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PubMed1995

Subcellular localization and kinetic properties of aromatase activity in rat brain.

The conversion of testosterone to estradiol is catalyzed by cytochrome P450 aromatase. In situ aromatization is required for the full expression of the effects of testosterone in the brain. This study examined the subcellular distribution and reaction kinetics of aromatase in the adult rat brain. Preoptic area, hypothalamus and amygdala were homogenized in isotonic sucrose buffered with potassium phosphate. Tissue homogenates were fractionated by ultracentrifugation. Aromatase activity was measured using a previously validated 3H2O assay. Marker enzymes were measured to identify organelles in the different subcellular fractions. Aromatase activity in all 3 tissues was enriched 10-fold in microsomes, but not in other subcellular fractions. The addition of either a NADPH-generating system or 1 mM NADPH to the reaction mixture stimulated aromatase activity in all subcellular fractions, whereas NADH was only minimally effective. In general, substrate affinity constants were equivalent in all brain areas and subcellular fractions (approximately 10 nM) suggesting that one predominant catalytic form of the enzyme is present in the rat brain. One week after castration, aromatase activity was significantly reduced in all subcellular fractions of preoptic area and in the whole homogenate and microsomal fraction of the hypothalamus. Castration did not significantly alter aromatase activity in any subcellular compartment of amygdala. To more critically evaluate its subcellular localization, aromatase activity was measured in purified synaptosomes. Aromatase activity was not enriched in these preparations suggesting that it is not substantially associated with nerve terminals in rat brain.

Amygdala↗

PubMed2004

Esub8: a novel tool to predict protein subcellular localizations in eukaryotic organisms.

BACKGROUND: Subcellular localization of a new protein sequence is very important and fruitful for understanding its function. As the number of new genomes has dramatically increased over recent years, a reliable and efficient system to predict protein subcellular location is urgently needed. RESULTS: Esub8 was developed to predict protein subcellular localizations for eukaryotic proteins based on amino acid composition. In this research, the proteins are classified into the following eight groups: chloroplast, cytoplasm, extracellular, Golgi apparatus, lysosome, mitochondria, nucleus and peroxisome. We know subcellular localization is a typical classification problem; consequently, a one-against-one (1-v-1) multi-class support vector machine was introduced to construct the classifier. Unlike previous methods, ours considers the order information of protein sequences by a different method. Our method is tested in three subcellular localization predictions for prokaryotic proteins and four subcellular localization predictions for eukaryotic proteins on Reinhardt's dataset. The results are then compared to several other methods. The total prediction accuracies of two tests are both 100% by a self-consistency test, and are 92.9% and 84.14% by the jackknife test, respectively. Esub8 also provides excellent results: the total prediction accuracies are 100% by a self-consistency test and 87% by the jackknife test. CONCLUSIONS: Our method represents a different approach for predicting protein subcellular localization and achieved a satisfactory result; furthermore, we believe Esub8 will be a useful tool for predicting protein subcellular localizations in eukaryotic organisms.

Computational Biology↗

PubMed1986

Subcellular distribution of leukotriene C4 binding units in cultured bovine aortic endothelial cells.

