Intracellular Trafficking of Guanylate-Binding Proteins Is Regulated by Heterodimerization in a Hierarchical Manner

Guanylate-binding proteins (GBPs) belong to the dynamin family of large GTPases and represent the major IFN-γ-induced proteins. Here we systematically investigated the mechanisms regulating the subcellular localization of GBPs. Three GBPs (GBP-1, GBP-2 and GBP-5) carry a C-terminal CaaX-prenylation signal, which is typical for small GTPases of the Ras family, and increases the membrane affinity of proteins. In this study, we demonstrated that GBP-1, GBP-2 and GBP-5 are prenylated in vivo and that prenylation is required for the membrane association of GBP-1, GBP-2 and GBP-5. Using co-immunoprecipitation, yeast-two-hybrid analysis and fluorescence complementation assays, we showed for the first time that GBPs are able to homodimerize in vivo and that the membrane association of GBPs is regulated by dimerization similarly to dynamin. Interestingly, GBPs could also heterodimerize. This resulted in hierarchical positioning effects on the intracellular localization of the proteins. Specifically, GBP-1 recruited GBP-5 and GBP-2 into its own cellular compartment and GBP-5 repositioned GBP-2. In addition, GBP-1, GBP-2 and GBP-5 were able to redirect non-prenylated GBPs to their compartment in a prenylation-dependent manner. Overall, these findings prove in vivo the ability of GBPs to dimerize, indicate that heterodimerization regulates sub-cellular localization of GBPs and underscore putative membrane-associated functions of this family of proteins

Modulation of the cost of pHi regulation during metabolic depression: a 31P-NMR study in invertebrate (Sipunculus nudus) isolated muscle

Extracellular acidosis has been demonstrated to play a key role in the process of metabolic depression under long-term environmental stress, exemplified in the marine invertebrate Sipunculus nudus. These findings led to the hypothesis that acid-base regulation is associated with a visible cost depending on the rate and mode of H+-equivalent ion exchange. To test this hypothesis, the effects of different ion-transport inhibitors on the rate of pH recovery during hypercapnia, on energy turnover and on steady-state acid-base variables were studied in isolated body wall musculature of the marine worm Sipunculus nudus under control conditions (pHe 7.90) and during steady-state extracellular acidosis (pHe 7.50 or 7.20) by in vivo 31P-NMR and oxygen consumption analyses. During acute hypercapnia (2 % CO2), recovery of pHi was delayed at pHe 7.5 compared with pHe 7.9. Inhibition of the Na+/H+-exchanger by 5-(N,N-dimethyl)-amiloride (DMA) at pHe 7.5 delayed recovery even further. This effect was much smaller at pHe 7.9. Inhibition of anion exchange by the addition of the transport inhibitor 4,4Ž-diisothiocyanatostilbene-2,2Ž-disulphonic acid (DIDS) prevented pH recovery at pHe 7.5 and delayed recovery at pHe 7.9, in accordance with an effect on Na+-dependent Cl-/HCO3- exchange. The effects of ouabain, DIDS and DMA on metabolic rate were reduced at low pHe, thereby supporting the conclusion that acidosis caused the ATP demand of Na+/K+-ATPase to fall. This reduction occurred via an inhibiting effect on both Na+/H+- and Na+-dependent Cl-/HCO3- (i.e. Na+/H+/Cl-/HCO3-) exchange in accordance with a reduction in the ATP demand for acid-base regulation during metabolic depression. Considering the ATP stoichiometries of the two exchangers, metabolic depression may be supported by the predominant use of Na+/H+/Cl-/HCO3- exchange under conditions of extracellular acidosis

A role for adenosine in metabolic depression in the marine invertebrate Sipunculus nudus

