Investigating the Role of the GET3-GET4/GET5 Interaction During Tail-Anchor Protein Targeting

The proper targeting of membrane proteins is essential to the viability of all cells. Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C-terminus, are post-translationally targeted to the endoplasmic reticulum (ER) membrane by the GET pathway (Guided Entry of TA proteins). In the yeast pathway, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 (Get4/5) complex, which tethers the co-chaperone Sgt2 to the central targeting factor, the Get3 ATPase. Although binding of Get4/5 to Get3 is critical for efficient TA targeting, the mechanisms by which Get4 regulates Get3 are unknown. To understand the molecular basis of Get4 function, we used a combination of structural biology, biochemistry, and cell biology. Get4/5 binds across the Get3 dimer interface, in an orientation only compatible with a closed Get3, providing insight into the role of nucleotide in complex formation. Additionally, this structure reveals two functionally distinct binding interfaces for anchoring and ATPase regulation, and loss of the regulatory interface leads to strong defects in vitro and in vivo. Additional crystal structures of the Get3-Get4/5 complex give rise to an alternate conformation, which represents an initial binding interaction mediated by electrostatics that facilitates the rate of subsequent inhibited complex formation. This interface is supported by an in-depth kinetic analysis of the Get3-Get4/5 interaction confirming the two-step complex formation. These results allow us to generate a refined model for Get4/5 function in TA targeting.

On the interaction of actinomycin and DNA

Experiments have been accomplished that (a) further define the nature of the strong, G-containing DNA binding sites for actinomycin D (AMD), and (b) quantitate the in vitro inhibition of E. coli RNA polymerase activity by T7 DNA-bound AMD.

Twenty-five to forty percent of the G's of crab dAT are disallowed as strong AMD binding sites. The G's are measured to be randomly distributed, and, therefore, this datum cannot be explained on the basis of steric interference alone. Poly dAC:TG binds as much AMD and as strongly as any natural DNA, so the hypothesis that the unique strong AMD binding sites are G and a neighboring purine is incorrect. The datum can be explained on the basis of both steric interference and the fact that TGA is a disallowed sequence for strong AMD binding.

Using carefully defined in vitro conditions, there is one RNA synthesized per T7 DNA by E. coli RNA polymerase. The rate of the RNA polymerase-catalyzed reaction conforms to the equation 1/rate = 1/k_A(ATP) + 1/K_G(GTP) + 1/K_C(CTP) + 1/K_U(UTP) T7 DNA-bound AMD has only modest effects on initiation and termination of the polymerase-catalyzed reaction, but a large inhibitory effect on propagation. In the presence of bound AMD, k_G and k_C are decreased, whereas k_A and k_U are unaffected. These facts are interpreted to mean that on the microscopic level, on the average, the rates of incorporation of ATP and UTP are the same in the absence or presence of bound AMD, but that the rates of incorporation of GTP and CTP are decreased in the presence of AMD.

Zooplankton - fish interaction in the littoral zone of Nyanza Gulf, Lake Victoria

The zooplankton community of the littoral zone of Nyanza Gulf, Lake Victoria, was studied between June 1998 and June 1999 to identify and quantify various zooplankton groups, and investigate the interactions that occur between them and the littoral fish through the food chain. Zooplankton samples were collected from five stations using a 83 micro-m mesh size plankton net hauled vertically through the water column. Fish samples were obtained by beach seine, except at Gingra (May 1999), where trawl samples were used. Gut/stomach analysis was carried out on the three major commercial species, Lates niloticus (L.), Oreochromis niloticus (L.) and Rastrineobola argentea (Pellegrin).

I. Nuclear spin-internal-rotation-coupling. II. Fluorine spin-rotation interaction and magnetic shielding in fluorobenzene

Part I.

The interaction of a nuclear magnetic moment situated on an internal top with the magnetic fields produced by the internal as well as overall molecular rotation has been derived following the method of Van Vleck for the spin-rotation interaction in rigid molecules. It is shown that the Hamiltonian for this problem may be written

H_SR = Ῑ · M · Ĵ + Ῑ · M” · Ĵ”

Where the first term is the ordinary spin-rotation interaction and the second term arises from the spin-internal-rotation coupling.

The F¹⁹ nuclear spin-lattice relaxation time (T₁) of benzotrifluoride and several chemically substituted benzotrifluorides, have been measured both neat and in solution, at room temperature by pulsed nuclear magnetic resonance. From these experimental results it is concluded that in benzotrifluoride the internal rotation is crucial to the spin relaxation of the fluorines and that the dominant relaxation mechanism is the fluctuating spin-internal-rotation interaction.

