Mass Spectrometry-based Methods for Phosphorylation Site Mapping of Hyperphosphorylated Proteins Applied to Net1, a Regulator of Exit from Mitosis in Yeast

Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1·Cdc14 complex in vitro by phosphorylation of Net1. We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies

A Numerical Study of the Superconducting Proximity Effect in Topological Surface States

We study the superconducting proximity effect induced in the surface states of the 3-d topological insulator Bi$_2$Se$_3$ by a singlet, s-wave superconductor deposited on its surface. To this effect, the $\mathbf{k}\cdot\mathbf{p}$-Hamiltonian of Bi$_2$Se$_3$ and the BCS-Hamiltonian are mapped onto tight-binding chains which we couple through a transfer-Hamiltonian at the interface. We then employ the Recursive Green's Function technique to obtain the local spectral function and infer the dispersion of the interface-states from it. In agreement with earlier microscopic studies of this problem, we find that the Fu-Kane model is a reasonable approximation at energies $\epsilon\ll \Delta_{\rm SC}$. However, for energies close to the SC bulk gap, the Fu-Kane model is expected to break down. Indeed, our numerical calculations show strong modifications of the interface-state dispersion for $\epsilon \gtrsim \Delta_{\rm SC}$. We find that the proximity effect can be strong enough to induce a gap in the surface state that is comparable to the superconducting gap. An analysis of the spatial profile of the states shows that their weight shifts towards the SC as the coupling strength increases. We conclude that an intermediate coupling is ideal for realising the Fu-Kane scenario.Comment: JOP: Conference Series (Proceedings of SCES 2011), accepte

A simple method for finite range decomposition of quadratic forms and Gaussian fields

We present a simple method to decompose the Green forms corresponding to a large class of interesting symmetric Dirichlet forms into integrals over symmetric positive semi-definite and finite range (properly supported) forms that are smoother than the original Green form. This result gives rise to multiscale decompositions of the associated Gaussian free fields into sums of independent smoother Gaussian fields with spatially localized correlations. Our method makes use of the finite propagation speed of the wave equation and Chebyshev polynomials. It improves several existing results and also gives simpler proofs.Comment: minor correction for t<

A regularisation approach to causality theory for C^{1,1}Lorentzian metrics

We show that many standard results of Lorentzian causality theory remain valid if the regularity of the metric is reduced to C^{1,1}. Our approach is based on regularisations of the metric adapted to the causal structure

Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex

BACKGROUND: In S. cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis. In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex. When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus. RESULTS: Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase. Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14. Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m') lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow normally and exhibit only minor defects in releasing Cdc14 during anaphase. However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene). CONCLUSIONS: We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets. Our study suggests that Cdc5/Polo is unusually promiscuous and highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments

ab initio modeling of open systems: charge transfer, electron conduction, and molecular switching of a C_{60} device

We present an {\it ab initio} analysis of electron conduction through a $C_{60}$ molecular device. Charge transfer from the device electrodes to the molecular region is found to play a crucial role in aligning the lowest unoccupied molecular orbital (LUMO) of the $C_{60}$ to the Fermi level of the electrodes. This alignment induces a substantial device conductance of $\sim 2.2 \times (2e^2/h)$. A gate potential can inhibit charge transfer and introduce a conductance gap near $E_F$, changing the current-voltage characteristics from metallic to semi-conducting, thereby producing a field effect molecular current switch

Compression Stress-Induced Internal Magnetic Field in Bulky TiO2 Photoanodes for Enhancing Charge-Carrier Dynamics

Enhancing charge-carrier dynamics is imperative to achieve efficient photoelectrodes for practical photoelectrochemical devices. However, a convincing explanation and answer for the important question which has thus far been absent relates to the precise mechanism of charge-carrier generation by solar light in photoelectrodes. Herein, to exclude the interference of complex multi-components and nanostructuring, we fabricate bulky TiO2 photoanodes through physical vapor deposition. Integrating photoelectrochemical measurements and in situ characterizations, the photoinduced holes and electrons are transiently stored and promptly transported around the oxygen-bridge bonds and 5-coordinated Ti atoms to form polarons on the boundaries of TiO2 grains, respectively. Most importantly, we also find that compressive stress-induced internal magnetic field can drastically enhance the charge-carrier dynamics for the TiO2 photoanode, including directional separation and transport of charge carriers and an increase of surface polarons. As a result, bulky TiO2 photoanode with high compressive stress displays a high charge-separation efficiency and an excellent charge-injection efficiency, leading to 2 orders of magnitude higher photocurrent than that produced by a classic TiO2 photoanode. This work not only provides a fundamental understanding of the charge-carrier dynamics of the photoelectrodes but also provides a new paradigm for designing efficient photoelectrodes and controlling the dynamics of charge carriers

Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5'-cap-structures of higher eukaryote mRNAs are ribose 2'-O-methylated. Likewise, a number of viruses replicating in the cytoplasm of eukayotes have evolved 2'-O-methyltransferases to modify autonomously their mRNAs. However, a defined biological role of mRNA 2'-O-methylation remains elusive. Here we show that viral mRNA 2'-O-methylation is critically involved in subversion of type-I-interferon (IFN-I) induction. We demonstrate that human and murine coronavirus 2'-O-methyltransferase mutants induce increased IFN-I expression, and are highly IFN-I sensitive. Importantly, IFN-I induction by 2'-O-methyltransferase-deficient viruses is dependent on the cytoplasmic RNA sensor melanoma differentiation-associated gene 5 (MDA5). This link between MDA5-mediated sensing of viral RNA and mRNA 2'-O-methylation suggests that RNA modifications, such as 2'-O-methylation, provide a molecular signature for the discrimination of self and non-self mRNA