The subcellular distribution of specific binding sites for [3H]leukotriene C4 ([3H]LTC4) was analyzed after sedimentation of organelles from disrupted bovine aortic endothelial cells on sucrose density gradients and was shown to be in membrane fractions I (20% sucrose) and IV (35% sucrose). Saturation binding studies of [3H]LTC4 on endothelial cell monolayers at 4 degrees C demonstrated high-affinity binding sites with a dissociation constant (Kd) of 6.8 +/- 2.2 nM (mean +/- SD) and a density of 0.12 +/- 0.02 pmol/10(6) cells. At 4 degrees C, the specific binding of [3H]LTC4 by each of the subcellular fractions reached equilibrium at 30 min and remained stable for an additional 60 min. After 30 min of incubation with [3H]LTC4, the addition of excess unlabeled LTC4 to each subcellular fraction reversed more than 70% of [3H]LTC4 binding in 10 min. The [3H]LTC4 binding activities of subcellular fractions were enhanced approximately twofold to fourfold in the presence of Ca2+, Mg2+, and Mn2+, whereas Na+, K+, and Li+ were without effect. As measured by saturation experiments, the Kd and density of LTC4 binding sites in fraction I were 4.8 +/- 1.6 nM and 16.5 +/- 1.9 pmol/mg of protein, respectively, and in fraction IV were 4.7 +/- 1.5 nM and 81.4 +/- 19 pmol/mg of protein, respectively. Inhibition of [3H]LTC4 binding in membrane-enriched subcellular fractions I and IV by LTC4 occurred with molar inhibition constant (Ki) values of 4.5 +/- 0.1 nM and 4.7 +/- 1.2 nM, respectively, whereas Ki values for LTD4 were 570 +/- 330 nM and 62.5 +/- 32.8 nM, respectively, and for LTE4 were greater than 1000 nM for each fraction; LTB4 and reduced glutathione were even less active. FPL55712, a putative antagonist of the sulfidopeptide LT components of slow reacting substance of anaphylaxis, had Ki values of 1520 +/- 800 nM and 1180 +/- 720 nM for [3H]LTC4 binding sites on membrane-enriched subcellular fractions I and IV, respectively. Thus as defined by Kd, Ki, and specificity, the LTC4 binding units that are distributed to the plasma membrane and the binding units in the subcellular fraction of greater density were similar to each other. Pretreatment of the isolated subcellular membrane fractions with trypsin abolished [3H]LTC4 binding by fraction I, enriched for the plasma membrane marker 5' nucleotidase, and that by fraction IV, enriched for the mitochondrial membrane marker succinate-cytochrome C reductase.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Maternal dietary fish oil enriches docosahexaenoate levels in brain subcellular fractions of offspring.

Prompted by the speculated essentiality of docosahexaenoic acid (DHA) for neural development, this study was undertaken to investigate the incorporation of (n-3) fatty acids in the maternal diet into various phospholipids of infant rat brain subcellular fractions: microsomes (Ms), synaptosomes (Sy), myelin (My), and mitochondria (Mt). Two groups of infant rats were nourished by dams fed diets containing 20% of either corn oil (CO) or menhaden oil (MO) from 2 until 12 days of age. DHA but not eicosapentaenoic acid (EPA) was distributed to all subcellular fractions of infant rats in the CO group. The levels of DHA were higher in Ms and Mt than Sy and My, and higher in phosphatidylethanolamine (PE) and phosphatidylserine (PS) than phosphatidylcholine (PC) and phosphatidylinositol (PI). The MO feeding enriched DHA in PE of all subcellular fractions, PS of all subcellular fractions, except My, PC of Sy, My and Mt, and PI of My. EPA was enriched in phospholipids in all subcellular fractions, except mitochondrial PS of the MO group. In the MO group, the ratios of EPA/DHA, ranging from 0.01 to 0.85, in all subcellular phospholipids were markedly lower than that found in the mother's milk (i.e., 1.5), suggesting an ability to elongate and desaturate EPA to DHA and/or disproportional uptake of the fatty acids by the brain. In PE of all subcellular fractions, the increased levels of DHA and EPA, with a concomitant reduction of arachidonic and/or linoleic acid, yielded higher ratios of total (n-3)/(n-6) fatty acids in the MO than the CO group. The inclusion of preformed DHA and EPA in the maternal diet provides an effective means to enrich these fatty acids in developing brains.

Animals↗

PubMed2005

New application of a subcellular fractionation method to kidney and testis for the determination of conjugated linoleic acid in selected cell organelles of healthy and cancerous human tissues.