Part II.

The radiofrequency spectrum corresponding to the reorientation of the F¹⁹ nuclear moment in flurobenzene has been studied by the molecular beam magnetic resonance method. A molecular beam apparatus with an electron bombardment detector was used in the experiments. The F¹⁹ resonance is a composite spectrum with contributions from many rotational states and is not resolved. A detailed analysis of the resonance line shape and width by the method of moments led to the following diagonal components of the fluorine spin-rotational tensor in the principal inertial axis system of the molecule:

F/Caa = -1.0 ± 0.5 kHz

F/Cbb = -2.7 ± 0.2 kHz

F/Ccc = -1.9 ± 0.1 kHz

From these interaction constants, the paramagnetic contribution to the F¹⁹ nuclear shielding in C₆H₅F was determined to be -284 ± ppm. It was further concluded that the F¹⁹ nucleus in this molecule is more shielded when the applied magnetic field is directed along the C-F bond axis. The anisotropy of the magnetic shielding tensor, σ_” - σ_⊥, is +160 ± 30 ppm.

Decomposition in lake sediments: bacterial action and interaction

This review discusses the processes involved in the decomposition of organic carbon derived initially from structural components of algae and other primary producers. It describes how groups of bacteria interact in time and space in a eutrophic lake. The relative importance of anaerobic and aerobic processes are discussed. The bulk of decomposition occurs within the sediment. The role of bacteria in the nitrogen cycle and the iron cycle, and in sulphate reduction and methanogenesis as the terminal metabolism of organic carbon are described.

I. Non-linear effects in a self-consistent calculation of SU_3 symmetry breaking in strong interaction coupling constants. II. Direct-channel reggeization of strong interaction scattering amplitudes

The thesis is divided into two parts. Part I generalizes a self-consistent calculation of residue shifts from SU₃ symmetry, originally performed by Dashen, Dothan, Frautschi, and Sharp, to include the effects of non-linear terms. Residue factorizability is used to transform an overdetermined set of equations into a variational problem, which is designed to take advantage of the redundancy of the mathematical system. The solution of this problem automatically satisfies the requirement of factorizability and comes close to satisfying all the original equations.

Part II investigates some consequences of direct channel Regge poles and treats the problem of relating Reggeized partial wave expansions made in different reaction channels. An analytic method is introduced which can be used to determine the crossed-channel discontinuity for a large class of direct-channel Regge representations, and this method is applied to some specific representations.

It is demonstrated that the multi-sheeted analytic structure of the Regge trajectory function can be used to resolve apparent difficulties arising from infinitely rising Regge trajectories. Also discussed are the implications of large collections of "daughter trajectories."

Two things are of particular interest: first, the threshold behavior in direct and crossed channels; second, the potentialities of Reggeized representations for us in self-consistent calculations. A new representation is introduced which surpasses previous formulations in these two areas, automatically satisfying direct-channel threshold constraints while being capable of reproducing a reasonable crossed channel discontinuity. A scalar model is investigated for low energies, and a relation is obtained between the mass of the lowest bound state and the slope of the Regge trajectory.

The oculomotor system: (1) Vertical-horizontal interaction and signal recognition, (2) Time delays and power spectra

In the first section of this thesis, two-dimensional properties of the human eye movement control system were studied. The vertical - horizontal interaction was investigated by using a two-dimensional target motion consisting of a sinusoid in one of the directions vertical or horizontal, and low-pass filtered Gaussian random motion of variable bandwidth (and hence information content) in the orthogonal direction. It was found that the random motion reduced the efficiency of the sinusoidal tracking. However, the sinusoidal tracking was only slightly dependent on the bandwidth of the random motion. Thus the system should be thought of as consisting of two independent channels with a small amount of mutual cross-talk.

These target motions were then rotated to discover whether or not the system is capable of recognizing the two-component nature of the target motion. That is, the sinusoid was presented along an oblique line (neither vertical nor horizontal) with the random motion orthogonal to it. The system did not simply track the vertical and horizontal components of motion, but rotated its frame of reference so that its two tracking channels coincided with the directions of the two target motion components. This recognition occurred even when the two orthogonal motions were both random, but with different bandwidths.

In the second section, time delays, prediction and power spectra were examined. Time delays were calculated in response to various periodic signals, various bandwidths of narrow-band Gaussian random motions and sinusoids. It was demonstrated that prediction occurred only when the target motion was periodic, and only if the harmonic content was such that the signal was sufficiently narrow-band. It appears as if general periodic motions are split into predictive and non-predictive components.