Involvement of neurotransmitters in metabolic depression under hypoxia and hypercapnia was examined in Sipunculus nudus. Concentration changes of several putative neurotransmitters in nervous tissue during anoxic or hypercapnic exposure or during combined anoxia and hypercapnia were determined. Among amino acids (gamma-aminobutyric acid, glutamate, glycine, taurine, serine, and aspartate) and monoamines (serotonin, dopamine, and norepinephrine), some changes were significant, but none were consistent with metabolic depression under all experimental conditions applied. Only the neuromodulator adenosine displayed concentration changes in accordance with metabolic depression under all experimental conditions. Levels increased during anoxia, during hypercapnia, and to an even greater extent during anoxic hypercapnia. Adenosine infusions into coelomic fluid via an indwelling catheter induced a significant depression of the normocapnic rate of O2 consumption from 0.36 +/- 0.04 to a minimum of 0.24 +/- 0.02 (SE) mumol.g-1.h-1 after 90 min (n = 6). Application of the adenosine antagonist theophylline caused a transient rise in O2 consumption 30 min after infusion during hypercapnia but not during normocapnia. Effects of adenosine and theophylline were observed in intact individuals but not in isolated body wall musculature. The results provide evidence for a role of adenosine in inducing metabolic depression in S. nudus, probably through the established effects of decreasing neuronal excitability and neurotransmitter release. In consideration of our previous finding that metabolic depression in isolated body wall musculature was elicited by extracellular acidosis, it is concluded that central and cellular mechanisms combine to contribute to the overall reduction in metabolic rate in S. nudus. </jats:p

Metabolic Depression During Environmental Stress: The Role of Extracellular <i>Versus</i> Intracellular pH in <i>Sipunculus Nudus</i>

ABSTRACT Environmental stresses such as hypoxia or hypercapnia are known to cause acid–base disturbances and in several organisms they lead to metabolic depression. The present study was undertaken to quantify the influence of these changes in acid–base parameters on metabolic rate. We determined the rate of oxygen consumption in a non-perfused preparation of the body wall musculature of the marine worm Sipunculus nudus at various levels of extra- and intracellular pH (pHe and pHi, respectively), and [HCO3-]. The acid–base status of the tissue was modified and clamped by long-term exposure to media set to specific values of extracellular pH, and [HCO3-]. At a pHe of 7.90, which is equivalent to the normoxic normocapnic in vivo extracellular pH, and an ambient of 0.03 kPa (control conditions), pHi was 7.26±0.02 (mean ± S.D., N=5). A reduction of extracellular pH from 7.90 to 7.20 resulted in a significant decrease of pHi to 7.17±0.05 at 0.03 kPa (normocapnia) and to 7.20±0.02 at 1.01 kPa (hypercapnia). At the same time, the rate of oxygen consumption of the tissue was significantly depressed by 18.7±4.7 % and 17.7±3.0 %, respectively. A significant depression of oxygen consumption by 13.7±4.7 % also occurred under hypercapnia at pHe 7.55 when pHi was elevated above control values (7.32±0.01). No significant changes in oxygen consumption were observed when pHe was either drastically elevated to 8.70 under normocapnia (pHi 7.36±0.05) or maintained at 7.90 during hypercapnia (pHi 7.37±0.03). ATP and phospho-L-arginine concentrations, as well as the Gibbs free energy change of ATP hydrolysis (dG/dξATP), were maintained at high levels during all treatments, indicating an equilibrium between energy supply and demand. We conclude that the depression of aerobic energy turnover in isolated body wall musculature of S. nudus is induced by low extracellular pH. A model is proposed which could explain a reduced ATP cost of pHi regulation during extracellular acidosis, thus contributing to metabolic depression.</jats:p

Modulation of the Cost of pHi Regulation During Metabolic Depression: A 31P-NMR Study in Invertebrate (<i>Sipunculus Nudus</i>) Isolated Muscle