To clarify the mechanism of the anticarcinogenic effect of conjugated linoleic acid (CLA), its intracellular distribution needs to be determined. Subcellular fractionation using centrifugation techniques is a method that is frequently used for isolation of cell organelles from different tissues. But as the size and density of the organelles differ, the method needs to be optimised for every type of tissue. The novelty of this study is the application of a subcellular fractionation method to human healthy and cancerous renal and testicular tissue. Separation of total tissue homogenate into nuclei, cytosol, and a mixture of mitochondria and plasma membranes was achieved by differential centrifugation. As mitochondria and plasma membranes seemed to be too similar in size and weight to be separated by differential centrifugation, discontinuous density-gradient centrifugation was carried out successfully. The purity of the subcellular fractions was checked by measuring the activity of marker enzymes. All fractions were highly enriched in their corresponding marker enzyme. However, the nuclear fractions of kidney and renal cell carcinoma were slightly contaminated with mitochondria and plasma membrane fractions of all tissues with lysosomes. The fraction designated the cytosolic fraction contained not only cytosol, but also microsomes and lysosomes. The CLA contents of the subcellular fractions were in the range 0.13-0.37% of total fatty acids and were lowest in the plasma membrane fractions of all types of tissue studied. C16:0, C18:0, C18:1 c9, C18:2 n-6, and C20:4 n-6 were found to be the major fatty acids in all the subcellular fractions studied. However, marked variations in fatty acid content between subcellular fractions and between types of tissue were detectable. Because of these differences between tissues, no general statement on characteristic fatty acid profiles of single subcellular fractions is possible.

Adipose Tissue↗

PubMed1991

Subcellular distribution of adenylyl cyclase and Gs alpha in rat brown adipose tissue.

The subcellular distribution of Gs alpha (the alpha-subunit of guanine nucleotide-binding stimulatory protein of adenylyl cyclase) was examined in interscapular brown adipose tissue (IBAT) to determine (1) if Gs alpha is completely colocalized with adenylyl cyclase in the plasma membrane, and (2) whether cold exposure, which increases adenylyl cyclase activity, changes the subcellular distribution of Gs alpha. Subcellular fractions were prepared from IBAT by differential centrifugation and analyzed for Gs alpha by immunoblotting. Adenylyl cyclase activity and Gs alpha were detected in all the subcellular fractions except the cytosol. The plasma membrane fraction showed the greatest enrichment of adenylyl cyclase and Gs alpha. However, the enrichment of adenylyl cyclase in the plasma membrane fraction was greater than that for Gs alpha, which was also associated to a large degree with the mitochondrial fraction. Thus, compared with the mitochondrial fraction, both 5' nucleotidase and adenylyl cyclase were enriched by over 200% in the plasma membrane fraction, but Gs alpha was enriched by only 50%. Exposure of rats to 4 degrees C for 3 days increased fluoride-stimulated adenylyl cyclase activity, but did not increase the amount of immunoreactive Gs alpha in any of the subcellular fractions examined. The above results demonstrate that not all Gs alpha in IBAT is colocalized with adenylyl cyclase in the plasma membrane. The finding that cold exposure did not change the subcellular distribution of Gs alpha indicates that the cold-induced increase in adenylyl cyclase activity is not due to translocation of Gs alpha from subcellular compartments to the plasma membrane.

5'-Nucleotidase↗

PubMed2005

Aging of human glyceraldehyde-3-phosphate dehydrogenase is dependent on its subcellular localization.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), long considered a traditional glycolytic protein, displays multiple activities independent of its role in energy generation. This functional diversity is dependent on its membrane, cytoplasmic or nuclear localization. GAPDH is encoded by one active gene and is synthesized as a single 37 kDa protein without alternate splicing. Accordingly, the identical protein would be present in each subcellular fraction. The accumulation of post-translational errors in protein structure as a function of oxidative stress is thought to provide a basic molecular mechanism for the aging process. Thus, during aging, the GAPDH protein should contain the identical degree of oxidative sequence alteration irrespective of its distribution. This would result in equivalent effects on GAPDH activity. However, conformational differences in GAPDH structure due to its subcellular protein, nucleic acid or membrane interactions could affect its degree of modification thereby selectively affecting its function. For that reason, we examined the subcellular expression and intracellular activity of GAPDH as a function of human aging. Subcellular GAPDH expression was quantitated by immunoblot analysis in fetal and senior human cells (postnuclear, nuclear, perinuclear). GAPDH activity was determined by in vitro assay. We now report that the aging of human GAPDH was subcellular dependent. Reductions of nuclear and postnuclear GAPDH activity in senior cells were twofold lower than that observed for the perinuclear protein. In contrast, the subcellular expression of the GAPDH protein was age-independent. These results suggest the possibility that subcellular interactions may mitigate oxidative stress-induced GAPDH modification in human aging. Such selective effects on GAPDH could affect its functional diversity.

