Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole

Abstract

Niemann-Pick type C is a storage disease caused by dysfunction of NPC proteins, which transport cholesterol from the lumen of lysosomes to the limiting membrane of that compartment. Using freeze fracture electron microscopy, we show here that the yeast NPC orthologs, Ncr1p and Npc2p, are essential for formation and expansion of raft-like domains in the vacuolar (lysosome) membrane, both in stationary phase and in acute nitrogen starvation. Moreover, the expanded raft-like domains engulf lipid droplets by a microautophagic mechanism. We also found that the multivesicular body pathway plays a crucial role in microautophagy in acute nitrogen starvation by delivering sterol to the vacuole. These data show that NPC proteins promote microautophagy in stationary phase and under nitrogen starvation conditions, likely by increasing sterol in the limiting membrane of the vacuole.DOI: http://dx.doi.org/10.7554/eLife.25960.001

Hallmarks of Reversible Phase Separation in Living, Unperturbed Cell Membranes

“…formation is linked to the availability of lipids and sterols, from lipid esters stored in cytoplasmic lipid droplets, through a process called microlipophagy. 89,90,111 Although all six of these findings are consistent with the hypothesis that vacuole domains arise through phase separation, none of them presents a direct test.…”

“…29 In synthetic GUVs with excess area (more area than necessary to enclose a spherical volume), domains in pseudohexagonal arrays are observed to bulge into or out of the vesicles and are hindered from colliding. 5,35,114,115 Similarly, the pseudohexagonal domains of yeast vacuoles have been observed to bulge inward toward the vacuole lumen, 88,90,111 and fusion of these domains is a rare event. We hypothesized that domain collision in vacuoles might be triggered by applying a hypoosmotic gradient to yeast cells, which causes the vacuoles inside to swell due to internal turgor pressure.…”

“…Fourth, only two types of domains are observed by both freeze-fracture and fluorescent labels (14 endogenous proteins and 2 lipid-sensitive dyes). 88,90,94,95 Finally, the fifth observation consistent with coexisting liquid phases is depletion of ergosterol from yeast reduces the number of vacuoles exhibiting domains. Sterols are a necessary membrane component to support coexisting liquid phases.…”

“…[86][87][88][89][90] Early freeze-fracture electron microscopy work indicated that the domains resulted from the membrane phase separating into coexisting liquid and gel phases. 91 More recent fluorescent microscopy work in unfixed, living cells, provides new evidence that the domains may be due to two coexisting liquid phases.…”

“…These rules apply equally well to simple bilayer membranes composed of only three types of lipids, to the complex bilayer membranes of giant plasma membrane vesicles blebbed from cells, and phase-separated cytoplasmic droplets recently implicated across a variety of cell biological activities. [88][89][90][91]94 In many cases the domains in vacuole membranes are ~200 nm or larger, and are therefore resolvable using conventional optical microscopy. were grown at 30 ˚C and imaged at ambient temperature (~22 ˚C) using either wide-field illumination on its own, wide-field illumination with z-z sectioning followed by iterative deconvolution, or structured illumination microscopy (3D-SIM) followed by iterative deconvolution.…”