Impact of Detergents on Membrane Protein Complex Isolation.

Detergents play an essential role during the isolation of membrane protein complexes. Inappropriate use of detergents may affect the native fold of the membrane proteins, their binding to antibodies, or their interaction with partner proteins. Here we used cadherin-11 (Cad11) as an example to examine the impact of detergents on membrane protein complex isolation. We found that mAb 1A5 could immunoprecipitate Cad11 when membranes were solubilized by dodecyl maltoside (DDM) but not by octylglucoside, suggesting that octylglucoside interferes with Cad11-mAb 1A5 interaction. Furthermore, we compared the effects of Brij-35, Triton X-100, cholate, CHAPSO, Zwittergent 3-12, Deoxy BIG CHAP, and digitonin on Cad11 solubilization and immunoprecipitation. We found that all detergents except Brij-35 could solubilize Cad11 from the membrane. Upon immunoprecipitation, we found that ß-catenin, a known cadherin-interacting protein, was present in Cad11 immune complex among the detergents tested except Brij-35. However, the association of p120 catenin with Cad11 varied depending on the detergents used. Using isobaric tag for relative and absolute quantitation (iTRAQ) to determine the relative levels of proteins in Cad11 immune complexes, we found that DDM and Triton X-100 were more efficient than cholate in solubilization and immunoprecipitation of Cad11 and resulted in the identification of both canonical and new candidate Cad11-interacting proteins.

TOR complex 2 localises to the cytokinetic actomyosin ring and controls the fidelity of cytokinesis.

The timing of cell division is controlled by the coupled regulation of growth and division. The target of rapamycin (TOR) signalling network synchronises these processes with the environmental setting. Here, we describe a novel interaction of the fission yeast TOR complex 2 (TORC2) with the cytokinetic actomyosin ring (CAR), and a novel role for TORC2 in regulating the timing and fidelity of cytokinesis. Disruption of TORC2 or its localisation results in defects in CAR morphology and constriction. We provide evidence that the myosin II protein Myp2 and the myosin V protein Myo51 play roles in recruiting TORC2 to the CAR. We show that Myp2 and TORC2 are co-dependent upon each other for their normal localisation to the cytokinetic machinery. We go on to show that TORC2-dependent phosphorylation of actin-capping protein 1 (Acp1, a known regulator of cytokinesis) controls CAR stability, modulates Acp1-Acp2 (the equivalent of the mammalian CAPZA-CAPZB) heterodimer formation and is essential for survival upon stress. Thus, TORC2 localisation to the CAR, and TORC2-dependent Acp1 phosphorylation contributes to timely control and the fidelity of cytokinesis and cell division.

Torin1-mediated TOR kinase inhibition reduces Wee1 levels and advances mitotic commitment in fission yeast and HeLa cells.

The target of rapamycin (TOR) kinase regulates cell growth and division. Rapamycin only inhibits a subset of TOR activities. Here we show that in contrast to the mild impact of rapamycin on cell division, blocking the catalytic site of TOR with the Torin1 inhibitor completely arrests growth without cell death in Schizosaccharomyces pombe. A mutation of the Tor2 glycine residue (G2040D) that lies adjacent to the key Torin-interacting tryptophan provides Torin1 resistance, confirming the specificity of Torin1 for TOR. Using this mutation, we show that Torin1 advanced mitotic onset before inducing growth arrest. In contrast to TOR inhibition with rapamycin, regulation by either Wee1 or Cdc25 was sufficient for this Torin1-induced advanced mitosis. Torin1 promoted a Polo and Cdr2 kinase-controlled drop in Wee1 levels. Experiments in human cell lines recapitulated these yeast observations: mammalian TOR (mTOR) was inhibited by Torin1, Wee1 levels declined and mitotic commitment was advanced in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by TOR signalling is a conserved mechanism that helps to couple cell cycle and growth controls.

Aurora promotes cell division during recovery from TOR-mediated cell cycle arrest by driving spindle pole body recruitment of Polo.

The coordination of cell division and growth in response to changes in nutrient supply is mediated by TOR signalling. In fission yeast, increased nutrient provision transiently delays mitotic onset without affecting growth. The result is an increase in cell size at division. We find that this block to cell division relies upon TOR and MAPK signalling and that mitotic entry during recovery from this block is regulated by the Aurora kinase Ark1. We show that Ark1 phosphorylation of polo kinase Plo1 within the linker region between the kinase domain and polo boxes drives Plo1 onto the spindle poles where it promotes mitosis. Interestingly, the use of Ark1 to phosphorylate Plo1 and promote mitotic entry is dependent on the environment.

Highly Synchronous Mitotic Progression in Schizosaccharomyces pombe Upon Relief of Transient Cdc2-asM17 Inhibition.

Synchronized progression of a cell population through the cell division cycle supports the biochemical and functional dissection of cell cycle controls and execution. The concerted behaviour of the population reflects the attributes of each cell within that population. The reversible imposition of a block to cell cycle progression at the G2-M boundary through transient inactivation of the Cdk1-Cyclin B activating phosphatase, Cdc25, with the temperature sensitive cdc25-22 mutant, has been widely used to study fission yeast mitosis and DNA replication. However, the biology of the compromised Cdc25-22 phosphatase generates significant division abnormalities upon release from mitotic arrest. We show how reversible inhibition of Cdc2-asM17, with the ATP analog 3-BrB-PP1, generates higher levels of synchrony with timing and morphology much more reminiscent of a normal division. We also describe a version of the H1 kinase assay of Cdk1-Cyclin B activity that is widely used to monitor mitotic progression which does not require radiolabeled ATP.

