Meira Weiss

Mutation in TECPR2 Reveals a Role for Autophagy in Hereditary Spastic Paraparesis

We studied five individuals from three Jewish Bukharian families affected by an apparently autosomal-recessive form of hereditary spastic paraparesis accompanied by severe intellectual disability, fluctuating central hypoventilation, gastresophageal reflux disease, wake apnea, areflexia, and unique dysmorphic features. Exome sequencing identified one homozygous variant shared among all affected individuals and absent in controls: a 1 bp frameshift TECPR2 deletion leading to a premature stop codon and predicting significant degradation of the protein. TECPR2 has been reported as a positive regulator of autophagy. We thus examined the autophagy-related fate of two key autophagic proteins, SQSTM1 (p62) and MAP1LC3B (LC3), in skin fibroblasts of an affected individual, as compared to a healthy control, and found that both protein levels were decreased and that there was a more pronounced decrease in the lipidated form of LC3 (LC3II). siRNA knockdown of TECPR2 showed similar changes, consistent with aberrant autophagy. Our results are strengthened by the fact that autophagy dysfunction has been implicated in a number of other neurodegenerative diseases. The discovered TECPR2 mutation implicates autophagy, a central intracellular mechanism, in spastic paraparesis.

Tightly bound sulpholipids in chloroplast CF0-CF1

Highly purified preparations of CF0-CF1 from chloroplasts contain a small amount of tightly bound lipids. Extraction and analysis of these lipids show that they are almost exclusively sulpholipids. The calculated amount of bound sulpholipids in spinach and in Dunaliella salina CF0-CF1 preparations are 5 and 20 mols/mol enzyme, respectively. Attempts to exchange the bound lipids with other lipids or with detergents have failed, indicating a very strong association with CF0-CF1.

Polyphosphate-hydrolysis--a protective mechanism against alkaline stress?

Different microorganisms, including yeast and algae, accumulate large amounts of polyphosphates. However, the physiological role of polyphosphates is largely unknown. In vivo 31P NMR studies, carried out in the unicellular alga, Dunaliella salina, demonstrate that cytoplasmic alkalization induces massive hydrolysis of polyphosphates, which is correlated kinetically with the recovery of cytoplasmic pH. Analysis of acid extracts of the cells indicates that long‐chain polyphosphates are hydrolysed mainly to tripolyphosphate. It is suggested that the hydrolysis of polyphosphates provides a pH‐stat mechanism to counterbalance alkaline stress.

The role of different thylakoid glycolipids in the function of reconstituted chloroplast ATP synthase

Abstract ATPase activity of CF 0 CF 1 from spinach chloroplasts is specifically stimulated by chloroplast lipids (Pick, U., Gounaris, K., Admon, A. and Barber, J. (1984) Biochim. Biophys. Acta 765, 12–20). The association of CF 0 -CF 1 with isolated lipids and their mixtures has been examined by analyzing the stimulation of ATPase and ATP-P i exchange activities, by binding studies and by measurement of proton conductance of reconstituted proteoliposomes. Monogalactosyldiacylglycerol is the only chloroplast lipid which by itself activates ATP hydrolysis. A mild saturation of the fatty acids of the lipid partially inhibits the activation. CF 0 -CF 1 has a higher binding capacity for monogalactosyldiacylglycerol (1.5 mg/mg protein) than for other thylakoid glycolipids. However, ATPase activation is not correlated with the amount of bound lipid but rather with its type. For the same amount of bound lipid, monogalactosyldiacylglycerol best activates ATP hydrolysis, while the acidic lipids phosphatidylglycerol and sulphoquinovosyldiacylglycerol inhibit ATPase activity. Optimal activation of ATP-P i exchange requires, in addition to monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerol at a ratio of 6:3:1, respectively. Correlations between proton conductance, ATP-P i exchange and uncoupler stimulation of ATPase activity indicate that sulphoquinovosyldiacylglycerol reduces the permeability of the proteoliposomes to protons. The results suggest that: (a) association of CF 0 -CF 1 with polyunsaturated monogalactosyldiacylglycerol greatly stimulates ATPase activity; (b) reconstitution of coupled CF 0 -CF 1 proteoliposomes requires a careful balance of the natural glycolipids of thylakoid membranes in similar proportions to their occurrence in chloroplasts, and (c) sulphoquinovosyldiacylglycerol may control the permeability of chloroplast membranes to protons.

