Wild-type and mutant alleles of the Aspergillus nidulans developmental regulator gene brlA: correlation of variant sites with protein function

The DNA sequences of two wild-type and eleven mutant alleles of the developmental regulator gene brlA from Aspergillus nidulans, which encodes a zinc-finger protein, were characterized. Variant sites were located on rescued plasmids or PCR products based either on their meiotic map position or the use of denaturing gradient gel electrophoresis. Mutations in three null mutants, one of which is partially suppressible, encode premature stop codons. Two environmentally sensitive mutants were characterised by substitution of leucines required for stabilisation of α-helices in each of the two putative zinc-finger domains. A third zinc-finger substitution is predicted to disrupt recognition of a guanine residue in the DNA target. The mutations in four other leaky mutants map C-terminal to the zinc fingers; one minimally leaky mutant has a premature stop codon, which results in the removal of the last 38 residues of the protein product. Received: 16 February 1999 / Accepted: 22 July 1999     

Calcium interacts with antifreeze proteins and chitinase from cold-acclimated winter rye

During cold acclimation, winter rye (Secale cereale) plants accumulate pathogenesis-related proteins that are also antifreeze proteins (AFPs) because they adsorb onto ice and inhibit its growth. Although they promote winter survival in planta, these dual-function AFPs proteins lose activity when stored at subzero temperatures in vitro, so we examined their stability in solutions containing CaCl2, MgCl2, or NaCl. Antifreeze activity was unaffected by salts before freezing, but decreased after freezing and thawing in CaCl2 and was recovered by adding a chelator. Ca2+ enhanced chitinase activity 3- to 5-fold in unfrozen samples, although hydrolytic activity also decreased after freezing and thawing in CaCl2. Native PAGE, circular dichroism, and Trp fluorescence experiments showed that the AFPs partially unfold after freezing and thawing, but they fold more compactly or aggregate in CaCl2. Ruthenium red, which binds to Ca(2+)-binding sites, readily stained AFPs in the absence of Ca2+, but less stain was visible after freezing and thawing AFPs in CaCl2. We conclude that the structure of AFPs changes during freezing and thawing, creating new Ca(2+)-binding sites. Once Ca2+ binds to those sites, antifreeze activity, chitinase activity and ruthenium red binding are all inhibited. Because free Ca2+ concentrations are typically low in the apoplast, antifreeze activity is probably stable to freezing and thawing in planta. Ca2+ may regulate chitinase activity if concentrations are increased locally by release from pectin or interaction with Ca(2+)-binding proteins. Furthermore, antifreeze activity can be easily maintained in vitro by including a chelator during frozen storage.     

3-D Structure of multilaminar lysosomes in antigen presenting cells reveals trapping of MHC II on the internal membranes

In late endosomes and lysosomes of antigen presenting cells major histocompatibility complex class II (MHC II) molecules bind peptides from degraded internalized pathogens. These compartments are called MHC class II compartments (MIICs), and from here peptide-loaded MHC II is transported to the cell surface for presentation to helper T-lymphocytes to generate an immune response. Recent studies from our group in mouse dendritic cells indicate that the MHC class II on internal vesicles of multivesicular late endosomes or multivesicular bodies is the main source of MHC II at the plasma membrane. We showed that dendritic cell activation triggers a back fusion mechanism whereby MHC II from the inner membranes is delivered to the multivesicular bodies' outer membrane. Another type of MIIC in B-lymphocytes and dendritic cells is more related to lysosomes and often appears as a multilaminar organelle with abundant MHC II-enriched internal membrane sheets. These multilaminar lysosomes have a functioning peptide-loading machinery, but to date it is not clear whether peptide-loaded MHC II molecules from the internal membranes can make their way to the cell surface and contribute to T cell activation. To obtain detailed information on the membrane organization of multilaminar lysosomes and investigate possible escape routes from the lumen of this organelle, we performed electron tomography on cryo-immobilized B-lymphocytes and dendritic cells. Our high-resolution 3-D reconstructions of multilaminar lysosomes indicate that their membranes are organized in such a way that MHC class II may be trapped on the inner membranes, without the possibility to escape to the cell surface.     

