Voltage-dependent-anion-channels (VDACs) in Arabidopsis have a dual localization in the cell but show a distinct role in mitochondria

In mammals, the Voltage-dependent anion channels (VDACs) are predominant proteins of the outer mitochondrial membrane (OMM) where they contribute to the exchange of small metabolites essential for respiration. They were shown to be as well associated with the plasma membrane (PM) and act as redox enzyme or are involved in ATP release for example. In Arabidopsis, we show that four out of six genomic sequences encode AtVDAC proteins. All four AtVDACs are ubiquitously expressed in the plant but each of them displays a specific expression pattern in root cell types. Using two complementary approaches, we demonstrate conclusively that the four expressed AtVDACs are targeted to both mitochondria and plasma membrane but in differential abundance, AtVDAC3 being the most abundant in PM, and conversely, AtVDAC4 almost exclusively associated with mitochondria. These are the first plant proteins to be shown to reside in both these two membranes. To investigate a putative function of AtVDACs, we analyzed T-DNA insertion lines in each of the corresponding genes. Knock-out mutants for AtVDAC1, AtVDAC2 and AtVDAC4 present slow growth, reduced fertility and yellow spots in leaves when atvdac3 does not show any visible difference compared to wildtype plants. Analyses of atvdac1 and atvdac4 reveal that yellow areas correspond to necrosis and the mitochondria are swollen in these two mutants. All these results suggest that, in spite of a localization in plasma membrane for three of them, AtVDAC1, AtVDAC2 and AtVDAC4 have a main function in mitochondria.     

Cholecystokinin-Induced Desensitization, Receptor Phosphorylation, and Internalization in the CHP212 Neuroblastoma Cell Line

Abstract: Agonist stimulation of cells often results in desensitization of the response, to protect the cell from overstimulation. We have previously shown that the type A cholecystokinin (CCK) receptor on the pancreatic acinar cell and on the model CHO-CCKR cell line undergoes desensitization in response to CCK, with receptor phosphorylation and internalization playing key roles. Although these mechanisms contribute in a cell-specific manner, no analogous information exists for the CCK receptor expressed on neuronal cells, where in vivo data demonstrate a particularly sensitive response to CCK. The present study was designed to explore CCK receptor desensitization in the CHP212 neuroblastoma cell line, focusing on inositol 1,4,5-trisphosphate (IP₃) responses to CCK and on recognized molecular and cellular mechanisms of desensitization. CCK promptly stimulated IP₃ responses in these cells, with hormonal responsiveness rapidly and completely desensitized. Both receptor phosphorylation and internalization were observed to occur, with the former occurring most rapidly and likely being responsible for the earliest desensitization observed. Although the time course of receptor phosphorylation and dephosphorylation, and the groups of kinases involved in the neuroblastoma cell line, were most similar to those in the pancreatic cell, the movement of the agonist-bound receptor in these cells was quite different from that in the pancreatic cell and most similar to that in the CHO-CCKR cell line. This hybrid response supports the cell-specific nature of CCK receptor regulation and provides an important system to explore the molecular determinants of these processes.     

Hepatocellular transport of cyclosomatostatins: evidence for a carrier system related to the multispecific bile acid transporter.

The uptake of the cyclopeptide c(Phe-Thr-Lys-Trp-Phe-D-Pro) (008), an analog of somatostatin with retro sequence, was studied in isolated hepatocytes. 008 is taken up by hepatocytes in a concentration-, time-, energy- and temperature- dependent manner. Since 008 is hydrophobic, it binds rapidly to liver cells. This is evident by the positive intercept at the gamma-axis in the uptake curves. At higher concentrations, a minor part of the transport occurs by diffusion at a rate of 8.307.10(-6) cm/s. This part of diffusion is measured at 4 degrees C and can be subtracted from the uptake at 37 degrees C resulting in the carrier mediated part of uptake which is saturable. Kinetic parameters for the saturable part of uptake are Km 1.5 microM and Vmax 40.0 pmol/mg per min. The transport is decreased in the absence of oxygen and in the presence of metabolic inhibitors. Uptake is accelerated at temperatures above 20 degrees C. The activation energy was determined to be 30.77 kJ/mol. The membrane potential and not a sodium gradient is the main driving force for 008 transport. Cholate (a typical substrate of the multispecific bile acid transporter) and taurocholate are mutual competitive inhibitors of 008 uptake. Phalloidin, antamanide and iodipamide, typical foreign substrates of the transporter, interfere with the uptake of 008. AS 30D ascites hepatoma cells, known to be unable to transport bile acids, phalloidin and iodipamide, are also unfit to transport 008. Interestingly, sulfobromophthalein (BSP) but not rifampicin, both foreign substrates of the bilirubin carrier, inhibits the transport of 008 in a competitive manner.     

