Thomas Beck

The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors

The rapamycin-sensitive TOR signalling pathway in Saccharomyces cerevisiae activates a cell-growth program in response to nutrients such as nitrogen and carbon. The TOR1 and TOR2 kinases (TOR) control cytoplasmic protein synthesis and degradation through the conserved TAP42 protein. Upon phosphorylation by TOR, TAP42 binds and possibly inhibits type 2A and type-2A-related phosphatases; however, the mechanism by which TOR controls nuclear events such as global repression of starvation-specific transcription is unknown. Here we show that TOR prevents transcription of genes expressed upon nitrogen limitation by promoting the association of the GATA transcription factor GLN3 with the cytoplasmic protein URE2. The binding of GLN3 to URE2 requires TOR-dependent phosphorylation of GLN3. Phosphorylation and cytoplasmic retention of GLN3 are also dependent on the TOR effector TAP42, and are antagonized by the type-2A-related phosphatase SIT4. TOR inhibits expression of carbon-source-regulated genes by stimulating the binding of the transcriptional activators MSN2 and MSN4 to the cytoplasmic 14-3-3 protein BMH2. Thus, the TOR signalling pathway broadly controls nutrient metabolism by sequestering several transcription factors in the cytoplasm.

The Yeast Phosphatidylinositol Kinase Homolog TOR2 Activates RHO1 and RHO2 via the Exchange Factor ROM2

The Saccharomyces cerevisiae phosphatidylinositol kinase homolog TOR2 is required for organization of the actin cytoskeleton. Overexpression of RHO1 or RHO2, encoding Rho-like GTPases, or ROM2, encoding a GDP/GTP exchange factor for RHO1 and RHO2, suppresses a tor2 mutation. Deletion of SAC7, a gene originally identified as a suppressor of an actin mutation, also suppresses a tor2 mutation. SAC7 is a novel GTPase-activating protein for RHO1. ROM2 exchange activity is reduced in a tor2 mutant, and overexpression of ROM2 lacking its PH domain can no longer suppress a tor2 mutation. Thus, TOR2 signals to the actin cytoskeleton through a GTPase switch composed of RHO1, RHO2, ROM2, and SAC7. TOR2 activates this switch via ROM2, possibly via the ROM2 PH domain.

The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease.

The Saccharomyces cerevisiae targets of rapamycin, TOR1 and TOR2, signal activation of cell growth in response to nutrient availability. Loss of TOR or rapamycin treatment causes yeast cells to arrest growth in early G1 and to express several other physiological properties of starved (G0) cells. As part of this starvation response, high affinity amino acid permeases such as the tryptophan permease TAT2 are targeted to the vacuole and degraded. Here we show that the TOR signalling pathway phosphorylates the Ser/Thr kinase NPR1 and thereby inhibits the starvation‐induced turnover of TAT2. Overexpression of NPR1 inhibits growth and induces the degradation of TAT2, whereas loss of NPR1 confers resistance to rapamycin and to FK506, an inhibitor of amino acid import. NPR1 is controlled by TOR and the type 2A phosphatase‐associated protein TAP42. First, overexpression of NPR1 is toxic only when TOR function is reduced. Secondly, NPR1 is rapidly dephosphorylated in the absence of TOR. Thirdly, NPR1 dephosphorylation does not occur in a rapamycin‐resistant tap42 mutant. Thus, the TOR nutrient signalling pathway also controls growth by inhibiting a stationary phase (G0) programme. The control of NPR1 by TOR is analogous to the control of p70 s6 kinase and 4E‐BP1 by mTOR in mammalian cells.

