Protein turnover in a psychrotrophic bacterium

Protein breakdown in pulse-labelled and longlabelled cells of Arthrobacter S ₁-55, a psychrotrophic bacterium, has been assessed at different temperatures. The temperature at which the cells were grown and labelled affected the breakdown of pulsed-labelled but not long-labelled proteins. Inhibitors of ATP synthesis inhibited proteolysis. Miscoding antibiotics stimulated the production of rapidly degradable proteins.     

Bombesin-like peptides stimulate phosphatidylinositol turnover in rat brain slices

The ability of bombesin (BN)-like peptides to stimulate phosphatidylinositol turnover in rat brain slices was investigated. BN (1 μM) significantly stimulated inositol-1-phosphate (IP₁) but not inositol-4,5-biphosphate (IP₂) or inositol-1,4,5-trisphosphate (IP₃) production using frontal cortex slices in the presence of LiCl (7.5 mM); BN had no effect on cAMP or cGMP levels. BN and the structurally-related gastrin releasing peptide (GRP) elevated IP₁ levels in a dose-dependent manner. Similarly, nanomolar concentrations of the GRP fragment (Ac-GRP^20–27) significantly elevated IP₁ levels, whereas micromolar concentrations of the inactive GRP^1–16 did not. BN significantly elevated IP₁ levels in those brain regions enriched in BN receptors such as the olfactory bulb, hippocampus, striatum, thalamus and frontal cortex, whereas IP₁ levels were not significantly increased in areas which have a low density of BN receptors such as the cerebellum, medulla/pons and midbrain. These data suggest that CNS BN receptors may utilize phosphatidylinositol as a second messenger.     

Life in harsh environments: carabid and spider trait types and functional diversity on a debris‐covered glacier and along its foreland

1. Patterns of species richness and species assemblage composition of ground‐dwelling arthropods in primary successions along glacier forelands are traditionally described using a taxonomic approach. On the other hand, the functional trait approach could ensure a better characterisation of their colonisation strategies in these types of habitat. 2. The functional trait approach was applied to investigate patterns of functional diversity and life‐history traits of ground beetles and spiders on an alpine debris‐covered glacier and along its forefield in order to describe their colonisation strategies. 3. Ground beetles and spiders were sampled at different successional stages, representing five stages of deglaciation. 4. The results show that the studied glacier hosts ground beetle and spider assemblages that are mainly characterised by the following traits: walking colonisers, ground hunters and small‐sized species. These traits are typical of species living in cold, wet, and gravelly habitats. The diversity of functional traits in spiders increased along the succession, and in both carabids and spiders, life‐history traits follow the ‘addition and persistence model’. Accordingly, there is no turnover but there is an addition of new traits and a variation in their proportion within each species assemblage along the succession. The distribution of ground beetles and spiders along the glacier foreland and on the glacier seems to be driven by dispersal ability and foraging strategy. 5. The proposed functional approach improves knowledge of the adaptive strategies of ground‐dwelling arthropods colonising glacier surfaces and recently deglaciated terrains, which represent landforms quickly changing due to global warming.     

Protein turnover in exponential and stationary phase Vibrio cells

Vibrio strain 14 supports phage alpha 3a growth in standing stationary phase cells but not in shaking (aerated) stationary phase cells. In exponential cells, protein was turned over at 1.8% h-1, and the rate was increased by starvation or inhibition of protein synthesis. In shaking stationary phase cells the rate of protein turnover was low (1.0% h-1) for proteins synthesised during growth but high (20% h-1) for recently synthesised proteins. In contrast recently synthesised proteins in standing stationary phase cells were stable over 60 min and proteins synthesised during growth were turned over at 2.9% h-1. ppGpp and pppGpp were detected in exponential cells, but were not detected in stationary phase cells.     

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A₂ to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a “sideways sliding” mechanism to explain how a conserved membrane-embedded α-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane.     

Post-transcriptional inhibition of ornithine decarboxylase induction by zinc in a difluoromethylornithine resistant cell line

Addition of Zn^2+_ to cell medium inhibited the induction of ornithine decarboxylase (ODC) activity in ODC overproducing L1210-DFMO^r cells. A significant effect was observed at a concentration as low as 0.01 mM, however a more marked inhibition was caused by the addition of 0.1 mM Zn²⁺. The inhibition of the induction of ODC activity was accompanied by a proportional decrease in the content of immunoreactive ODC protein, whereas the level of ODC mRNA, detemined by a solution hybridization RNase protection assay, was not affected signigicantly. Instead, some acceleration of ODC turnover was observed. the addition of 0.1 mM Co²⁺ or Mn²⁺, but not of other divalent metal ions, also inhibited ODC induction; differently from Zn²⁺ however, these metals affected cell viability and/or cell growth. Removal of endogenous Zn²⁺ by a chelator also provoked a strong decrease of ODC induction, which was reversed by Zn²⁺. However, addition of Zn²⁺ in excess of the chelator proved to be markedly inhibitory. These results indicate that both a restricted Zn²⁺ availability and an enhanced presence of the metal can inhibit the induction of ODC in L1210-DFMO^r cells.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Electrophoretic patterns of protein synthesis and turnover in apical plasma membrane and outer bilayer of Schistosoma mansoni

Polypeptide fractions labelled with [¹⁴C]leucine and associated with fractioned inner plasma membrane and outer bilayer (envelope) from the apical double bilayer complex of the surface epithelium of the human blood fluke, Schistosoma mansoni, were analyzed by two-dimensional electrophoresis and fluorography. In contrast to the distribution of alkaline phosphatase, the polypeptide profiles of the two bilayer fractions were similar due to cross contamination between one membrane containing larger amounts of protein (inner) and the second bilayer having more heavily labelled proteins (outer bilayer). Convincing evidence for only two of 35 polypeptides could be provided for localization to the outer bilayer. These results suggest that the marker enzyme used for the inner bilayer, alkaline phosphatase, may not be homogeneously distributed in this membrane. In pulse-chase studies a correction factor for cross-contamination was derived. The rate to turnover of the polypeptide fractions was twice as fast for the outer compared to the inner membrane, this difference being consistent with the view that multilamellar bodies are the precursors of the apical double bilayer complex. Comparing the rates of surface renewal in adult and juvenile schistosomes leads to the suggestion that membrane turnover can be correlated with susceptibility to host immune effector mechanisms.     

A Two-Layered Targeting Mechanism Underlies Nuclear RNA Sorting by the Human Exosome

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