ngLOC: software and web server for predicting protein subcellular localization in prokaryotes and eukaryotes

ABSTRACT: BACKGROUND: Understanding protein subcellular localization is a necessary component toward understanding the overall function of a protein. Numerous computational methods have been published over the past decade, with varying degrees of success. Despite the large number of published methods in this area, only a small fraction of them are available for researchers to use in their own studies. Of those that are available, many are limited by predicting only a small number of major organelles in the cell. Additionally, the majority of methods predict only a single location, even though it is known that a large fraction of the proteins in eukaryotic species shuttle between locations to carry out their function. FINDINGS: We present a software package and a web server for predicting subcellular localization of protein sequences based on the ngLOC method. ngLOC is an n-gram-based Bayesian classifier that predicts subcellular localization of proteins both in prokaryotes and eukaryotes. The overall prediction accuracy varies from 89.8% to 91.4% across species. This program can predict 11 distinct locations each in plant and animal species. ngLOC also predicts 4 and 5 distinct locations on gram-positive and gram-negative bacterial datasets, respectively. CONCLUSIONS: ngLOC is a generic method that can be trained by data from a variety of species or classes for predicting protein subcellular localization. The standalone software is freely available for academic use under GNU GPL, and the ngLOC web server is also accessible at http://ngloc.unmc.edu.     

Localized RNAs and their functions

The eukaryotic cell is partitioned by membranes into spatially and functionally discrete subcellular organelles. In addition, the cytoplasm itself is partitioned into discrete subregions that carry out specific functions. Such compartmentation can be achieved by localizing proteins and RNAs to different subcellular regions. This review will focus on localized RNAs, with a particular emphasis on RNA localization mechanisms and on the possible biological functions of localization of these RNAs. In recent years, an increasing number of localized RNAs have been identified in a variety of cell types among many animal species. Emphasis here will be on localized RNAs in the most intensively studied systems – Drosophila and Xenopus eggs and early embryos.     

Subcellular Localization of Catalase in Sea Urchin (Tetrapigus niger) Gametes: Implications for Peroxisome Biogenesis

Peroxisomes are essential subcellular organelles that appear to be derived from pre-existing organelles. To test the presence of peroxisomes in sea urchin (Tetrapigus niger) sperm and eggs, we performed biochemical and morphological experiments to evaluate the subcellular distribution of catalase as the typical peroxisomal marker. In sea urchin sperm, we found that catalase is localized in the cell cytosol. In contrast, sea urchin eggs contain sedimentable catalase, presumably contained in peroxisome-like structures detected by immunomicroscopy and by cytochemistry. Our results show, for the first time, evidence for the presence of peroxisome-like structures in sea urchin eggs and provide evidence for the peroxisome biogenesis hypothesis by division of pre-existing organelles.     

A Systematic Cell‐Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins

Peroxisomes are membrane‐bound organelles found in almost all eukaryotic cells. They perform specialized biochemical functions that vary with organism, tissue or cell type. Mutations in human genes required for the assembly of peroxisomes result in a spectrum of diseases called the peroxisome biogenesis disorders. A previous sequence‐based comparison of the predicted proteome of Drosophila melanogaster (the fruit fly) to human proteins identified 82 potential homologues of proteins involved in peroxisomal biogenesis, homeostasis or metabolism. However, the subcellular localization of these proteins relative to the peroxisome was not determined. Accordingly, we tested systematically the localization and selected functions of epitope‐tagged proteins in Drosophila Schneider 2 cells to determine the subcellular localization of 82 potential Drosophila peroxisomal protein homologues. Excluding the Pex proteins, 34 proteins localized primarily to the peroxisome, 8 showed dual localization to the peroxisome and other structures, and 26 localized exclusively to organelles other than the peroxisome. Drosophila is a well‐developed laboratory animal often used for discovery of gene pathways, including those linked to human disease. Our work establishes a basic understanding of peroxisome protein localization in Drosophila. This will facilitate use of Drosophila as a genetically tractable, multicellular model system for studying key aspects of human peroxisome disease.      

