The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.
The molecular mechanisms underlying microtubule participation in autophagy are not known. In this study, we show that starvation-induced autophagosome formation requires the most dynamic microtubule subset. Upon nutrient deprivation, labile microtubules specifically recruit markers of autophagosome formation like class III-phosphatidylinositol kinase, WIPI-1, the Atg12-Atg5 conjugate, and LC3-I, whereas mature autophagosomes may bind to stable microtubules. We further found that upon nutrient deprivation, tubulin acetylation increases both in labile and stable microtubules and is required to allow autophagy stimulation. Tubulin hyperacetylation on lysine 40 enhances kinesin-1 and JIP-1 recruitment on microtubules and allows JNK phosphorylation and activation. JNK, in turn, triggers the release of Beclin 1 from Bcl-2-Beclin 1 complexes and its recruitment on microtubules where it may initiate autophagosome formation. Finally, although kinesin-1 functions to carry autophagosomes in basal conditions, it is not involved in motoring autophagosomes after nutrient deprivation. Our results show that the dynamics of microtubules and tubulin post-translational modifications play a major role in the regulation of starvation-induced autophagy.Macroautophagy (simply referred to here as autophagy) occurs at low basal levels to perform homeostatic functions such as protein and organelle turnover. It is also an adaptive catabolic response to different metabolic stresses, including nutrient deprivation, growth factor depletion, or hypoxia (1, 2). Newly assembled multilayer membranes expand and sequester parts of the cytoplasm to form autophagosomes that subsequently fuse with lysosomes to degrade their content (1, 2). When cells lack nutrients, inhibition of the mammalian target of rapamycin activates the ULK complex (ULK1 and ULK2 are the mammalian orthologs of the yeast Atg1) to initiate the cascade of events leading to the formation of autophagosomes (reviewed in Ref.3). Building of isolation membranes or phagophores involves a complex comprising Beclin 1 (Beclin 1 is the mammalian homolog of the yeast Atg6) and the class III PI3K 4 (PI3K(III), also called hVps34) (4). Newly synthesized phosphatidylinositol 3-phosphate then recruits effectors such as WIPI-1, the homolog of yeast Atg18 (5, 6), to allow the recruitment of other autophagosomal building bricks (4). Atg9 contributes to membrane shuttling that elongates the pre-autophagosomal membrane (reviewed in Ref. 7). Such elongation also involves ubiquitin-like machineries, which in turn allow Atg12-to-Atg5 conjugation, and the modification of LC3 (LC3 is the mammalian ortholog of the yeast Atg8) (8) prior to their recruitment to autophagosomal membranes. LC3 is a light chain of MAP1 first identified in neurons (9, 10). After autophagy induction, the C-terminal region of native LC3 (pro-LC3) is cleaved by Atg4, yielding LC3-I. Atg7 and Atg3 then conjugate LC3-I to phosphatidylethanolamine on its C-terminal glycine, yielding LC3-II that attaches to the autophagosomal membrane ...
Autophagy is initiated by multimembrane vesicle (autophagosome) formation upon mammalian target of rapamycin inhibition and phosphatidylinositol 3-phosphate [PtdIns(3)P] generation. Upstream of microtubule-associated protein 1 light chain 3 (LC3), WD-repeat proteins interacting with phosphoinositides (WIPI proteins) specifically bind PtdIns(3)P at forming autophagosomal membranes and become membrane-bound proteins of generated autophagosomes. Here, we applied automated high-throughput WIPI-1 puncta analysis, paralleled with LC3 lipidation assays, to investigate Ca -dependent signaling, including CaMKI independent of AMPK␣ 1 /␣ 2 . Our data also suggest that AMPK␣ 1 /␣ 2 might differentially contribute to the regulation of WIPI-1 at the onset of autophagy.
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