Modeling Sustainability of Arctic Communities: An Interdisciplinary Collaboration of Researchers and Local Knowledge Holders

How will climate change affect the sustainability of Arctic villages over the next 40 years? This question motivated a collaboration of 23 researchers and four Arctic communities (Old Crow, Yukon Territory, Canada; Aklavik, Northwest Territories, Canada; Fort McPherson, Northwest Territories, Canada; and Arctic Village, Alaska, USA) in or near the range of the Porcupine Caribou Herd. We drew on existing research and local knowledge to examine potential effects of climate change, petroleum development, tourism, and government spending cutbacks on the sustainability of four Arctic villages. We used data across eight disciplines to develop an Arctic Community Synthesis Model and a Web-based, interactive Possible Futures Model. Results suggested that climate warming will increase vegetation biomass within the herd’s summer range. However, despite forage increasing, the herd was projected as likely to decline with a warming climate because of increased insect harassment in the summer and potentially greater winter snow depths. There was a strong negative correlation between hypothetical, development-induced displacement of cows and calves from utilized calving grounds and calf survival during June. The results suggested that climate warming coupled with petroleum development would cause a decline in caribou harvest by local communities. Because the Synthesis Model inherits uncertainties associated with each component model, sensitivity analysis is required. Scientists and stakeholders agreed that (1) although simulation models are incomplete abstractions of the real world, they helped bring scientific and community knowledge together, and (2) relationships established across disciplines and between scientists and communities were a valuable outcome of the study. Additional project materials, including the Web-based Possible Futures Model, are available at http://www.taiga.net/sustain.     

The carboxyl-terminal domain of bacteriophage T7 single-stranded DNA-binding protein modulates DNA binding and interaction with T7 DNA polymerase

Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein (gp2.5). Previous studies have demonstrated that the acidic carboxyl terminus of the protein is essential and that it mediates multiple protein-protein interactions. A screen for lethal mutations in gene 2.5 uncovered a variety of essential amino acids, among which was a single amino acid substitution, F232L, at the carboxyl-terminal residue. gp2.5-F232L exhibits a 3-fold increase in binding affinity for single-stranded DNA and a slightly lower affinity for T7 DNA polymerase when compared with wild type gp2.5. gp2.5-F232L stimulates the activity of T7 DNA polymerase and, in contrast to wild-type gp2.5, promotes strand displacement DNA synthesis by T7 DNA polymerase. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta 26C, binds single-stranded DNA 40-fold more tightly than the wild-type protein and cannot physically interact with T7 DNA polymerase. gp2.5-Delta 26C is inhibitory for DNA synthesis catalyzed by T7 DNA polymerase on single-stranded DNA, and it does not stimulate strand displacement DNA synthesis at high concentration. The biochemical and genetic data support a model in which the carboxyl-terminal tail modulates DNA binding and mediates essential interactions with T7 DNA polymerase.     

TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution

The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.     

A novel yeast expression system for the overproduction of quality-controlled membrane proteins

Saturation of the cell's protein folding capacity and accumulation of inactive incompletely folded protein often accompanying the overexpression of membrane proteins (MPs) presents an obstacle to their efficient purification in a functional form for structural studies. We present a novel strategy for optimization of functional MP expression in Saccharomyces cerevisiae. This approach exploits the unfolded protein response (UPR) pathway, a stress signaling mechanism that senses the accumulation of unfolded proteins in the endoplasmic reticulum. We demonstrate that a high level of UPR induction upon expression of a MP reflects impaired functional expression of that protein. Tuning the expression level of the protein so as to avoid or minimize UPR induction results in its increased functional expression. UPR status can therefore serve as a proxy variable for the extent of impaired expression of a MP that may even be applicable in the absence of knowledge of the protein's biological function.     

Prokaryote multidrug efflux proteins of the major facilitator superfamily: amplified expression, purification and characterisation

In bacterial genomes 3-12% of open reading frames are predicted to encode membrane transport proteins. These proteins can be vital for antibiotic efflux, protein/ toxin secretion, cell nutrition, environmental sensing, ATP synthesis, and other functions. Some, such as the multidrug efflux proteins, are potential targets for the development of new antibacterials and also for applications in biotechnology. In general membrane transport proteins are poorly understood, because of the technical difficulties involved in isolating sufficient protein for elucidation of their structure-activity relationships. We describe a general strategy for the amplified expression, purification and characterisation of prokaryotic multidrug efflux proteins of the 'Major facilitator superfamily' of transport proteins, using the Bacillus subtilis multidrug resistance protein, 'Bmr', as example.