Vacuolar-type H<Superscript>+</Superscript>-ATPase with the <Emphasis Type="Italic">a</Emphasis>3 isoform is the proton pump on premature melanosomes

The melanosome, an organelle specialized for melanin synthesis, is one of the lysosome-related organelles. Its lumen is reported to be acidified by vacuolar-type H⁺-ATPase (V-ATPase). Mammalian V-ATPase exhibits structural diversity in its subunit isoforms; with regard to membrane intrinsic subunit a, four isoforms (a1–a4) have been found to be localized to distinct subcellular compartments. In this study, we have shown that the a3 isoform is co-localized with a melanosome marker protein, Pmel17, in mouse melanocytes. Acidotropic probes (LysoSensor and DAMP) accumulate in non-pigmented Pmel17-positive melanosomes, and DAMP accumulation is sensitive to bafilomycin A1, a specific inhibitor of V-ATPase. However, none of the subunit a isoforms is associated with highly pigmented mature melanosomes, in which the acidotropic probes are also not accumulated. oc/oc mice, which have a null mutation at the a3 locus, show no obvious defects in melanogenesis. In the mutant melanocytes, the expression of the a2 isoform is modestly elevated, and a considerable fraction of this isoform is localized to premature melanosomes. These observations suggest that the V-ATPase keeps the lumen of premature melanosomes acidic, whereas melanosomal acidification is less significant in mature melanosomes. Ge-Hong Sun-Wada and Yoh Wada contributed equally to this study. This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Hayashi and Noda Foundations.     

Expression of the bacterial heavy metal transporter MerC fused with a plant SNARE,SYP121, in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> increases cadmium accumulation and tolerance

The bacterial merC gene from the Tn21-encoded mer operon is a potential molecular tool for improving the efficiency of metal phytoremediation. Arabidopsis SNARE molecules, including SYP111, SYP121, and AtVAM3 (SYP22), were attached to the C-terminus of MerC to target the protein to various organelles. The subcellular localization of transiently expressed GFP-fused MerC-SYP111, MerC-SYP121, and MerC-AtVAM3 was examined in Arabidopsis suspension-cultured cells. We found that GFP-MerC-SYP111 and GFP-MerC-SYP121 localized to the plasma membrane, whereas GFP-AtVAM3 localized to the vacuolar membranes. These results demonstrate that SYP111/SYP121 and AtVAM3 target foreign molecules to the plasma membrane and vacuolar membrane, respectively. To enhance the efficiency and potential of plants to sequester and accumulate cadmium from contaminated sites, transgenic Arabidopsis plants expressing MerC, MerC-SYP111, MerC-SYP121, or MerC-AtVAM3 were generated. The transgenic plants that expressed MerC, MerC-SYP121, or MerC-AtVAM3 appeared to be normal, whereas the transgenic that expressed MerC-SYP111 exhibited severe growth defects. The transgenic plants expressing merC-SYP121 were more resistant to cadmium than the wild type and accumulated significantly more cadmium. Thus, the expression of MerC-SYP121 in the plant plasma membrane may provide an ecologically compatible approach for the phytoremediation of cadmium pollution.     

Cold‐induced organelle relocation in the liverwort Marchantia polymorpha L.

Organelles change their subcellular positions in response to various environmental conditions. Recently, we reported that cold treatments alter the intracellular position of chloroplasts and nuclei (cold positioning) in the fern Adiantum capillus‐veneris; chloroplasts and nuclei localized to the periclinal cell wall relocated to anticlinal cell wall after cold treatments. To further understand organelle positioning under cold conditions, we studied cold‐induced organelle relocation in the liverwort Marchantia polymorpha L. When sporelings and gemmmalings were treated under low temperature (5 °C), chloroplast cold positioning response was successfully induced both in the sporelings and the gemmmalings of M. polymorpha. Using a genetic transformation, nuclei, mitochondria or peroxisomes were visualized with a fluorescent protein, and the transgenic gemmmalings were incubated under the cold condition. Nuclei and peroxisomes, but not mitochondria, clearly relocated from the periclinal cell wall to the anticlinal cell wall after cold treatments. Our findings suggest that several organelles concurrently change their positions in the liverwort cell to cope with cold temperature.     