Differential regulation in tobacco cell suspensions of genes involved in plant-bacteria interactions by pathogen-related signals

Six cDNA clones whose corresponding mRNAs accumulate early during the hypersensitive reaction in tobacco leaves have been classified into 2 groups according to their maximum levels of accumulation in an incompatible versus a compatible interaction withPseudomonas solanacearum. We present evidence that, at least in the first stages of the interaction, tobacco cell suspensions retain the ability to respond differentially to compatible and incompatible isolates ofP. solanacearum.In addition, studies on the effect of a fungal elicitor on the accumulation of the mRNAs corresponding to the cDNA clones in cell suspensions indicate that only one group of genes responds to this treatment.     

Preferential Transfer of 2-Docosahexaenoyl-1-Lysophosphatidylcholine Through an In Vitro Blood-Brain Barrier Over Unesterified Docosahexaenoic Acid

Abstract : The passage of either unesterified docosahexaenoic acid (DHA) or lysophosphatidylcholine-containing DHA (lysoPC-DHA) through an in vitro model of the blood-brain barrier was investigated. The model was constituted by a brain capillary endothelial cell monolayer set over the medium of an astrocyte culture. Cells were incubated for 4 h with a medium devoid of serum, then the endothelial cell medium was replaced by the same medium containing labeled DHA or lysoPC-DHA and incubations were performed for 2 h. DHA uptake by cells and its transfer to the lower medium (astrocyte medium when they were present) were measured. When the lower medium from preincubation and astrocytes were maintained during incubation, the passage of lysoPC-DHA was higher than that of unesterified DHA. The passage of both forms decreased when astrocytes were removed. The preference for lysoPC-DHA was not seen when the lower medium from preincubation was replaced by fresh medium, and was reversed when albumin was added to the lower medium. A preferential lysoPC-DHA passage also occurred after 2 h with brain endothelial cells cultured without astrocytes but not with aortic endothelial cells cultured and incubated under the same conditions. Altogether, these results suggest that the blood-brain barrier cells released components favoring the DHA transfer and exhibit a preference for lysoPC-DHA.     

Mosquito (Aedes aegypti ) aquaporin, present in tracheolar cells, transports water, not glycerol, and forms orthogonal arrays in Xenopus oocyte membranes.

Previous results showed that mRNA encoding a putative aquaporin (AQP) (GenBank accession number AF218314) is present in the tracheolar cells associated with female Aedes aegypti Malpighian tubules. In this study, immunohistochemistry detected the protein, AeaAQP, also in tracheolar cells, suggesting its involvement in water movement in the respiratory system. When expressed in Xenopus oocytes, AeaAQP increased the osmotic water permeability from 15 x 10(-6) to 150 x 10(-6) m x s-1, which was inhibited by mercury ions. No permeability to glycerol or other solute was observed. AeaAQP expressed in oocytes was solubilized as a homotetramer in nondenaturing detergent as deduced from velocity centrifugation on density gradients. Phylogenetic analysis of MIP (major intrinsic protein) family sequences shows that AeaAQP clusters with other native orthogonal array forming proteins. Specific orthogonal arrays were detected by freeze-fracture analysis of AeaAQP oocyte membranes. We conclude that, in tracheolar cells of A. aegypti, AeaAQP is probably a highly water-permeable homotetrameric MIP which natively can form 2D crystals.     

Molecular characterization of a second copy of holocarboxylase synthetase gene (hcs2) in Arabidopsis thaliana.

Holocarboxylase synthetase (HCS), catalyzing the covalent attachment of biotin, is ubiquitously represented in living organisms. Indeed, the biotinylation is a post-translational modification that allows the transformation of inactive biotin-dependent carboxylases, which are committed in fundamental metabolisms such as fatty acid synthesis, into their active holo form. Among other living organisms, plants present a peculiarly complex situation. In pea, HCS activity has been detected in three subcellular compartments and the systematic sequencing of the Arabidopsis genome revealed the occurrence of two hcs genes (hcs1 and hcs2). Hcs1 gene product had been previously characterized at molecular and biochemical levels. Here, by PCR amplification, we cloned an hcs2 cDNA from Arabidopsis thaliana (Ws ecotype) mRNA. We observed the occurrence of multiple cDNA forms which resulted from the alternative splicing of hcs2 mRNA. Furthermore, we evidenced a nucleotide polymorphism at the hcs2 gene within the Ws ecotype, which affected splicing of hcs2 mRNA. This contrasted sharply with the situation at hcs1 locus. However, this polymorphism had no apparent effect on total HCS activity in planta. Finally, hcs2 mRNAs were found 4-fold less abundant than hcs1 mRNA and the most abundant hcs2 mRNA spliced variant should code for a truncated protein. We discuss the possible role of such a multiplicity of putative HCS proteins in plants and discuss the involvement of each of hcs genes in the correct realization of biotinylation.