Aging↗

PubMed2006

Large-scale predictions of gram-negative bacterial protein subcellular locations.

Many species of Gram-negative bacteria are pathogenic bacteria that can cause disease in a host organism. This pathogenic capability is usually associated with certain components in Gram-negative cells. Therefore, developing an automated method for fast and reliable prediction of Gram-negative protein subcellular location will allow us to not only timely annotate gene products, but also screen candidates for drug discovery. However, protein subcellular location prediction is a very difficult problem, particularly when more location sites need to be involved and when unknown query proteins do not have significant homology to proteins of known subcellular locations. PSORT-B, a recently updated version of PSORT, widely used for predicting Gram-negative protein subcellular location, only covers five location sites. Also, the data set used to train PSORT-B contains many proteins with high degrees of sequence identity in a same location group and, hence, may bear a strong homology bias. To overcome these problems, a new predictor, called "Gneg-PLoc", is developed. Featured by fusing many basic classifiers each being trained with a stringent data set containing proteins with strictly less than 25% sequence identity to one another in a same location group, the new predictor can cover eight subcellular locations; that is, cytoplasm, extracellular space, fimbrium, flagellum, inner membrane, nucleoid, outer membrane, and periplasm. In comparison with PSORT-B, the new predictor not only covers more subcellular locations, but also yields remarkably higher success rates. Gneg-PLoc is available as a Web server at http://202.120.37.186/bioinf/Gneg. To support the demand of people working in the relevant areas, a downloadable file is provided at the same Web site to list the results identified by Gneg-PLoc for 49 907 Gram-negative protein entries in the Swiss-Prot database that have no subcellular location annotations or are annotated with uncertain terms. The large-scale results will be updated twice a year to cover the new entries of Gram-negative bacterial proteins and reflect the new development of Gneg-PLoc.

Bacterial Proteins↗

PubMed2005

Dynamics of metal subcellular distribution and its relationship with metal uptake in marine mussels.

We examined the dynamics of subcellular distribution of metals (Cd, Ag, and Zn) in the marine green mussel Perna viridis by partitioning the metals into the insoluble fraction (IF), heat-sensitive proteins (HSP), and metallothionein-like proteins (MTLP) during metal uptake and elimination. Variations in metal uptake and elimination then were correlated with the subcellular distributions of these metals. The IF and HSP were the first ligands to bind with the metals during the dissolved exposure, and more metals were found in the HSP when the metal influx rate was higher. However, to minimize toxicity, metals were redistributed from HSP to MTLP afterwards. The subcellular distribution of metals was dependent of the exposure route in the mussels. During dietary metal exposure, the metals attained equilibrium before they were assimilated and the metal assimilation efficiency was independent of the metal partitioning in different subcellular fractions. During the efflux, metals in the soluble fraction mediated depuration, whereas metals in the insoluble fraction acted as a final storage pool. Redistribution also may occur between the metal-sensitive and inactive pools without significant depuration as a secondary protective mechanism. We further demonstrated that the higher efflux rate of Ag and Cd was related to a higher partitioning in the MTLP and a lower partitioning in the IF. Our study shows that subcellular pools other than MTLP were involved in immediate metal handling in the bivalves. The wide dynamics of subcellular metal distribution suggests that the relevance of individual subcellular fractions is dependent on the exposure pathway.

Animals↗

PubMed2006

Prediction of protein subcellular localization.