Variable subcellular localization of glycosphingolipids

Although most glycosphingolipids (GSLs) are thought to be locatedin the outer leaflet of the plasma membrane, recent evidenceindicates that GSLs are also associated with intracellular organelles.We now report that the subcellular localization of GSLs variesdepending on the GSL structure and cell type. GSL localizationwas determined by indirect immunofluorescence microscopy offixed permeabilized cells. A single GSL exhibited variable subcellularlocalization in different cells. For example, antibody to GalCeris localized primarily to the plasma membrane of HaCaT II-3keratinocytes, but to intracellular organelies in other epithelialcells. GalCer is localized to small vesicles and tubulovesicularstructures in MDCK cells, and to the surface of phase-denselipid droplets in HepG2 hepatoma cells. Furthermore, withina single cell type, individual GSLs were found to exhibit differentpatterns of subcellular localization. In HepG2 cells, LacCerwas associated with small vesicles, which differed from thephase-dense vesicles stained by anti-GalCer, and Gb4Cer wasassociated with the intermediate filaments of the cytoskeleton.Both anti-GalCer and monoclonal antibody A2B5, which binds polysialogangliosides,localized to mitochondria. The distinct subcellular localizationpatterns of GSLs raise interesting questions about their functionsin different organelles. Together with published data on theenrichment of GSLs in specific organelles and in apical plasmamembrane, these findings indicate the existence of specificsorting mechanisms that regulate the intracellular transportand localization of GSLs. cytoskeleton glycosphingolipid intracellular organelles mitochondria subcellular localization     

Functional analysis and subcellular localization of two geranylgeranyl diphosphate synthases from <Emphasis Type="Italic">Penicillium paxilli</Emphasis>

The filamentous fungus Penicillium paxilli contains two distinct geranylgeranyl diphosphate (GGPP) synthases, GgsA and GgsB (PaxG). PaxG and its homologues in Neotyphodium lolii and Fusarium fujikuroi are associated with diterpene secondary metabolite gene clusters. The genomes of other filamentous fungi including Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae and Fusarium graminearum also contain two or more copies of GGPP synthase genes, although the diterpene metabolite capability of these fungi is not known. The objective of this study was to understand the biological significance of the presence of two copies of GGPP synthases in P. paxilli by investigating their subcellular localization. Using a carotenoid complementation assay and gene deletion analysis, we show that P. paxilli GgsA and PaxG have GGPP synthase activities and that paxG is required for paxilline biosynthesis, respectively. In the ΔpaxG mutant background ggsA was unable to complement paxilline biosynthesis. A GgsA-EGFP fusion protein was localized to punctuate organelles and the EGFP-GRV fusion protein, containing the C-terminus tripeptide GRV of PaxG, was localized to peroxisomes. A truncated PaxG mutant lacking the C-terminus tripeptide GRV was unable to complement a ΔpaxG mutant demonstrating that the tripeptide is functionally important for paxilline biosynthesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Subcellular localization of aflatoxin biosynthetic enzymes Nor-1, Ver-1, and OmtA in time-dependent fractionated colonies of<Emphasis Type="Italic"> Aspergillus parasiticus</Emphasis>

