In this issue, Takahara and Maeda (2012) discover that together, Pbp1 and sequestration of the TORC1 complex in cytoplasmic mRNP stress granules provides a negative regulatory mechanism for TORC1 signaling during stress.
To address the role of sequence in the folding of homologous proteins, the folding and unfolding kinetics of the all-helical bacterial immunity proteins Im2 and Im9 were characterised, together with six chimeric derivatives of these proteins. We show that both Im2 and Im9 fold rapidly (kUNH2O ≈ 2000 s-1 at pH 7.0, 25°C) in apparent two-state transitions, through rate-limiting transition states that are highly compact (βTS 0.93 and 0.96, respectively). Whilst the folding and unfolding properties of three of the chimeras (Im2 (1-44)Im9, Im2 (1-64)Im9 and Im2 (25-44)Im9) are similar to their parental counterparts, in other chimeric proteins the introduced sequence variation results in altered kinetic behaviour. At low urea concentrations, Im2 (1-29)Im9 and Im2 (56-64)Im9 fold in two-state transitions via transition states that are significantly less compact (βTS ≈ 0.7) than those characterised for the other immunity proteins presented here. At higher urea concentrations, however, the rate-limiting transition state for these two chimeras switches or moves to a more compact species (βTS ≈ 0.9). Surprisingly, Im2 (30-64)Im9 populates a highly collapsed species (βI = 0.87) in the dead-time (2.5 ms) of stopped flow measurements. These data indicate that whilst topology may place significant constraints on the folding process, specific inter-residue interactions, revealed here through multiple sequence changes, can modulate the ruggedness of the folding energy landscape. © 2001 Academic Press.
The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60% sequence identity. At pH 7.0 and 10°C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10°C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins. © 2001 Academic Press.
Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of ∼150 μs, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.
The Target of Rapamycin kinase Complex I (TORC1) is a master regulator of cell growth and metabolism in eukaryotes. Studies in yeast and human cells have shown that nitrogen/amino acid starvation signals act through Npr2/3 and the small GTPases Gtr1/2 (Rags in humans) to inhibit TORC1. However, it is unclear how other stress and starvation stimuli inhibit TORC1, and/or act in parallel with the TORC1 pathway, to control cell growth. To help answer these questions, we developed a novel automated pipeline and used it to measure the expression of a TORC1 dependent ribosome biogenesis gene (NSR1) during osmotic stress in 4700 Saccharomyces cerevisiae strains from the yeast knock-out collection. This led to the identification of 440 strains with significant and reproducible defects in NSR1 repression. The cell growth control and stress response proteins deleted in these strains form a highly connected network, including; 56 proteins involved in vesicle trafficking and vacuolar function; 53 proteins that act downstream of TORC1 according to a rapamycin assay--including components of the HDAC Rpd3L, Elongator, and the INO80, CAF-1 and SWI/SNF chromatin remodeling complexes; over 100 proteins involved in signaling and metabolism; and 17 proteins that directly interact with TORC1. These data provide an important resource for labs studying cell growth control and stress signaling, and demonstrate the utility of our new, and easily adaptable, method for mapping gene regulatory networks.