Activation of the Dunaliella acidophila Plasma Membrane H+-ATPase by Trypsin Cleavage of a Fragment That Contains a Phosphorylation Site

Trypsin treatment of purified H+-ATPase from plasma membranes of the extreme acidophilic alga Dunaliella acidophila enhances ATP hydrolysis and H+ pumping activities. The activation is associated with an alkaline pH shift, an increase in Vmax, and a decrease in Km(ATP). The activation is correlated with cleavage of the 100-kD ATPase polypeptide to a fragment of approximately 85 kD and the appearance of three minor hydrophobic fragments of 7 to 8 kD, which remain associated with the major 85-kD polypeptide. The N-terminal sequence of the small fragments has partial homology to residues 713 to 741 of Arabidopsis thaliana plasma membrane H+-ATPases. Incubation of cells with 32P-labeled orthophosphate (32Pi) results in incorporation of 32P into the ATPase 100-kD polypeptide. Trypsin treatment of the 32Pi-labeled ATPase leads to complete elimination of label from the approximately 85-kD polypeptide. Cleavage of the phosphorylated enzyme with endoproteinase Glu-C (V-8) yields a phosphorylated 12-kD fragment. Peptide mapping comparison between the 100-kD and the trypsinized 85-kD polypeptides shows that the 12-kD fragment is derived from the trypsin-cleaved part of the enzyme. The N-terminal sequence of the 12-kD fragment closely resembles a C-terminal stretch of an ATPase from another Dunaliella species. It is suggested that trypsin activation of the D. acidophila plasma membrane H+-ATPase results from elimination of an autoinhibitory domain at the C-terminal end of the enzyme that carries a vicinal phosphorylation site.

Characterization of soluble and membrane-bound forms of a vanadate-sensitive ATPase from plasma membranes of the halotolerant alga Dunaliella salina

A plasma-membrane preparation, obtained by an osmotic lysis of cells of the halotolerant alga Dunaliella salina ( Sheffer and Avron, 1986 ) was further purified on glycerol gradients. Two fractions of a vanadate-sensitive ATPase activity were resolved on the gradients: a soluble and a membrane-bound fraction. The enzymes exhibit identical sensitivities to vanadate, dicyclohexylcarbodiimide, diethylstilbestrol, SH reagents and phloridzin but are insensitive to molybdate, nitrate, cyanide, azide and quercetin. The two ATPase activities also have an identical K m value for Mg-ATP (0.9 mM), optimal activity at pH 7, are stimulated by 200 mM KCl or NaCl but inhibited by high salt concentrations. Antibodies against yeast plasma membrane H + -ATPase cross-react with 92 kDa or with 60 kDa polypeptides in the plasma membrane and soluble ATPase fractions, respectively. Treatment of the purified plasma membranes with trypsin releases a soluble vanadate-sensitive ATPase from the membranes, and the release is protectable by ATP. It is suggested that the soluble ATPase, which is resolved on the glycerol gradients, is a proteolytic product of the plasma membrane ATPase which is a vanadate-sensitive H + -ATPase.

Effects of Iron Deficiency on Iron Binding and Internalization into Acidic Vacuoles in Dunaliella salina

Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe3+ ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability.