The biosynthesis of aflatoxin in Aspergillus parasiticus is a complex process that involves the activities of at least 18 pathway enzymes. The distribution of these enzymes within fungal colonies and fungal cells is not clearly understood. The objective of this study was to investigate the distribution and subcellular location of Nor-1, Ver-1, and OmtA, which represent early, middle, and late enzymatic activities, respectively, in the aflatoxin biosynthetic pathway. The distribution of these three enzymes within A. parasiticus SU-1 was analyzed in time-fractionated, 72-h fungal colonies (fraction 1, 48–72 h; fraction 2, 24–48 h; fraction 3, 0–24 h). Western blot analysis and immunofluorescence microscopy demonstrated the highest abundance of Nor-1, Ver-1, and OmtA in colony fraction 2. Fungal tissues in this fraction were analyzed by immunoelectron microscopy. Nor-1 and Ver-1 were primarily localized to the cytoplasm, suggesting that they are cytosolic enzymes. OmtA was also detected in the cytoplasm. However, in cells located near the basal (substrate) surface of the colony, OmtA was predominantly detected in organelles tentatively identified as vacuoles. The role of this organelle in toxin biosynthesis is unclear. The relative distribution of OmtA to the cytoplasm or to vacuole-like organelles may depend on the age and/or physiological condition of the fungal cells.     

Localization of replication forks in wild-type and <Emphasis Type="Italic">mukB</Emphasis> mutant cells of <Emphasis Type="Italic">Escherichia coli</Emphasis>

To examine the subcellular localization of the replication machinery in Escherichia coli, we have developed an immunofluorescence method that allows us to determine the subcellular location of newly synthesized DNA pulse-labeled with 5-bromo-2′-deoxyuridine (BrdU). Using this technique, we have analyzed growing cells. In wild-type cells that showed a single BrdU fluorescence signal, the focus was located in the middle of the cell; in cells with two signals, the foci were localized at positions equivalent to 1/4 and 3/4 of the cell length. The formation of BrdU foci was dependent upon ongoing chromosomal replication. A mutant lacking MukB, which is required for proper partitioning of sister chromosomes, failed to maintain the ordered localization of BrdU foci: (1) a single BrdU focus tended to be localized at a pole-proximal region of the nucleoid, and (2) a focus was often found to consist of two replicating chromosomes. Thus, the positioning of replication forks is affected by the disruption of the mukB gene.     

Mapping the subcellular protein distribution in three human cell lines

The subcellular locations of proteins are closely related to their function and constitute an essential aspect for understanding the complex machinery of living cells. A systematic effort has been initiated to map the protein distribution in three functionally different cell lines with the aim to provide a subcellular localization index for at least one representative protein from all human protein-encoding genes. Here, we present the results of more than 3500 proteins mapped to 16 subcellular compartments. The results indicate a ubiquitous protein expression with a majority of the proteins found in all three cell lines and a large portion localized to two or more compartments. The inter-relationships between the subcellular compartments are visualized in a protein-compartment network based on all detected proteins. Hierarchical clustering was performed to determine how closely related the organelles are in terms of protein constituents and compare the proteins detected in each cell type. Our results show distinct organelle proteomes, well conserved across the cell types, and demonstrate that biochemically similar organelles are grouped together.     

The N‐Terminal Sequences of Four Major Hydrogenosomal Proteins Are Not Essential for Import into Hydrogenosomes of Trichomonas vaginalis

The human pathogen Trichomonas vaginalis harbors hydrogenosomes, organelles of mitochondrial origin that generate ATP through hydrogen‐producing fermentations. They contain neither genome nor translation machinery, but approximately 500 proteins that are imported from the cytosol. In contrast to well‐studied organelles like Saccharomyces mitochondria, very little is known about how proteins are transported across the two membranes enclosing the hydrogenosomal matrix. Recent studies indicate that—in addition to N‐terminal transit peptides—internal targeting signals might be more common in hydrogenosomes than in mitochondria. To further characterize the extent to which N‐terminal and internal motifs mediate hydrogenosomal protein targeting, we transfected Trichomonas with 24 hemagglutinin (HA) tag fusion constructs, encompassing 13 different hydrogenosomal and cytosolic proteins of the parasite. Hydrogenosomal targeting of these proteins was analyzed by subcellular fractionation and independently by immunofluorescent localization. The investigated proteins include some of the most abundant hydrogenosomal proteins, such as pyruvate ferredoxin oxidoreductase (PFO), which possesses an amino‐terminal targeting signal that is processed on import into hydrogenosomes, but is shown here not to be required for import into hydrogenosomes. Our results demonstrate that the deletion of N‐terminal signals of hydrogenosomal precursors generally has little, if any, influence upon import into hydrogenosomes. Although the necessary and sufficient signals for hydrogenosomal import recognition appear complex, targeting to the organelle is still highly specific, as demonstrated by the finding that six HA‐tagged glycolytic enzymes, highly expressed under the same promoter as other constructs studied here, localized exclusively to the cytosol and did not associate with hydrogenosomes.     