Because the protein's function is usually related to its subcellular localization, the ability to predict subcellular localization directly from protein sequences will be useful for inferring protein functions. Recent years have seen a surging interest in the development of novel computational tools to predict subcellular localization. At present, these approaches, based on a wide range of algorithms, have achieved varying degrees of success for specific organisms and for certain localization categories. A number of authors have noticed that sequence similarity is useful in predicting subcellular localization. For example, Nair and Rost (Protein Sci 2002;11:2836-2847) have carried out extensive analysis of the relation between sequence similarity and identity in subcellular localization, and have found a close relationship between them above a certain similarity threshold. However, many existing benchmark data sets used for the prediction accuracy assessment contain highly homologous sequences-some data sets comprising sequences up to 80-90% sequence identity. Using these benchmark test data will surely lead to overestimation of the performance of the methods considered. Here, we develop an approach based on a two-level support vector machine (SVM) system: the first level comprises a number of SVM classifiers, each based on a specific type of feature vectors derived from sequences; the second level SVM classifier functions as the jury machine to generate the probability distribution of decisions for possible localizations. We compare our approach with a global sequence alignment approach and other existing approaches for two benchmark data sets-one comprising prokaryotic sequences and the other eukaryotic sequences. Furthermore, we carried out all-against-all sequence alignment for several data sets to investigate the relationship between sequence homology and subcellular localization. Our results, which are consistent with previous studies, indicate that the homology search approach performs well down to 30% sequence identity, although its performance deteriorates considerably for sequences sharing lower sequence identity. A data set of high homology levels will undoubtedly lead to biased assessment of the performances of the predictive approaches-especially those relying on homology search or sequence annotations. Our two-level classification system based on SVM does not rely on homology search; therefore, its performance remains relatively unaffected by sequence homology. When compared with other approaches, our approach performed significantly better. Furthermore, we also develop a practical hybrid method, which combines the two-level SVM classifier and the homology search method, as a general tool for the sequence annotation of subcellular localization.

Algorithms↗

PubMed2006

Predicting eukaryotic protein subcellular location by fusing optimized evidence-theoretic K-Nearest Neighbor classifiers.

Facing the explosion of newly generated protein sequences in the post genomic era, we are challenged to develop an automated method for fast and reliably annotating their subcellular locations. Knowledge of subcellular locations of proteins can provide useful hints for revealing their functions and understanding how they interact with each other in cellular networking. Unfortunately, it is both expensive and time-consuming to determine the localization of an uncharacterized protein in a living cell purely based on experiments. To tackle the challenge, a novel hybridization classifier was developed by fusing many basic individual classifiers through a voting system. The "engine" of these basic classifiers was operated by the OET-KNN (Optimized Evidence-Theoretic K-Nearest Neighbor) rule. As a demonstration, predictions were performed with the fusion classifier for proteins among the following 16 localizations: (1) cell wall, (2) centriole, (3) chloroplast, (4) cyanelle, (5) cytoplasm, (6) cytoskeleton, (7) endoplasmic reticulum, (8) extracell, (9) Golgi apparatus, (10) lysosome, (11) mitochondria, (12) nucleus, (13) peroxisome, (14) plasma membrane, (15) plastid, and (16) vacuole. To get rid of redundancy and homology bias, none of the proteins investigated here had >/=25% sequence identity to any other in a same subcellular location. The overall success rates thus obtained via the jack-knife cross-validation test and independent dataset test were 81.6% and 83.7%, respectively, which were 46 approximately 63% higher than those performed by the other existing methods on the same benchmark datasets. Also, it is clearly elucidated that the overwhelmingly high success rates obtained by the fusion classifier is by no means a trivial utilization of the GO annotations as prone to be misinterpreted because there is a huge number of proteins with given accession numbers and the corresponding GO numbers, but their subcellular locations are still unknown, and that the percentage of proteins with GO annotations indicating their subcellular components is even less than the percentage of proteins with known subcellular location annotation in the Swiss-Prot database. It is anticipated that the powerful fusion classifier may also become a very useful high throughput tool in characterizing other attributes of proteins according to their sequences, such as enzyme class, membrane protein type, and nuclear receptor subfamily, among many others. A web server, called "Euk-OET-PLoc", has been designed at http://202.120.37.186/bioinf/euk-oet for public to predict subcellular locations of eukaryotic proteins by the fusion OET-KNN classifier.

Amino Acids↗

PubMedDate not supplied

Subcellular proteomics.

