66 research outputs found

    Physiological modulation of BiP activity by trans-protomer engagement of the interdomain linker.

    Get PDF
    DnaK/Hsp70 chaperones form oligomers of poorly understood structure and functional significance. Site-specific proteolysis and crosslinking were used to probe the architecture of oligomers formed by the endoplasmic reticulum (ER) Hsp70, BiP. These were found to consist of adjacent protomers engaging the interdomain linker of one molecule in the substrate binding site of another, attenuating the chaperone function of oligomeric BiP. Native gel electrophoresis revealed a rapidly-modulated reciprocal relationship between the burden of unfolded proteins and BiP oligomers and slower equilibration between oligomers and inactive, covalently-modified BiP. Lumenal ER calcium depletion caused rapid oligomerization of mammalian BiP and a coincidental diminution in substrate binding, pointing to the relative inertness of the oligomers. Thus, equilibration between inactive oligomers and active monomeric BiP is poised to buffer fluctuations in ER unfolded protein load on a rapid timescale attainable neither by inter-conversion of active and covalently-modified BiP nor by the conventional unfolded protein response.Supported by grants from the Wellcome Trust (Wellcome 084812/Z/08/Z) the European Commission (EU FP7 Beta-Bat No: 277713), a Wellcome Trust Strategic Award for core facilities to the Cambridge Institute for Medical Research (Wellcome 100140) and a US Public Health Service grant NIH-GM54068 (to LH). DR is a Wellcome Trust Principal Research Fellow.This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.7554/eLife.0896

    The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco

    Get PDF
    The ER-resident molecular chaperone BiP (binding protein) was overexpressed in soybean. When plants growing in soil were exposed to drought (by reducing or completely withholding watering) the wild-type lines showed a large decrease in leaf water potential and leaf wilting, but the leaves in the transgenic lines did not wilt and exhibited only a small decrease in water potential. During exposure to drought the stomata of the transgenic lines did not close as much as in the wild type, and the rates of photosynthesis and transpiration became less inhibited than in the wild type. These parameters of drought resistance in the BiP overexpressing lines were not associated with a higher level of the osmolytes proline, sucrose, and glucose. It was also not associated with the typical drought-induced increase in root dry weight. Rather, at the end of the drought period, the BiP overexpressing lines had a lower level of the osmolytes and root weight than the wild type. The mRNA abundance of several typical drought-induced genes [NAC2, a seed maturation protein (SMP), a glutathione-S-transferase (GST), antiquitin, and protein disulphide isomerase 3 (PDI-3)] increased in the drought-stressed wild-type plants. Compared with the wild type, the increase in mRNA abundance of these genes was less (in some genes much less) in the BiP overexpressing lines that were exposed to drought. The effect of drought on leaf senescence was investigated in soybean and tobacco. It had previously been reported that tobacco BiP overexpression or repression reduced or accentuated the effects of drought. BiP overexpressing tobacco and soybean showed delayed leaf senescence during drought. BiP antisense tobacco plants, conversely, showed advanced leaf senescence. It is concluded that BiP overexpression confers resistance to drought, through an as yet unknown mechanism that is related to ER functioning. The delay in leaf senescence by BiP overexpression might relate to the absence of the response to drought

    FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP

    Get PDF
    Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoan-specific FIC-domain-containing ER-localized enzyme (FICD) rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here we show that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD/_{-/-} hamster cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation-defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase in vitro\textit{in vitro}. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD in vivo\textit{in vivo}, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.Supported by Wellcome Trust Principal Research Fellowship to D.R. (Wellcome 200848/Z/16/Z), a UK Medical Research Council PhD studentship to L.A.P. and a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research (Wellcome 100140)

    Pulmonary endoplasmic reticulum stress-scars, smoke, and suffocation.

    Get PDF
    Protein misfolding within the endoplasmic reticulum (ER stress) can be a cause or consequence of pulmonary disease. Mutation of proteins restricted to the alveolar type II pneumocyte can lead to inherited forms of pulmonary fibrosis, but even sporadic cases of pulmonary fibrosis appear to be strongly associated with activation of the unfolded protein response and/or the integrated stress response. Inhalation of smoke can impair protein folding and may be an important cause of pulmonary ER stress. Similarly, tissue hypoxia can lead to impaired protein homeostasis (proteostasis). But the mechanisms linking smoke and hypoxia to ER stress are only partially understood. In this review, we will examine the role of ER stress in the pathogenesis of lung disease by focusing on fibrosis, smoke, and hypoxia

    A componential analysis of the knowledge -strategy interaction in analogical problem -solving

    No full text
    The purpose of this research was to investigate the influence of prior knowledge on an analogy problem solving strategy organizing the investigation around the subprocesses of the strategy. A sample of 112 high school freshman and sophomores participated in the study. All of the students were male and participation was voluntary. The students were initially assigned to 1 of 4 treatment groups that differed with respect to the vocabulary and relationship familiarity of a set of analogy problems they were asked to solve. All students received training via a computer based tutorial that taught them a strategy for solving analogy problems of the form A : B :: C : D. The strategy was based on a theory of analogy problem solving that decomposed the process into a series of subprocesses or components. After solving this set of problems they were given a short questionnaire asking them about their perceptions of the solving by components strategy. All students were then asked to solve a second common set of analogy problems then given a second questionnaire to complete. Results indicated that vocabulary and relationship familiarity significantly influenced performance and strategy use. Students who were given problems with less familiar vocabulary and relationships took longer to solve the problems and produced more errors than students given more familiar vocabulary and relationships. These findings were also found at the component level where vocabulary familiarity had a significant effect on encoding (a vocabulary intensive component) whereas relationship familiarity had no effect on encoding. Similar results were found with respect to the relationship familiarity on the three other components that were considered relationship intensive. In terms of strategy use students who were given the less familiar vocabulary and relationships where less likely to use the strategy on a common set of items and found the strategy they used more effortful, less more difficult to use, and more useless that those given the more familiar vocabulary and relationships. The results seemed to indicate that the subprocesses of a problem solving strategy could be decomposed into its component processes and used to make predictions about strategy behavior
    corecore