Abstract
BackgroundAutophagy is a cytoprotective, lysosomal degradation system regulated upon induced phosphatidylinositol 3-phosphate (PtdIns(3)P) generation by phosphatidylinositol 3-kinase class III (PtdIns3KC3) downstream of mTORC1 inhibition. The human PtdIns(3)P-binding β-propeller protein WIPI-1 accumulates at the initiation site for autophagosome formation (phagophore), functions upstream of the Atg12 and LC3 conjugation systems, and localizes at both the inner and outer membrane of generated autophagosomes. In addition, to a minor degree WIPI-1 also binds PtdIns(3,5)P2. By homology modelling we earlier identified 24 evolutionarily highly conserved amino acids that cluster at two opposite sites of the open Velcro arranged WIPI-1 β-propeller.ResultsBy alanine scanning mutagenesis of 24 conserved residues in human WIPI-1 we define the PtdIns-binding site of human WIPI-1 to critically include S203, S205, G208, T209, R212, R226, R227, G228, S251, T255, H257. These amino acids confer PtdIns(3)P or PtdIns(3,5)P2 binding. In general, WIPI-1 mutants unable to bind PtdIns(3)P/PtdIns(3,5)P2 lost their potential to localize at autophagosomal membranes, but WIPI-1 mutants that retained PtdIns(3)P/PtdIns(3,5)P2 binding localized at Atg12-positive phagophores upon mTORC1 inhibition. Both, downregulation of mTOR by siRNA or cellular PtdIns(3)P elevation upon PIKfyve inhibition by YM201636 significantly increased the localization of WIPI-1 at autophagosomal membranes. Further, we identified regulatory amino acids that influence the membrane recruitment of WIPI-1. Exceptional, WIPI-1 R110A localization at Atg12-positive membranes was independent of autophagy stimulation and insensitive to wortmannin. R112A and H185A mutants were unable to bind PtdIns(3)P/PtdIns(3,5)P2 but localized at autophagosomal membranes, although in a significant reduced number of cells when compared to wild-type WIPI-1.ConclusionsWe identified amino acids of the WIPI-1 β-propeller that confer PtdIns(3)P or PtdIns(3,5)P2 binding (S203, S205, G208, T209, R212, R226, R227, G228, S251, T255, H257), and that regulate the localization at autophagosomal membranes (R110, R112, H185) downstream of mTORC1 inhibition.
Highlights
Autophagy is a cytoprotective, lysosomal degradation system regulated upon induced phosphatidylinositol 3-phosphate (PtdIns(3)P) generation by phosphatidylinositol 3-kinase class III (PtdIns3KC3) downstream of Mammalian target of rapamycin complex 1 (mTORC1) inhibition
We identified amino acids of the with phosphoinositide (WIPI)-1 β-propeller that confer PtdIns(3)P or PtdIns(3,5)P2 binding (S203, S205, G208, T209, R212, R226, R227, G228, S251, T255, H257), and that regulate the localization at autophagosomal membranes (R110, R112, H185) downstream of mTORC1 inhibition
As we employed rapamycin-mediated mTORC1 inhibition to induce autophagy for the characterization of the generated WIPI-1 mutants, we functionally addressed if siRNA-mediated downregulation of mTOR (Figure 10A) would likewise result in an increase of WIPI-1 punctapositive cells
Summary
Lysosomal degradation system regulated upon induced phosphatidylinositol 3-phosphate (PtdIns(3)P) generation by phosphatidylinositol 3-kinase class III (PtdIns3KC3) downstream of mTORC1 inhibition. The human PtdIns(3)P-binding β-propeller protein WIPI-1 accumulates at the initiation site for autophagosome formation (phagophore), functions upstream of the Atg and LC3 conjugation systems, and localizes at both the inner and outer membrane of generated autophagosomes. Macroautophagy (hereafter referred to as autophagy) is defined as a stochastic lysosomal bulk degradation pathway for cytoplasmic material, and characterized by membrane rearrangements from autophagosome formation to fusion events with the lysosomal compartment. Stochastic and selective autophagy control both turnover and clearance of the cytoplasm, thereby critically contributing to eukaryotic cell homeostasis The autophagosomal membrane is of as yet uncertain origin [5], but recent reports provide evidence that multiple membrane systems, such as the endoplasmic reticulum [6,7] or the plasma membrane [8] contribute to autophagosome formation from initial template membranes (phagophore). Autophagosomes sequester the cytoplasmic cargo and communicate or fuse with the lysosomal compartment to permit final degradation [10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
