Wolfgang Oppliger

Two TOR Complexes, Only One of which Is Rapamycin Sensitive, Have Distinct Roles in Cell Growth Control

The target of rapamycin (TOR) proteins in Saccharomyces cerevisiae, TOR1 and TOR2, redundantly regulate growth in a rapamycin-sensitive manner. TOR2 additionally regulates polarization of the actin cytoskeleton in a rapamycin-insensitive manner. We describe two functionally distinct TOR complexes. TOR Complex 1 (TORC1) contains TOR1 or TOR2, KOG1 (YHR186c), and LST8. TORC2 contains TOR2, AVO1 (YOL078w), AVO2 (YMR068w), AVO3 (YER093c), and LST8. FKBP-rapamycin binds TORC1, and TORC1 disruption mimics rapamycin treatment, suggesting that TORC1 mediates the rapamycin-sensitive, TOR-shared pathway. FKBP-rapamycin fails to bind TORC2, and TORC2 disruption causes an actin defect, suggesting that TORC2 mediates the rapamycin-insensitive, TOR2-unique pathway. Thus, the distinct TOR complexes account for the diversity, specificity, and selective rapamycin inhibition of TOR signaling. TORC1 and possibly TORC2 are conserved from yeast to man.

Activation of mTORC2 by association with the ribosome.

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 investigated the upstream regulation of mTORC2. A genetic screen in yeast and subsequent studies in mammalian cells revealed that ribosomes, but not protein synthesis, are required for mTORC2 signaling. Active mTORC2 was physically associated with the ribosome, and insulin-stimulated PI3K signaling promoted mTORC2-ribosome binding, suggesting that ribosomes activate mTORC2 directly. Findings with melanoma and colon cancer cells suggest that mTORC2-ribosome association is important in oncogenic PI3K signaling. Thus, TORC2-ribosome interaction is a likely conserved mechanism of TORC2 activation that is physiologically relevant in both normal and cancer cells. As ribosome content determines growth capacity of a cell, this mechanism of TORC2 regulation ensures that TORC2 is active only in growing cells.

Glutaminolysis Activates Rag-mTORC1 Signaling

Amino acids control cell growth via activation of the highly conserved kinase TORC1. Glutamine is a particularly important amino acid in cell growth control and metabolism. However, the role of glutamine in TORC1 activation remains poorly defined. Glutamine is metabolized through glutaminolysis to produce α-ketoglutarate. We demonstrate that glutamine in combination with leucine activates mammalian TORC1 (mTORC1) by enhancing glutaminolysis and α-ketoglutarate production. Inhibition of glutaminolysis prevented GTP loading of RagB and lysosomal translocation and subsequent activation of mTORC1. Constitutively active Rag heterodimer activated mTORC1 in the absence of glutaminolysis. Conversely, enhanced glutaminolysis or a cell-permeable α-ketoglutarate analog stimulated lysosomal translocation and activation of mTORC1. Finally, cell growth and autophagy, two processes controlled by mTORC1, were regulated by glutaminolysis. Thus, mTORC1 senses and is activated by glutamine and leucine via glutaminolysis and α-ketoglutarate production upstream of Rag. This may provide an explanation for glutamine addiction in cancer cells.

The Tim9p-Tim10p complex binds to the transmembrane domains of the ADP/ATP carrier.

The soluble Tim9p–Tim10p (Tim, translocase of inner membrane) complex of the mitochondrial intermembrane space mediates the import of the carrier proteins and is a component of the TIM22 import system. The mechanism by which the Tim9p–Tim10p complex assembles and binds the carriers is not well understood, but previous studies have proposed that the conserved cysteine residues in the ‘twin CX3C’ motif coordinate zinc and potentially generate a zinc‐finger‐like structure that binds to the matrix loops of the carrier proteins. Here we have purified the native and recombinant Tim9p–Tim10p complex, and show that both complexes resemble each other and consist of three Tim9p and three Tim10p. Results from inductively coupled plasma–mass spectrometry studies failed to detect zinc in the Tim9p–Tim10p complex. Instead, the cysteine residues seemingly formed disulfide linkages. The Tim9p–Tim10p complex bound specifically to the transmembrane domains of the ADP/ATP carrier, but had no affinity for Tim23p, an inner membrane protein that is inserted via the TIM22 complex. The chaperone‐like Tim9p–Tim10p complex thus may prevent aggregation of the unfolded carrier proteins in the aqueous intermembrane space.

Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins

Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p co‐purify in successive chromatographic fractionations and co‐immunoprecipitated with each other. Tim9p can be cross‐linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser67→Cys67 mutation in Tim9p suppresses the temperature‐sensitive growth defect of tim10‐1 and tim12‐1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.

Import of proteins into yeast mitochondria: the purified matrix processing protease contains two subunits which are encoded by the nuclear MAS1 and MAS2 genes.

We have purified the metalloprotease which is localized in the soluble matrix space of Saccharomyces cerevisiae mitochondria and cleaves the amino‐terminal matrix‐targeting sequences from imported mitochondrial precursor proteins. The enzyme consists of two loosely associated non‐identical subunits of mol. wt 48,000 and 51,000, respectively. Attempts to separate the two subunits from each other caused loss of activity. The smaller subunit had been identified as the product of the nuclear MAS1 gene (Witte et al., 1988). The larger subunit is now identified as the product of the nuclear MAS2 gene.

Sequential action of two hsp70 complexes during protein import into mitochondria

The mitochondrial chaperone mhsp70 mediates protein transport across the inner membrane and protein folding in the matrix. These two reactions are effected by two different mhsp70 complexes. The ADP conformation of mhsp70 favors formation of a complex on the inner membrane; this ‘import complex’ contains mhsp70, its membrane anchor Tim44 and the nucleotide exchange factor mGrpE. The ATP conformation of mhsp70 favors formation of a complex in the matrix; this ‘folding complex’ contains mhsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE. A precursor protein entering the matrix interacts first with the import complex and then with the folding complex. A chaperone can thus function as part of two different complexes within the same organelle.

Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported proteins into the yeast mitochondrial inner membrane.

ABSTRACT Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.

Dynamic interaction of the protein translocation systems in the inner and outer membranes of yeast mitochondria.

Mitochondria contain two distinct protein import systems, one in the outer and the other in the inner membrane. These systems can act independently of one another in submitochondrial fractions of if a protein is transported to the outer membrane or to the intermembrane space. It has been proposed that the two systems associate reversibly when a protein is transported across both membranes, but this hypothesis has remained unproven. In order to address this question, we have checked whether antibodies against a subunit of one system can co‐immunoprecipitate subunits of the other system. We find that the two systems associate stably if a matrix‐targeted precursor is arrested during import; no association is seen in the absence of a stuck precursor. These experiments provide direct evidence that protein import into the mitochondrial matrix is mediated by the reversible interaction of the two translocation systems.

Hepatic mTORC1 controls locomotor activity, body temperature, and lipid metabolism through FGF21.

Significance The mammalian target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to nutrients, growth factors, and cellular energy. Aberrant mTORC1 signaling is implicated in human diseases such as diabetes, obesity, and cancer. Our results reveal that ectopic mTORC1 activation in the liver controls the stress hormone fibroblast growth factor 21 (FGF21) in a peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α)–dependent manner via glutamine depletion, which in turn affects whole-body behavior and metabolism. mTORC1 signaling correlates with FGF21 expression in human liver tumors, suggesting that our findings in mice may have physiological relevance in glutamine-addicted human cancers. Thus, treatment with the anticancer drug rapamycin may have beneficial effects by blocking tumor growth and by preventing deregulation of whole-body physiology due to FGF21 expression. The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of growth and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α)–dependent fibroblast growth factor 21 (FGF21) expression in the liver. Injection of glutamine or knockdown of PGC-1α or FGF21 in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1α and FGF21. Finally, mTORC1 signaling correlated with FGF21 expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level.