The Ubiquitous Distribution of Late Embryogenesis Abundant Proteins across Cell Compartments in Arabidopsis Offers Tailored Protection against Abiotic Stress

Late embryogenesis abundant (LEA) proteins are hydrophilic, mostly intrinsically disordered proteins, which play major roles in desiccation tolerance. In Arabidopsis thaliana, 51 genes encoding LEA proteins clustered into nine families have been inventoried. To increase our understanding of the yet enigmatic functions of these gene families, we report the subcellular location of each protein. Experimental data highlight the limits of in silico predictions for analysis of subcellular localization. Thirty-six LEA proteins localized to the cytosol, with most being able to diffuse into the nucleus. Three proteins were exclusively localized in plastids or mitochondria, while two others were found dually targeted to these organelles. Targeting cleavage sites could be determined for five of these proteins. Three proteins were found to be endoplasmic reticulum (ER) residents, two were vacuolar, and two were secreted. A single protein was identified in pexophagosomes. While most LEA protein families have a unique subcellular localization, members of the LEA_4 family are widely distributed (cytosol, mitochondria, plastid, ER, and pexophagosome) but share the presence of the class A α-helix motif. They are thus expected to establish interactions with various cellular membranes under stress conditions. The broad subcellular distribution of LEA proteins highlights the requirement for each cellular compartment to be provided with protective mechanisms to cope with desiccation or cold stress.     

Distribution of carbonic anhydrase, K+-ATPase and K+-phosphatase in subcellular fractions of gastric mucosa]

The distribution of carbonic anhydrase, K+-ATPase and K+-phosphatase in the subcellular fractions of gastric mucosa was studied. It was found that 90% of carbonic anhydrase are localized in the hyaloplasm, whereas K+-ATPase and K+-phosphatase are predominantly localized in the microsomal fraction. Subfractionation of the microsomal fraction in a sucrose density gradient showed that the membrane-bound carbonic anhydrase (5% of total content) and K+-ATPase are bound to various cell organelles. It is concluded that carbonic anhydrase functions as an intracellular pH-stat and is not directly involved in proton generation by the cell.     

Peroxisomal proteins in rat gametes

Peroxisomes are essential subcellular organelles, which appear to be derived from pre-existing organelles. This biogenetic mechanism assumes the presence of peroxisomes in either or both mammalian gametes (sperms and/or oocytes). In order to test the presence and subcellular localization of peroxisomal proteins in rat sperms and oocytes, the authors carried out fractionation and immunofluorescence experiments. The results showed that rat oocytes contain peroxisomelike structures, which were detected by indirect immunofluorescence, using an antisera against total peroxisomal proteins. In contrast, such structures were not detected in rat sperms, which appear to contain catalase localized in the cell cytosol. The results reported herein show evidence for the first time of the presence of peroxisome-like structures in mammalian oocytes, and provide evidence for the peroxisome biogenesis hypothesis, by division of pre-existing organelles.     