For unpredictable motions, the relationship between the time delay and the average speed of the retinal image was linear. Based on this I proposed a model explaining the time delays for both random and periodic motions. My experiments did not prove that the system is sampled data, or that it is continuous. However, the model can be interpreted as representative of a sample data system whose sample interval is a function of the target motion.

It was shown that increasing the bandwidth of the low-pass filtered Gaussian random motion resulted in an increase of the eye movement bandwidth. Some properties of the eyeball-muscle dynamics and the extraocular muscle "active state tension" were derived.

1. Ultrasonic studies of binary liquid structure in the critical region. Theory and experiment for the 2,6-lutidine/water system. 2. Hartree-Fock calculations of electric polarizabilities of some simple atoms and molecules, and their practicality. 3. Calculation of vibrational transition probabilities in collinear atom-diatom and diatom-diatom collisions with Lennard-Jones interaction

Part 1. Many interesting visual and mechanical phenomena occur in the critical region of fluids, both for the gas-liquid and liquid-liquid transitions. The precise thermodynamic and transport behavior here has some broad consequences for the molecular theory of liquids. Previous studies in this laboratory on a liquid-liquid critical mixture via ultrasonics supported a basically classical analysis of fluid behavior by M. Fixman (e. g., the free energy is assumed analytic in intensive variables in the thermodynamics)--at least when the fluid is not too close to critical. A breakdown in classical concepts is evidenced close to critical, in some well-defined ways. We have studied herein a liquid-liquid critical system of complementary nature (possessing a lower critical mixing or consolute temperature) to all previous mixtures, to look for new qualitative critical behavior. We did not find such new behavior in the ultrasonic absorption ascribable to the critical fluctuations, but we did find extra absorption due to chemical processes (yet these are related to the mixing behavior generating the lower consolute point). We rederived, corrected, and extended Fixman's analysis to interpret our experimental results in these more complex circumstances. The entire account of theory and experiment is prefaced by an extensive introduction recounting the general status of liquid state theory. The introduction provides a context for our present work, and also points out problems deserving attention. Interest in these problems was stimulated by this work but also by work in Part 3.

Part 2. Among variational theories of electronic structure, the Hartree-Fock theory has proved particularly valuable for a practical understanding of such properties as chemical binding, electric multipole moments, and X-ray scattering intensity. It also provides the most tractable method of calculating first-order properties under external or internal one-electron perturbations, either developed explicitly in orders of perturbation theory or in the fully self-consistent method. The accuracy and consistency of first-order properties are poorer than those of zero-order properties, but this is most often due to the use of explicit approximations in solving the perturbed equations, or to inadequacy of the variational basis in size or composition. We have calculated the electric polarizabilities of H₂, He, Li, Be, LiH, and N₂ by Hartree-Fock theory, using exact perturbation theory or the fully self-consistent method, as dictated by convenience. By careful studies on total basis set composition, we obtained good approximations to limiting Hartree-Fock values of polarizabilities with bases of reasonable size. The values for all species, and for each direction in the molecular cases, are within 8% of experiment, or of best theoretical values in the absence of the former. Our results support the use of unadorned Hartree-Pock theory for static polarizabilities needed in interpreting electron-molecule scattering data, collision-induced light scattering experiments, and other phenomena involving experimentally inaccessible polarizabilities.

Part 3. Numerical integration of the close-coupled scattering equations has been carried out to obtain vibrational transition probabilities for some models of the electronically adiabatic H₂-H₂ collision. All the models use a Lennard-Jones interaction potential between nearest atoms in the collision partners. We have analyzed the results for some insight into the vibrational excitation process in its dependence on the energy of collision, the nature of the vibrational binding potential, and other factors. We conclude also that replacement of earlier, simpler models of the interaction potential by the Lennard-Jones form adds very little realism for all the complication it introduces. A brief introduction precedes the presentation of our work and places it in the context of attempts to understand the collisional activation process in chemical reactions as well as some other chemical dynamics.

The interaction of color and luminance in stereoscopic vision

Experiments are described using the random dot stereo patterns devised by Julesz, but substituting various colors and luminances for the usual black and white random squares. The ability to perceive the patterns in depth depends on a luminance difference between the colors used. If two colors are the same luminance, then depth is not perceived although each of the individual squares which make up the patterns is easily seen due to the color difference. This is true for any combination of different colors. If different colors are used for corresponding random squares between the left and right eye patterns, stereopsis is possible for all combinations of binocular rivalry in color, provided the luminance difference is large enough. Rivalry in luminance always precludes stereopsis, regardless of the colors involved.