and autophagic bodies, this suggests that, within the vacuole, Atg15 can lyse vesicles where it is not embedded. In-depth topological analyses showed that Atg15 is a single membrane-spanning protein with the amino-terminus in the cytosol and the rest, including the active site motif, in the ER lumen. Remarkably, only membrane-embedded Atg15 variants affected growth when overexpressed. The growth defects depended on its active site serine 332, showing that it was linked to the enzymatic activity of Atg15. Interestingly, the growth defects were independent of vacuolar proteinase A and vacuolar acidification.
ISSN:2073-4409
2073-4409
DOI:10.3390/cells12162056