Primary Structure and Effect of pH on the Expression of the Plasma Membrane H+-ATPase from Dunaliella acidophila and Dunaliella salina

The plasma membrane H+-ATPase gene was cloned and sequenced from the extremely acidophilic green alga Dunaliella acidophila and from the extremely halotolerant Dunaliella salina. A special feature of the Dunaliella H+-ATPases is an extended C-terminal domain. The deduced amino acid sequences of the two proteins are 75% identical but differ in their C terminus. A hydrophilic loop within this domain in D. salina, which presumably faces the cell exterior, has a high ratio of acidic over basic amino acids, typical of halophilic proteins. The amount of the ATPase protein in plasma membranes and the level of its mRNA transcript in D. acidophila are far higher than in D. salina, suggesting that D. acidophila overexpresses the enzyme. A pH shift from 9.0 to 7.0 induces in D. salina a large increase in the level of the H+-ATPase mRNA and in the amount of the H+-ATPase protein. This suggests that the expression of the H+-ATPase in D. salina is pH-regulated at the transcriptional level. The implications of these findings are discussed with respect to the adaptive pressures imposed on these algal species by their exceptional environmental conditions.

Transient Na+ flux following hyperosmotic shock in the halotolerant alga Dunaliella salina : a response to intracellular pH changes

Summary The mechanism of the transient Na + influx that is induced by hyperosmotic shock in the halotolerant alga Dunaliella salina and its relationship to glycerol production has been investigated. It is demonstrated that: (1) The rapid Na + influx is independent on metabolic energy but is affected by temperature, external pH and the transmembrane electrical potential. Na + elimination depends on metabolic energy but not on external pH suggesting that it is catalyzed by a different mechanism. (2) Intracellular acidification stimulates whereas intracellular alkalinization inhibits Na + influx following hyperosmotic shocks. Na + influx can also be induced by artificial intracellular acidification under isoosmotic conditions. These results suggest that Na + influx following hyperosmotic shocks may result from internal acidification. (3) Lithium and vanadate ions, inhibitors of the plasma membrane Na + /H + antiporter and H + -ATPase, respectively, inhibit Na + influx. These treatments as well as internal alkalinization by ammonium ions also delay glycerol synthesis and volume recovery from hyperosmotic shock. The results suggest a connection between intracellular pH and glycerol synthesis. (4) Extracellular Na + is not obligatory for recovery from hyperosmotic shocks, and does not provide any advantage for resisting lysis by extreme osmotic changes to Dunaliella cells. It is suggested that Na + influx following hyperosmotic shock in D. salina is a secondary response to intracellular acidification and is catalysed by a Na + /H + antiporter at the plasma membrane. The results imply that Na + /H + antiport is activated by internal acidification or by hyperpolarization and inhibited by internal alkalinization or by depolarization. The possible role of intracellular pH in glycerol synthesis is discussed.

Phosphate and sulfate uptake in the halotolerant alga Dunaliella are driven by Na+-symport mechanism

Summary Phosphate and sulfate uptake were measured in the halotolerant alga Dunaliella salina under anion-sufficient or anion-deficient conditions. Both phosphate and sulfate uptake were strictly and specifically dependent on Na+ ions. Pi accumulation was inhibited by Na+ ionophores and by inhibitors of Na+ extrusion. Pi uptake was associated with a stoichiometric influx of Na+ ions at a ratio of 1 Na+/1 Pi. Depletion of phosphate or sulfate from growth media induced a 20-fold or a 2.5-fold stimulation of the corresponding rate of uptake, respectively. The enhancements were associated with an increase in Vmax values, but not in the Km for anion substrates (Km(Pi)∼1 μmol/L, Km(SO4)∼15 μmol/L) or for Na+ (Km(Na+)∼35 mmol/L for Pi, ∼170 mmol/L for SO42-). These results suggest that uptake of phosphate and of sulfate in D. salina are mediated by Na+/anion symporters and are driven by δμNa+ across the plasma membrane. The implications of these results on the availability of phosphate and sulfate in hypersaline solutions are discussed.