Competition between neighbouring topogenic signals during membrane protein insertion into the ER membrane

To better define the mechanism of membrane protein insertion into the membrane of the endoplasmic reticulum, we measured the kinetics of translocation across microsomal membranes of the N-terminal lumenal tail and the lumenal domain following the second transmembrane segment (TM2) in the multispanning mouse protein Cig30. In the wild-type protein, the N-terminal tail translocates across the membrane before the downstream lumenal domain. Addition of positively charged residues to the N-terminal tail dramatically slows down its translocation and allows the downstream lumenal domain to translocate at the same time as or even before the N-tail. When TM2 is deleted, or when the loop between TM1 and TM2 is lengthened, addition of positively charged residues to the N-terminal tail causes TM1 to adopt an orientation with its N-terminal end in the cytoplasm. We suggest that the topology of the TM1-TM2 region of Cig30 depends on a competition between TM1 and TM2 such that the transmembrane segment that inserts first into the ER membrane determines the final topology

Distant downstream sequence determinants can control N-tail translocation during protein insertion into the Endoplasmic Reticulum membrane

We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic N-terminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes. Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the N-tail remains mostly nontranslocated and the majority of the molecules adopt an 'inverted' topology where normally nontranslocated parts are translocated and vice versa. N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charged residues immediately downstream of the first trasnmembrane segment. We conclude that as many as four consecutive transmembrane segments may be collectively involved in determining membrane protein topology in the ER and that the effects of downstream sequence determinants may vary depending on the size and charge of the N-tail. We also provide evidence to suggest that the ProW N-tail is translocated across the ER membrane in a C-to-N-terminal direction

Influence of the C‐terminus of the glycophorin A transmembrane fragment on the dimerization process

The monomer-dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix-helix packing in a membrane‐mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C‐terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two‐stage model for membrane protein folding and oligomerization

Murchison Widefield Array limits on radio emission from Antares neutrino events

We present a search, using the Murchison Widefield Array (MWA), for electromagnetic (EM) counterparts to two candidate high-energy neutrino events detected by the ANTARES neutrino telescope in 2013 November and 2014 March. These events were selected by ANTARES because they are consistent, within 0°.4, with the locations of galaxies within 20 Mpc of Earth. Using MWA archival data at frequencies between 118 and 182 MHz, taken ~20 days prior to, at the same time as, and up to a year after the neutrino triggers, we look for transient or strongly variable radio sources that are consistent with the neutrino positions. No such counterparts are detected, and we set a 5s upper limit for low-frequency radio emission of ~1037 erg s-1 for progenitors at 20 Mpc. If the neutrino sources are instead not in nearby galaxies, but originate in binary neutron star coalescences, our limits place the progenitors at z 0.2. While it is possible, due to the high background from atmospheric neutrinos, that neither event is astrophysical, the MWA observations are nevertheless among the first to follow up neutrino candidates in the radio, and illustrate the promise of wide-field instruments like MWA for detecting EM counterparts to such events

A search for neutrino emission from the Fermi bubbles with the ANTARES telescope

Analysis of the Fermi-LAT data has revealed two extended structures above and below the Galactic Centre emitting gamma rays with a hard spectrum, the so-called Fermi bubbles. Hadronic models attempting to explain the origin of the Fermi bubbles predict the emission of high-energy neutrinos and gamma rays with similar fluxes. The ANTARES detector, a neutrino telescope located in the Mediterranean Sea, has a good visibility to the Fermi bubble regions. Using data collected from 2008 to 2011 no statistically significant excess of events is observed and therefore upper limits on the neutrino flux in TeV range from the Fermi bubbles are derived for various assumed energy cutoffs of the source

Correlation between intramolecular base composition heterogeneity of DNA and control of transcriptional expression in E. coli temperate phage P2

Typescript.Thesis (Ph. D.)--University of Hawaii at Manoa, 1972.Bibliography: leaves 91-96.ix, 96 l illusIntramolecular base compositional heterogeneity has been demonstrated in the DNA of the phage P2 by following the optical density of solutions at 260 nm as a function of increasing temperature. First derivative curves of the functions thus generated have been obtained for P2 DNA and the DNA of two closely related phages. Comparison of these curves reveals similarities at the high temperature (high GC) end and differences at the low temperature (low GC) end. All three of these phages can, by supplying the genes concerned with phage particle maturation, serve as helper for the defective phage P4. The strands of P2 DNA have been separated on the basis of their buoyant densities in CsCl solutions when complexed with poly UG. In vivo transcription patterns from these strands in cells infected with various P2 mutants have demonstrated that the "heavy" strand is the one predominantly transcribed, that some "light" strand transcription originates from an operon coding for proteins involved in phage head assembly (thus fuling out a "read-through" mechanism for late gene activation), and that early "light" strand transcription does not originate from DNA deleted in two mutants which is in the right half of the physical map. There is, however, some "light" strand transcription early in infection. P2 DNA has been sheared in: half and the halves separated on the basis of Hg++ binding in Cs2SO4 density gradients. Electron microscopic analysis of partially denatured molecules in these preparations have fixed them with respect to the physical map. In ~ transcription originates primarily from the right, or low GC half early in infection and then shifts to the left, or high GC half. Mutants in genes A and B, which are located in the right half of the genetic map, are defective in both DNA replication and in effecting this shift. Infection with one polar amber mutant under non-permissive conditions has demonstrated that this operon lies :in the right half of the DNA and thus helps to fix the physical map with respect to the genetic map. Two of those regions of the phage DNA which are known to be transcribed from a repressed genome, namely the prophage, appear to be of quite low GC content