ABSTRACT Extracellular acidosis has been demonstrated to play a key role in the process of metabolic depression under long-term environmental stress, exemplified in the marine invertebrate Sipunculus nudus. These findings led to the hypothesis that acid–base regulation is associated with a visible cost depending on the rate and mode of H+-equivalent ion exchange. To test this hypothesis, the effects of different ion-transport inhibitors on the rate of pH recovery during hypercapnia, on energy turnover and on steady-state acid–base variables were studied in isolated body wall musculature of the marine worm Sipunculus nudus under control conditions (pHe 7.90) and during steady-state extracellular acidosis (pHe 7.50 or 7.20) by in vivo31P-NMR and oxygen consumption analyses. During acute hypercapnia (2 % CO2), recovery of pHi was delayed at pHe 7.5 compared with pHe 7.9. Inhibition of the Na+/H+-exchanger by 5-(N,N-dimethyl)-amiloride (DMA) at pHe 7.5 delayed recovery even further. This effect was much smaller at pHe 7.9. Inhibition of anion exchange by the addition of the transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) prevented pH recovery at pHe 7.5 and delayed recovery at pHe 7.9, in accordance with an effect on Na+-dependent Cl−/HCO3− exchange. The effects of ouabain, DIDS and DMA on metabolic rate were reduced at low pHe, thereby supporting the conclusion that acidosis caused the ATP demand of Na+/K+-ATPase to fall. This reduction occurred via an inhibiting effect on both Na+/H+- and Na+-dependent Cl−/HCO3− (i.e. Na+/H+/Cl−/HCO3−) exchange in accordance with a reduction in the ATP demand for acid–base regulation during metabolic depression. Considering the ATP stoichiometries of the two exchangers, metabolic depression may be supported by the predominant use of Na+/H+/Cl−/HCO3− exchange under conditions of extracellular acidosis.</jats:p

Acid–Base Regulation, Metabolism and Energetics in <i>Sipunculus Nudus</i> As a Function of Ambient Carbon Dioxide Level

ABSTRACT Changes in the rates of oxygen consumption and ammonium excretion, in intra- and extracellular acid–base status and in the rate of H+-equivalent ion transfer between animals and ambient water were measured during environmental hypercapnia in the peanut worm Sipunculus nudus. During exposure to 1% CO2 in air, intracellular and coelomic plasma values rose to levels above those expected from the increase in ambient CO2 tension. Simultaneously, coelomic plasma was reduced below control values. The rise in also induced a fall in intra- and extracellular pH, but intracellular pH was rapidly and completely restored. This was achieved during the early period of hypercapnia at the expense of a non-respiratory increase in the extracellular acidosis. The pH of the extracellular space was only partially compensated (by 37%) during long-term hypercapnia. The net release of basic equivalents under control conditions turned to a net release of protons to the ambient water before a net, albeit reduced, rate of base release was re-established after a new steady state had been achieved with respect to acid–base parameters. Hypercapnia also affected the mode and rate of metabolism. It caused the rate of oxygen consumption to fall, whereas the rate of ammonium excretion remained constant or even increased, reflecting a reduction of the O/N ratio in both cases. The transient intracellular acidosis preceded a depletion of the phosphagen phospho-L-arginine, an accumulation of free ADP and a decrease in the level of Gibbs free energy change of ATP hydrolysis, before replenishment of phosphagen and restoration of pHi and energy status occurred in parallel. In conclusion, long-term hypercapnia in vivo causes metabolic depression, a parallel shift in acid–base status and increased gas partial pressure gradients, which are related to a reduction in ventilatory activity. The steady-state rise in H+-equivalent ion transfer to the environment reflects an increased rate of production of protons by metabolism. This observation and the reduction of the O/N ratio suggest that a shift to protein/amino acid catabolism has taken place. Metabolic depression prevails, with completely compensated intracellular acidosis during long-term hypercapnia eliminating intracellular pH as a significant factor in the regulation of metabolic rate in vivo. Fluctuating levels of the phosphagen, of free ADP and in the ATP free energy change values independent of pH are interpreted as being correlated with oscillating ATP turnover rates during early hypercapnia and as reflecting a tight coupling of ATP turnover and energy status via the level of free ADP.</jats:p