Cristina Prescianotto-Baschong

Quantitative Phosphoproteomics Reveal mTORC1 Activates de Novo Pyrimidine Synthesis

Coordinating Metabolism Growth factors help to coordinate metabolism with growth in part by stimulating the activity of the protein kinase mTORC1 (mechanistic target of rapamycin complex 1). Ben-Sahra et al. (p. 1323, published online 21 February) and Robitaille et al. (p. 1320, published online 21 February) independently identified a key target of mTORC1—carbamolyl-phosphate synthase 2, or CAD, the rate-limiting enzyme for de novo synthesis of pyrimidines. Metabolomic profiling and phosphoproteomic analyses of normal cells and cells lacking signaling by mTORC1 converged on CAD as a key point at which growth-promoting signals also ramp up production of nucleic acids. In addition to its role in stimulating protein and lipid synthesis, the kinase mammalian target of rapamycin stimulates nucleotide biosynthesis. The Ser-Thr kinase mammalian target of rapamycin (mTOR) controls cell growth and metabolism by stimulating glycolysis and synthesis of proteins and lipids. To further understand the central role of mTOR in cell physiology, we used quantitative phosphoproteomics to identify substrates or downstream effectors of the two mTOR complexes. mTOR controlled the phosphorylation of 335 proteins, including CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase). CAD catalyzes the first three steps in de novo pyrimidine synthesis. mTORC1 indirectly phosphorylated CAD-S1859 through S6 kinase (S6K). CAD-S1859 phosphorylation promoted CAD oligomerization and thereby stimulated de novo synthesis of pyrimidines and progression through S phase of the cell cycle in mammalian cells. Thus, mTORC1 also stimulates the synthesis of nucleotides to control cell proliferation.

Feature Article: mTOR complex 2-Akt signaling at mitochondria-associated endoplasmic reticulum membranes (MAM) regulates mitochondrial physiology.

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP3 receptor (IP3R)-Grp75–voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.

The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1–1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1–1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

Ordering of Compartments in the Yeast Endocytic Pathway

We have characterized the morphology of the yeast endocytic pathway leading from the plasma membrane to the vacuole by following the trafficking of positively charged nanogold in combination with compartment identification using immunolocalization of t‐SNARE proteins. The first endocytic compartment, termed the early/recycling endosome, contains the t‐SNARE, Tlg1p. The next compartment, the prevacuolar compartment, contains Pep12p. After transport to the prevacuolar compartment, where vacuolar enzymes are seen on their way to the vacuole, endocytic content is delivered to the late endosome and on to the vacuole, both of which are devoid of Pep12p immunolabel. Traffic to the prevacuolar compartment is reduced in strains mutant for the Rab5 homologs, Vps21p, Ypt52p, and Ypt53p and in vps27 mutant cells. On the other hand, traffic to the early recycling endosome is less dependent on Rab5 homologs and does not require Vps27p.

Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold

Positively charged nanogold was used as a probe to trace the internalization of plasma membrane (PM) domains carrying negatively charged residues at an ultrastructural level. The probe revealed distinct endocytic pathways within tobacco protoplasts and allowed the morphology of the organelles involved in endocytosis to be characterized in great detail. Putative early endosomes with a tubulo-vesicular structure, similar to that observed in animal cells, are described and a new compartment, characterized by interconnected vesicles, was identified as a late endosome using the Arabidopsis anti-syntaxin family Syp-21 antibody. Endocytosis dissection using Brefeldin A (BFA), pulse chase, temperature- and energy-dependent experiments combined with quantitative analysis of nanogold particles in different compartments, suggested that recycling to the PM predominated with respect to degradation. Further experiments using ikarugamycin (IKA), an inhibitor of clathrin-dependent endocytosis, and negatively charged nanogold confirmed that distinct endocytic pathways coexist in tobacco protoplasts.

mTOR complex 2-Akt signaling at mitochondria-associated endoplasmic reticulum membranes (MAM) regulates mitochondrial physiology

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP3 receptor (IP3R)-Grp75–voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.

Dominant pro-vasopressin mutants that cause diabetes insipidus form disulfide-linked fibrillar aggregates in the endoplasmic reticulum

Autosomal dominant neurohypophyseal diabetes insipidus results from mutations in the precursor protein of the antidiuretic hormone arginine vasopressin. Mutant prohormone is retained in the endoplasmic reticulum of vasopressinergic neurons and causes their progressive degeneration by an unknown mechanism. Here, we show that several dominant pro-vasopressin mutants form disulfide-linked homo-oligomers and develop large aggregations visible by immunofluorescence and immunogold electron microscopy, both in a fibroblast and a neuronal cell line. Double-labeling showed the pro-vasopressin aggregates to colocalize with the chaperone calreticulin, indicating that they originated from the endoplasmic reticulum. The aggregates revealed a remarkable fibrillar substructure. Bacterially expressed and purified mutant pro-vasopressin spontaneously formed fibrils under oxidizing conditions. Mutagenesis experiments showed that the presence of cysteines, but no specific single cysteine, is essential for disulfide oligomerization and aggregation in vivo. Our findings assign autosomal dominant diabetes insipidus to the group of neurodegenerative diseases associated with the formation of fibrillar protein aggregates.

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells.

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.

Defects in the Secretory Pathway and High Ca2+ Induce Multiple P-bodies

This manuscript demonstrates for the first time that P-body (PB) formation in response to stress is a regulated process, and that at least two different pathways drive PB assembly. We provide evidence that PB formation and translation attenuation are not strictly linked.

The polysome-associated proteins Scp160 and Bfr1 prevent P body formation under normal growth conditions.

ABSTRACT Numerous mRNAs are degraded in processing bodies (P bodies) in Saccharomyces cerevisiae. In logarithmically growing cells, only 0–1 P bodies per cell are detectable. However, the number and appearance of P bodies change once the cell encounters stress. Here, we show that the polysome-associated mRNA-binding protein Scp160 interacts with P body components, such as the decapping protein Dcp2 and the scaffold protein Pat1, presumably, on polysomes. Loss of either Scp160 or its interaction partner Bfr1 caused the formation of Dcp2-positive structures. These Dcp2-positive foci contained mRNA, because their formation was inhibited by the presence of cycloheximide. In addition, Scp160 was required for proper P body formation because only a subset of bona fide P body components could assemble into the Dcp2-positive foci in &Dgr;scp160 cells. In either &Dgr;bfr1 or &Dgr;scp160 cells, P body formation was uncoupled from translational attenuation as the polysome profile remained unchanged. Collectively, our data suggest that Bfr1 and Scp160 prevent P body formation under normal growth conditions.