Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s)alpha protein: identification of cytosolic forms of G(s)alpha

Distribution of the alpha subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in beta-adrenoceptor density and adenylyl cyclase activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) variants of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alphaL was the dominant isoform of G(s)alpha in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound G(s)alphaS also elevated during post-natal development, but more pronounced changes were found in cytosolic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller than G(s)alphaL) was detected among soluble proteins shortly after birth, G(s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc(-) reconstitutive assay of cytosolic G(s)alpha indicated that only approximately 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of beta-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.     

Identification of cytosolic Mg2+-dependent soluble inorganic pyrophosphatases in potato and phylogenetic analysis

Using polyclonal antibodies raised against a previously cloned potato Mg2+-dependent soluble inorganic pyrophosphatase (ppa1 gene) [8], a second gene, called ppa2, could be isolated. A single locus homologous to ppa2 was mapped on potato chromosomes, unlinked to the two loci identified for ppa1. From a phylogenetic and structural point of view, the PPA1 and PPA2 polypeptides are more closely related to prokaryotic than to eukaryotic Mg2+-dependent soluble inorganic pyrophosphatases (soluble PPases). Subcellular localization by immunogold electron microscopy, using sections from leaf parenchyma cells, showed that PPA1 and PPA2 are localized to the cytosol. Based on these observations, the likely phylogenetic origin and the physiological significance of the cytosolic soluble pyrophosphatases are discussed.     

Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions

Gram-negative bacteria have five major subcellular localization sites: the cytoplasm, the periplasm, the inner membrane, the outer membrane, and the extracellular space. The subcellular location of a protein can provide valuable information about its function. With the rapid increase of sequenced genomic data, the need for an automated and accurate tool to predict subcellular localization becomes increasingly important. We present an approach to predict subcellular localization for Gram-negative bacteria. This method uses the support vector machines trained by multiple feature vectors based on n-peptide compositions. For a standard data set comprising 1443 proteins, the overall prediction accuracy reaches 89%, which, to the best of our knowledge, is the highest prediction rate ever reported. Our prediction is 14% higher than that of the recently developed multimodular PSORT-B. Because of its simplicity, this approach can be easily extended to other organisms and should be a useful tool for the high-throughput and large-scale analysis of proteomic and genomic data.     

In silico prediction of the peroxisomal proteome in fungi,plants and animals

In an attempt to improve our abilities to predict peroxisomal proteins, we have combined machine-learning techniques for analyzing peroxisomal targeting signals (PTS1) with domain-based cross-species comparisons between eight eukaryotic genomes. Our results indicate that this combined approach has a significantly higher specificity than earlier attempts to predict peroxisomal localization, without a loss in sensitivity. This allowed us to predict 430 peroxisomal proteins that almost completely lack a localization annotation. These proteins can be grouped into 29 families covering most of the known steps in all known peroxisomal pathways. In general, plants have the highest number of predicted peroxisomal proteins, and fungi the smallest number.     

A new hybrid approach to predict subcellular localization of proteins by incorporating gene ontology

Based on the recent development in the gene ontology and functional domain databases, a new hybridization approach is developed for predicting protein subcellular location by combining the gene product, functional domain, and quasi-sequence-order effects. As a showcase, the same prokaryotic and eukaryotic datasets, which were studied by many previous investigators, are used for demonstration. The overall success rate by the jackknife test for the prokaryotic set is 94.7% and that for the eukaryotic set 92.9%. These are so far the highest success rates achieved for the two datasets by following a rigorous cross-validation test procedure, suggesting that such a hybrid approach may become a very useful high-throughput tool in the area of bioinformatics, proteomics, as well as molecular cell biology. The very high success rates also reflect the fact that the subcellular localization of a protein is closely correlated with: (1). the biological objective to which the gene or gene product contributes, (2). the biochemical activity of a gene product, and (3). the place in the cell where a gene product is active.     