A TOR (target of rapamycin) and nutritional phosphoproteome of fission yeast reveals novel targets in networks conserved in humans.

Fluctuations in TOR, AMPK and MAP-kinase signalling maintain cellular homeostasis and coordinate growth and division with environmental context. We have applied quantitative, SILAC mass spectrometry to map TOR and nutrient-controlled signalling in the fission yeast Schizosaccharomyces pombe. Phosphorylation levels at more than 1000 sites were altered following nitrogen stress or Torin1 inhibition of the TORC1 and TORC2 networks that comprise TOR signalling. One hundred and thirty of these sites were regulated by both perturbations, and the majority of these (119) new targets have not previously been linked to either nutritional or TOR control in either yeasts or humans. Elimination of AMPK inhibition of TORC1, by removal of AMPKα (ssp2::ura4+), identified phosphosites where nitrogen stress-induced changes were independent of TOR control. Using a yeast strain with an ATP analogue-sensitized Cdc2 kinase, we excluded sites that were changed as an indirect consequence of mitotic control modulation by nitrogen stress or TOR signalling. Nutritional control of gene expression was reflected in multiple targets in RNA metabolism, while significant modulation of actin cytoskeletal components points to adaptations in morphogenesis and cell integrity networks. Reduced phosphorylation of the MAPKK Byr1, at a site whose human equivalent controls docking between MEK and ERK, prevented sexual differentiation when resources were sparse but not eliminated.

Ste12/Fab1 phosphatidylinositol-3-phosphate 5-kinase is required for nitrogen-regulated mitotic commitment and cell size control.

Tight coupling of cell growth and cell cycle progression enable cells to adjust their rate of division, and therefore size, to the demands of proliferation in varying nutritional environments. Nutrient stress promotes inhibition of Target Of Rapamycin Complex 1 (TORC1) activity. In fission yeast, reduced TORC1 activity advances mitotic onset and switches growth to a sustained proliferation at reduced cell size. A screen for mutants, that failed to advance mitosis upon nitrogen stress, identified a mutant in the PIKFYVE 1-phosphatidylinositol-3-phosphate 5-kinase fission yeast homolog Ste12. Ste12PIKFYVE deficient mutants were unable to advance the cell cycle to reduce cell size after a nitrogen downshift to poor nitrogen (proline) growth conditions. While it is well established that PI(3,5)P2 signalling is required for autophagy and that Ste12PIKFYVE mutants have enlarged vacuoles (yeast lysosomes), neither a block to autophagy or mutants that independently have enlarged vacuoles had any impact upon nitrogen control of mitotic commitment. The addition of rapamycin to Ste12PIKFYVE deficient mutants reduced cell size at division to suggest that Ste12PIKFYVE possibly functions upstream of TORC1. ste12 mutants display increased Torin1 (TOR inhibitor) sensitivity. However, no major impact on TORC1 or TORC2 activity was observed in the ste12 deficient mutants. In summary, Ste12PIKFYVE is required for nitrogen-stress mediated advancement of mitosis to reduce cell size at division.

Phosphorylation of the TOR ATP binding domain by AGC kinase constitutes a novel mode of TOR inhibition.

TOR (target of rapamycin) signaling coordinates cell growth, metabolism, and cell division through tight control of signaling via two complexes, TORC1 and TORC2. Here, we show that fission yeast TOR kinases and mTOR are phosphorylated on an evolutionarily conserved residue of their ATP-binding domain. The Gad8 kinase (AKT homologue) phosphorylates fission yeast Tor1 at this threonine (T1972) to reduce activity. A T1972A mutation that blocked phosphorylation increased Tor1 activity and stress resistance. Nitrogen starvation of fission yeast inhibited TOR signaling to arrest cell cycle progression in G1 phase and promoted sexual differentiation. Starvation and a Gad8/T1972-dependent decrease in Tor1 (TORC2) activity was essential for efficient cell cycle arrest and differentiation. Experiments in human cell lines recapitulated these yeast observations, as mTOR was phosphorylated on T2173 in an AKT-dependent manner. In addition, a T2173A mutation increased mTOR activity. Thus, TOR kinase activity can be reduced through AGC kinase-controlled phosphorylation to generate physiologically significant changes in TOR signaling.

TORC2 and the AGC kinase Gad8 regulate phosphorylation of the ribosomal protein S6 in fission yeast.

TOR (Target Of Rapamycin) signalling coordinates cell growth and division in response to changes in the nutritional environment of the cell. TOR kinases form two distinct complexes: TORC1 and TORC2. In mammals, the TORC1 controlled S6K1 kinase phosphorylates the ribosomal protein S6 thereby co-ordinating cell size and nutritional status. We show that the Schizosaccharomyces pombe AGC kinase Gad8 co-immunoprecipitates with the ribosomal protein S6 (Rps6) and regulates its phosphorylation status. It has previously been shown that Gad8 is phosphorylated by TORC2. Consistent with this, we find that TORC2 as well as TORC1 modulates Rps6 phosphorylation. Therefore, S6 phosphorylation in fission yeast actually represents a read-out of the combined activities of TORC1 and TORC2. In contrast, we find that the in vivo phosphorylation status of Maf1 (a repressor of RNA polymerase III) specifically correlates with TORC1 activity.