The step from the analysis of the genome to the analysis of the proteome is not just a matter of numerical complexity in terms of variants of gene products that can arise from a single gene. A significant further level of complexity is introduced by the supramolecular organization of gene products because of protein-protein interactions or targeting of proteins to specific subcellular structures. There is currently no single proteome analysis strategy that can sufficiently address all levels of the organization of the proteome. To approach an appropriate analytical complement for the interrogation of the proteome at all of the levels at which it is organized, there emerges the need for a whole arsenal of proteomics strategies. The proteome analysis at the level of subcellular structures (that can be enriched by subcellular fractionation) represents an analytical strategy that combines classic biochemical fractionation methods and tools for the comprehensive identification of proteins. Among the key potentials of this strategy is the capability to screen not only for previously unknown gene products but also to assign them, along with other known, but poorly characterized gene products, to particular subcellular structures. Furthermore, the analysis at the subcellular level is a prerequisite for the detection of important regulatory events such as protein translocation in comparative studies. This review is meant to give an overview on recent key studies in the field of proteome analysis at the level of subcellular structures, and to highlight potentials and requirements.

Animals↗

PubMed1986

A comparison of lipids from liver and hepatoma subcellular membranes.

Subcellular fractions of nuclei, mitochondria, endoplasmic reticulum, plasma membrane and cytosol were prepared from liver and hepatoma 7288CTC. Marker enzyme activities, biochemical compositions and electron microscopy were used to establish purity. Hepatoma NADH: cytochrome C reductase and 5'-nucleotidase exhibited abnormal subcellular distributions. The lipids from the subcellular fractions were examined in detail. Mitochondria and plasma membranes were characterized by elevated percentages of diphosphatidylglycerol and sphingomyelin, respectively, in both tissues. All hepatoma subcellular fractions contained dramatically elevated levels of sphingomyelin and cholesterol, two components that form preferential strong complexes in vitro. The fatty acid composition of hepatoma sphingomyelin differed markedly from liver and, unlike liver, did not exhibit organelle specific compositions. Some hepatoma lipid classes contained reduced percentages of palmitate while others contained higher levels. Hepatoma phosphatidylcholine and phosphatidylethanolamine from organelles contained lower percentages of long chain polyunsaturated fatty acids than liver. Generally, unique fatty acid profiles exhibited by individual phospholipid classes of liver subcellular fractions were absent or much reduced in the hepatoma. The ratios of oleate to vaccenate were near one for most of the phospholipid classes of most liver fractions, but all hepatoma classes, with few exceptions, contained a much higher percentage of oleate in all subcellular fractions. The hypothesis is proposed that the origin of some acyl moieties for the biosynthesis of various hepatoma lipid classes differs from liver sources. The possible changes in acyl pools, sources and compartments for complex lipid biosynthesis could result in change in the quantities of molecular species that could contribute to the abnormal properties of the hepatoma membranes.

Animals↗

PubMed1988

Phosphatidylinositol kinase, phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions.

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [gamma-32P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150-200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4-11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40-70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP2 in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.

1-Phosphatidylinositol 4-Kinase↗

PubMed2003

Proteome analysis at the level of subcellular structures.

The targeting of proteins to particular subcellular sites is an important principle of the functional organization of cells at the molecular level. In turn, knowledge about the subcellular localization of a protein is a characteristic that may provide a hint as to the function of the protein. The combination of classic biochemical fractionation techniques for the enrichment of particular subcellular structures with the large-scale identification of proteins by mass spectrometry and bioinformatics provides a powerful strategy that interfaces cell biology and proteomics, and thus is termed 'subcellular proteomics'. In addition to its exceptional power for the identification of previously unknown gene products, the analysis of proteins at the subcellular level is the basis for monitoring important aspects of dynamic changes in the proteome such as protein transloction. This review summarizes data from recent subcellular proteomics studies with an emphasis on the type of data that can retrieved from such studies depending on the design of the analytical strategy.

Animals↗

PubMed2005

Combining experimental and predicted datasets for determination of the subcellular location of proteins in Arabidopsis.