Both the hydrophobicity and a positively charged region flanking the C-terminal region of the transmembrane domain of signal-anchored proteins play critical roles in determining their targeting specificity to the endoplasmic reticulum or endosymbiotic organelles in Arabidopsis cells

Proteins localized to various cellular and subcellular membranes play pivotal roles in numerous cellular activities. Accordingly, in eukaryotic cells, the biogenesis of organellar proteins is an essential process requiring their correct localization among various cellular and subcellular membranes. Localization of these proteins is determined by either cotranslational or posttranslational mechanisms, depending on the final destination. However, it is not fully understood how the targeting specificity of membrane proteins is determined in plant cells. Here, we investigate the mechanism by which signal-anchored (SA) proteins are differentially targeted to the endoplasmic reticulum (ER) or endosymbiotic organelles using in vivo targeting, subcellular fractionation, and bioinformatics approaches. For targeting SA proteins to endosymbiotic organelles, the C-terminal positively charged region (CPR) flanking the transmembrane domain (TMD) is necessary but not sufficient. The hydrophobicity of the TMD in CPR-containing proteins also plays a critical role in determining targeting specificity; TMDs with a hydrophobicity value >0.4 on the Wimley and White scale are targeted primarily to the ER, whereas TMDs with lower values are targeted to endosymbiotic organelles. Based on these data, we propose that the CPR and the hydrophobicity of the TMD play a critical role in determining the targeting specificity between the ER and endosymbiotic organelles.     

砷在药用植物三七根部组织及其亚细胞分布特征

采用同步辐射X射线荧光分析(SRXRF)与亚细胞分布研究相结合的方法,从细胞组织微区及亚细胞分布层面首次揭示了药用植物三七(Panax notoginseng)受砷(As)的毒害作用。研究结果表明,三七根部的As元素多集中在表皮组织中,且有向维管束运转的趋势;细胞液是As主要富集的亚细胞组分,用20 mg·L~(-1)As处理的三七细胞液中As含量约为不加As的200倍;分析三七主根亚细胞组分的As含量与营养液As浓度的曲线拟合方程,确定了营养液As浓度直接影响细胞液组分的As含量;各组分所占比例从大到小表现为:细胞液细胞壁细胞质,20 mg·L~(-1)As处理的细胞液中砷含量约占三组分总量的65.78%,达到最高比例,细胞壁和细胞器中则始终维持较低的砷浓度。     

Quantitative assessment of cell damagein situ: Electron microprobe X-ray analysis of model organisms treated with noxious species

The existing set of methods for assessing toxicity of noxas, based on experiments with whole animals (subclinical toxicity, toxicokinetics, carcinogenity, teratogenity, neurotoxicologyetc.) does not provide much information about cellular and subcellular effects such compounds may exert. We suggest to complement the current methodology by combining a traditional morphological observation in an electron microscope with a spectroscopic method of electron microprobe X-ray analysis (or X-ray microanalysis). The latter makes it possible to measure concentrations of chemical elements in invidividual cells and organelles and effects of noxas can thus be assessed (i) at subcellular level, (ii) directlyin situ and (iii) quantitatively. Concentrations of biologically important elements such as phosphorus, sulfur or zinc were measured in individual organelles in both intact and noxa-treated tissues, thus offering a possibility of comparing the effects of various noxious species at subcellular level (with the noxa previously applied to whole tissue or animal). The suggested correlation of analytical and morphological information may also provide new insights into cellular targeting of noxas (and potentially also drugs) as some organelles appear to be much more susceptible to damage than others. Presented at the 4th Mini-Symposium on Biosorption and Microbial Degradation, Prague, Czech Republic, November 26–29, 1996.     

Isolation of cytoplasmic enzymes from pollen

The cytoplasmic isozyme of many cytoplasmic-organelle isozyme pairs, as well as other cytoplasmic enzymes in plants, can be readily obtained from pollen by soaking it in an appropriate buffer for 4 hours. Enzymes localized in subcellular organelles appear not to be released during the soaking period, although they are released if the pollen is crushed. The technique is a useful initial step in studies of subcellular localization of enzymes or for obtaining small quantities of cytoplasmic enzymes free of organellar contaminants.