The differential palmitoylation states of N-Ras and H-Ras determine their distinct Golgi subcompartment localizations.

Despite a high degree of structural homology and shared exchange factors, effectors and GTPase activating proteins, a large body of evidence suggests functional heterogeneity among Ras isoforms. One aspect of Ras biology that may explain this heterogeneity is the differential subcellular localizations driven by the C-terminal hypervariable regions of Ras proteins. Spatial heterogeneity has been documented at the level of organelles: palmitoylated Ras isoforms (H-Ras and N-Ras) localize on the Golgi apparatus whereas K-Ras4B does not. We tested the hypothesis that spatial heterogeneity also exists at the sub-organelle level by studying the localization of differentially palmitoylated Ras isoforms within the Golgi apparatus. Using confocal, live-cell fluorescent imaging and immunogold electron microscopy we found that, whereas the doubly palmitoylated H-Ras is distributed throughout the Golgi stacks, the singly palmitoylated N-Ras is polarized with a relative paucity of expression on the trans Golgi. Using palmitoylation mutants, we show that the different sub-Golgi distributions of the Ras proteins are a consequence of their differential degree of palmitoylation. Thus, the acylation state of Ras proteins controls not only their distribution between the Golgi apparatus and the plasma membrane, but also their distribution within the Golgi stacks.

Inhibition of ATPIF1 ameliorates severe mitochondrial respiratory chain dysfunction in mammalian cells.

Mitochondrial respiratory chain disorders are characterized by loss of electron transport chain (ETC) activity. Although the causes of many such diseases are known, there is a lack of effective therapies. To identify genes that confer resistance to severe ETC dysfunction when inactivated, we performed a genome-wide genetic screen in haploid human cells with the mitochondrial complex III inhibitor antimycin. This screen revealed that loss of ATPIF1 strongly protects against antimycin-induced ETC dysfunction and cell death by allowing for the maintenance of mitochondrial membrane potential. ATPIF1 loss protects against other forms of ETC dysfunction and is even essential for the viability of human ρ° cells lacking mitochondrial DNA, a system commonly used for studying ETC dysfunction. Importantly, inhibition of ATPIF1 ameliorates complex III blockade in primary hepatocytes, a cell type afflicted in severe mitochondrial disease. Altogether, these results suggest that inhibition of ATPIF1 can ameliorate severe ETC dysfunction in mitochondrial pathology.

Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival.

The mechanistic target of rapamycin complex 1 (mTORC1) pathway regulates organismal growth in response to many environmental cues, including nutrients and growth factors. Cell-based studies showed that mTORC1 senses amino acids through the RagA-D family of GTPases (also known as RRAGA, B, C and D), but their importance in mammalian physiology is unknown. Here we generate knock-in mice that express a constitutively active form of RagA (RagA(GTP)) from its endogenous promoter. RagA(GTP/GTP) mice develop normally, but fail to survive postnatal day 1. When delivered by Caesarean section, fasted RagA(GTP/GTP) neonates die almost twice as rapidly as wild-type littermates. Within an hour of birth, wild-type neonates strongly inhibit mTORC1, which coincides with profound hypoglycaemia and a decrease in plasma amino-acid concentrations. In contrast, mTORC1 inhibition does not occur in RagA(GTP/GTP) neonates, despite identical reductions in blood nutrient amounts. With prolonged fasting, wild-type neonates recover their plasma glucose concentrations, but RagA(GTP/GTP) mice remain hypoglycaemic until death, despite using glycogen at a faster rate. The glucose homeostasis defect correlates with the inability of fasted RagA(GTP/GTP) neonates to trigger autophagy and produce amino acids for de novo glucose production. Because profound hypoglycaemia does not inhibit mTORC1 in RagA(GTP/GTP) neonates, we considered the possibility that the Rag pathway signals glucose as well as amino-acid sufficiency to mTORC1. Indeed, mTORC1 is resistant to glucose deprivation in RagA(GTP/GTP) fibroblasts, and glucose, like amino acids, controls its recruitment to the lysosomal surface, the site of mTORC1 activation. Thus, the Rag GTPases signal glucose and amino-acid concentrations to mTORC1, and have an unexpectedly key role in neonates in autophagy induction and thus nutrient homeostasis and viability.

Presenilin deficiency or lysosomal inhibition enhances Wnt signaling through relocalization of GSK3 to the late-endosomal compartment.

Sustained canonical Wnt signaling requires the inhibition of glycogen synthase kinase 3 (GSK3) activity by sequestration of GSK3 inside multivesicular endosomes (MVEs). Here, we show that Wnt signaling is increased by the lysosomal inhibitor chloroquine, which causes accumulation of MVEs. A similar MVE expansion and increased Wnt responsiveness was found in cells deficient in presenilin, a protein associated with Alzheimer's disease. The Wnt-enhancing effects were entirely dependent on the functional endosomal sorting complex required for transport (ESCRT), which is needed for the formation of intraluminal vesicles in MVEs. We suggest that accumulation of late endosomal structures leads to enhanced canonical Wnt signaling through increased Wnt-receptor/GSK3 sequestration. The decrease in GSK3 cytosolic activity stabilized cytoplasmic GSK3 substrates such as ß-catenin, the microtubule-associated protein Tau, and other proteins. These results underscore the importance of the endosomal pathway in canonical Wnt signaling and reveal a mechanism for regulation of Wnt signaling by presenilin deficiency.