Clofibrate-induced relocation of phosphatidylcholine transfer protein to mitochondria in endothelial cells

The phosphatidylcholine transfer protein (PC-TP) is a specific transporter of phosphatidylcholine (PC) between membranes. To get more insight into its physiological function, we have studied the localization of PC-TP by microinjection of fluorescently labeled PC-TP in foetal bovine heart endothelial (FBHE) cells and by expression of an enhanced yellow fluorescent protein-PC-TP fusion protein in FBHE cells, human umbilical vein endothelial cells, and HepG2 cells. Analysis by confocal laser scanning microscopy showed that PC-TP was evenly distributed throughout the cytosol with an apparently elevated level in nuclei. By measuring the fluorescence recovery after bleaching it was established that PC-TP is highly mobile throughout the cell, with its transport into the nucleus being hindered by the nuclear envelope. Given the proposed function of PC-TP in lipid metabolism, we have tested a number of compounds (phorbol ester, bombesin, A23187, thrombin, dibutyryl cyclic AMP, oleate, clofibrate, platelet-derived growth factor, epidermal growth factor, and hydrogen peroxide) for their ability to affect intracellular PC-TP distribution. Only clofibrate (100 microM) was found to have an effect, with PC-TP moving to mitochondria within 5 min of stimulation. This relocation did not occur with PC-TP(S110A), lacking the putative protein kinase C (PKC)-dependent phosphorylation site, and was restricted to the primary endothelial cells. Relocation did not occur in HepG2 cells, possibly due to the fact that clofibrate does not induce PKC activation in these cells.     

Rat liver acyl-CoA synthetase 4 is a peripheral-membrane protein located in two distinct subcellular organelles,peroxisomes, and mitochondrial-associated membrane

Obesity and non-insulin-dependent diabetes favor storage of fatty acids in triacylglycerol over oxidation. Recently, individual acyl-CoA synthetase (ACS) isoforms have been implicated in the channeling of fatty acids either toward lipid synthesis or toward oxidation. Although ACS1 had been localized to three different subcellular regions in rat liver, endoplasmic reticulum, mitochondria, and peroxisomes, the study had used an antibody raised against the full-length ACS1 protein which cross-reacts with other isoforms, probably because all ACS family members contain highly conserved amino acid sequences. Therefore, we examined the subcellular location of ACS1, ACS4, and ACS5 in rat liver to determine which isoform was present in peroxisomes, whether the ACSs were intrinsic membrane proteins, and which ACS isoforms were up-regulated by PPAR alpha ligands. Non-cross-reacting ACS1, ACS4, and ACS5 peptide antibodies showed that ACS4 was the only ACS isoform present in peroxisomes isolated from livers of gemfibrozil-treated rats. ACS4 was also present in fractions identified as mitochondria-associated membrane (MAM). ACS1 was present in endoplasmic reticulum fractions and ACS5 was present in mitochondrial fractions. Incubation with troglitazone, a specific inhibitor of ACS4, decreased ACS activity in the MAM fractions 30-45% and in the peroxisomal fractions about 30%. Because the signal for ACS4 protein in peroxisomes was so strong compared to the MAM fraction, we examined ACS4 mRNA abundance in livers of rats treated with the PPAR alpha agonist GW9578. Treatment with GW9578 increased ACS4 mRNA abundance 40% and ACS1 mRNA 25%. Although we had originally proposed that ACS4 is linked to triacylglycerol synthesis, it now appears that ACS4 may also be important in activating fatty acids destined for peroxisomal oxidation. We also determined that, unlike ACS1 and 5, ACS4 is not an intrinsic membrane protein. This suggests that ACS4 is probably targeted and linked to MAM and peroxisomes by interactions with other proteins.     

Astrocytic but not neuronal increased expression and redistribution of parkin during unfolded protein stress

Parkin is a ubiquitin ligase that facilitates proteasomal protein degradation and is involved in a common autosomal recessive form of Parkinson's disease. Its expression is part of the unfolded protein response in cell lines where its overexpression protects against unfolded protein stress. How parkin expression is regulated in brain primary cells under stress situations is however, less well established. Here, the cellular and subcellular localization of parkin under basal conditions and during unfolded protein stress was investigated in primary cultures of rat astrocytes and hippocampal neurons. Immunofluorescense microscopy and biochemical analysis demonstrated that parkin is mainly associated with the endoplasmic reticulum (ER) in hippocampal neurons while it is associated with Golgi membranes, the nuclei and light vesicles in astrocytes. The constitutive parkin expression was high in neurons as compared with astrocytes. However, unfolded protein stress elicited a selective increase in astrocytic parkin expression and a change in distribution, whereas neuronal parkin remained largely unmodified. The cell specific differences argue in favour of different cellular binding sites and substrates for the protein and a pathogenic role for astrocytes in Parkinson's disease caused by parkin dysfunction.     

Predicting protein subcellular localisation from amino acid sequence information

Predicting the subcellular localisation of proteins is an important part of the elucidation of their functions and interactions. Here, the amino acid sequence motifs that direct proteins to their proper subcellular compartment are surveyed, different methods for localisation prediction are discussed, and some benchmarks for the more commonly used predictors are presented.