Activation of the RAS/Cyclic AMP Pathway Suppresses a TOR Deficiency in Yeast

ABSTRACT The TOR (target of rapamycin) and RAS/cyclic AMP (cAMP) signaling pathways are the two major pathways controlling cell growth in response to nutrients in yeast. In this study we examine the functional interaction between TOR and the RAS/cAMP pathway. First, activation of the RAS/cAMP signaling pathway confers pronounced resistance to rapamycin. Second, constitutive activation of the RAS/cAMP pathway prevents several rapamycin-induced responses, such as the nuclear translocation of the transcription factor MSN2 and induction of stress genes, the accumulation of glycogen, the induction of autophagy, the down-regulation of ribosome biogenesis (ribosomal protein gene transcription and RNA polymerase I and III activity), and the down-regulation of the glucose transporter HXT1. Third, many of these TOR-mediated responses are independent of the previously described TOR effectors TAP42 and the type 2A-related protein phosphatase SIT4. Conversely, TOR-controlled TAP42/SIT4-dependent events are not affected by the RAS/cAMP pathway. Finally, and importantly, TOR controls the subcellular localization of both the protein kinase A catalytic subunit TPK1 and the RAS/cAMP signaling-related kinase YAK1. Our findings suggest that TOR signals through the RAS/cAMP pathway, independently of TAP42/SIT4. Therefore, the RAS/cAMP pathway may be a novel TOR effector branch.

Muscle strength in overwintering bears

Unlike humans, bears retain their muscle tone when moribund for long periods.

Protein Use and Muscle-Fiber Changes in Free-Ranging, Hibernating Black Bears

Studies of the metabolic and physiological changes that bears undergo during hibernation have, for the most part, supported the paradigm that bears use only fatty tissues as a metabolic substate during hibemation. This study was performed to document the extent of protein loss and alteration of muscle‐fiber characteristics of selected muscles in black bears during winter dormanc. Muscle biopises were removed from the gas‐trocnemius and biceps femoris from seven free‐ranging female black bears on the Uncompahgre Plateau in west‐central Colo‐rado. Six of the seven bears produced cubs during the hibernat‐ing season. Muscle samples were collected from the left hind limb shortly after bears entered their dens (fall), and additional samples were collected from the right hind limb Just prior to bears leaving their dens (spring). Protein concentration, fast‐and slow‐twitch muscle‐fiber rations and muscle‐fiber cross‐sectional areas, and citrate synthase activity were measured in the laboratory. While Protein concentration decreased in both muscles during the hibernation period, it was lower than pre‐dicted for lactating females, In addition, muscle‐fiber number and cross‐sectional area were unchanged in these muscles, sug‐gesting only limited muscle atrophy. In support of these obser‐vations, there were a moderate but significant increase in the proportion of fast‐twitch fibers only in the biceps femoris, with a concomitant decrese in citrate synthase activity, but no alteration of the fiber ratio in the gastrocnemius during hiber‐natio. The findings suggest that hibernating bears, particu

Hibernating Bears Conserve Muscle Strength and Maintain Fatigue Resistance

Black bears spend several months each winter confined to a small space within their den without food or water. In nonhibernating mammals, these conditions typically result in severe muscle atrophy, causing a loss of strength and endurance. However, an initial study indicated that bears appeared to conserve strength while denning. We conducted an in vivo, nonsubjective measurement of strength, resistance to fatigue, and contractile properties on the tibialis anterior muscle of six hibernating bears during both early and late winter using a rigid leg brace and foot force plate. After 110 d of anorexia and confinement, skeletal muscle strength loss in hibernating bears was about one‐half that in humans confined to bed rest. Bears lost 29% of muscle strength over 110 d of denning without food, while humans on a balanced diet but confined to bed for 90 d have been reported to lose 54% of their strength. Additionally, muscle contractile properties, including contraction time, half‐relaxation time, half–maximum value time, peak rate of development and decay, time to peak force development, and time to peak force decay did not change, indicating that no small‐scale alterations in whole‐muscle function occurred over the winter. This study further supports our previous findings that black bears have a high resistance to atrophy despite being subjected to long‐term anorexia and limited mobility.

Control of the Actin Cytoskeleton by Extracellular Signals

Rho-type GTPases play a central role in linking extracellular signals to the organization of the actin cytoskeleton, from yeast to mammals (Van Aelst and D’Souza-Schorey 1997; Hall 1998b; Schmidt and Hall 1998). In addition to their role in actin organization, Rho-type GTPases also control signalling pathways that activate transcription factors. The Rho proteins thus regulate a broad variety of cellular events, such as shape changes, migration, attachment, proliferation, differentiation, and survival. The Rho family is a subclass of the Ras superfamily of GTPases. These small GTPases function as binary switches, cycling between an active, GTP-bound form and an inactive, GDP-bound form.