Substantial experimental datasets defining the subcellular location of Arabidopsis (Arabidopsis thaliana) proteins have been reported in the literature in the form of organelle proteomes built from mass spectrometry data (approximately 2,500 proteins). Subcellular location for specific proteins has also been published based on imaging of chimeric fluorescent fusion proteins in intact cells (approximately 900 proteins). Further, the more diverse history of biochemical determination of subcellular location is stored in the entries of the Swiss-Prot database for the products of many Arabidopsis genes (approximately 1,800 proteins). Combined with the range of bioinformatic targeting prediction tools and comparative genomic analysis, these experimental datasets provide a powerful basis for defining the final location of proteins within the wide variety of subcellular structures present inside Arabidopsis cells. We have analyzed these published experimental and prediction data to answer a range of substantial questions facing researchers about the veracity of these approaches to determining protein location and their interrelatedness. We have merged these data to form the subcellular location database for Arabidopsis proteins (SUBA), providing an integrated understanding of protein location, encompassing the plastid, mitochondrion, peroxisome, nucleus, plasma membrane, endoplasmic reticulum, vacuole, Golgi, cytoskeleton structures, and cytosol (www.suba.bcs.uwa.edu.au). This includes data on more than 4,400 nonredundant Arabidopsis protein sequences. We also provide researchers with an online resource that may be used to query protein sets or protein families and determine whether predicted or experimental location data exist; to analyze the nature of contamination between published proteome sets; and/or for building theoretical subcellular proteomes in Arabidopsis using the latest experimental data.

Arabidopsis↗

PubMed1988

Protein tyrosine kinase activity and its endogenous substrates in rat brain: a subcellular and regional survey.

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.

Animals↗

PubMed1996

Subcellular properties of triggered Ca2+ waves in isolated citrate-loaded guinea-pig atrial myocytes characterized by ratiometric confocal microscopy.

1. Spatiotemporal aspects of subcellular Ca2+ signalling were studied in cultured adult guinea-pig atrial myocytes. A mixture of the Ca2+ indicators fluo-3 and Fura Red in combination with laser-scanning confocal microscopy was used for [Ca2+]i measurements while membrane currents were recorded simultaneously. 2. In citrate-loaded atrial myocytes not every Ca2+ current (ICa) could trigger Ca2+ release from the sarcoplasmic reticulum (SR). Two types of Ca2+ signals could be observed: Ca2+ transients resulting from (i) Ca2+ influx alone and (ii) additional Ca2+ release. 3. Ca2+ release elicited by voltage steps of 100-150 ms duration was either apparently homogeneous or propagated as Ca2+ waves through the entire cell. With brief ICa (50-75 ms), Ca2+ waves with limited subcellular propagation were observed frequently. These waves always originated from either end of the myocyte. 4. The time course of changes in Na(+)-Ca2+ exchange current (INaCa) depended on the subcellular properties of the underlying Ca2+ transient and on the particular cell geometry. Apparently homogeneous Ca2+ release was accompanied by an inward change of INaCa the onset phase of which was fused with ICa. Changes in INaCa caused by a Ca2+ wave propagating through the entire cell showed a W shape, which could be attributed to differences of the fractional surface-to-volume ratio in different cell segments during propagation of the Ca2+ wavefront. Those waves with limited spreading only activated a small component of INaCa. 5. The different subcellular patterns of Ca2+ release signals can be explained by spatial inhomogeneities in the positive feedback of the SR. This depends on the local SR Ca2+ loading state under the control of the local Ca2+ influx during activation of ICa. Due to the higher surface-to-volume ratio at the two ends of the myocyte, SR loading and therefore the positive feedback in Ca(2+)-induced Ca2+ release may be higher at the ends, locations where Ca2+ waves are preferentially triggered. 6. We conclude that the individual cell geometry may be an important determinant of subcellular Ca2+ signalling not only in cardiac muscle cells but presumably also in other types of cells that depend on Ca2+ signalling. In addition, the cell geometry in combination with varying subcellular Ca2+ release patterns can greatly affect the time course of Ca(2+)-activated membrane currents.

Animals↗