Abstract
The lipid kinase VPS34 orchestrates diverse processes, including autophagy, endocytic sorting, phagocytosis, anabolic responses and cell division. VPS34 forms various complexes that help adapt it to specific pathways, with complexes I and II being the most prominent ones. We found that physicochemical properties of membranes strongly modulate VPS34 activity. Greater unsaturation of both substrate and non-substrate lipids, negative charge and curvature activate VPS34 complexes, adapting them to their cellular compartments. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of complexes I and II on membranes elucidated structural determinants that enable them to bind membranes. Among these are the Barkor/ATG14L autophagosome targeting sequence (BATS), which makes autophagy-specific complex I more active than the endocytic complex II, and the Beclin1 BARA domain. Interestingly, even though Beclin1 BARA is common to both complexes, its membrane-interacting loops are critical for complex II, but have only a minor role for complex I.
Highlights
Lipids form membranes that delimit cellular compartments, but they can act as signalling molecules that recruit proteins to membranes
To assess how membrane properties modify the activities of VPS34 complexes, we have developed a quantitative confocal microscopy assay to measure the activity of the human Class III PI3K complexes on giant unilamellar vesicles (GUVs), which have a radius of ~ 1–30 mm
We examined whether the difference in activity between complexes I and II could be because complex II, which is normally active on early endosomes, was not assayed on endosome-mimicking membranes
Summary
Lipids form membranes that delimit cellular compartments, but they can act as signalling molecules that recruit proteins to membranes. It forms two principal complexes, complexes I and II Both complexes have core components VPS34, VPS15 and Beclin 1 (Vps in yeast) bound to a specific component, which is either ATG14L (Atg in yeast) in complex I or UVRAG (Vps in yeast) in complex II. This single subunit difference between these two complexes localizes them to different membrane compartments. Complex II is mainly active in endocytic pathways by partially co-localizing in mammalian cells with Rab5- and Rab7-positive endolysosomal compartments as well as with Rab9-positive
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