Organelle Markers Research Articles

Responses of Nontransformed Human Hepatocytes to Conditional Expression of Full-Length Hepatitis C Virus Open Reading Frame

Hepatitis C virus (HCV) is a major cause of chronic hepatitis that can lead to cirrhosis and hepatocellular carcinoma. To study the effects of HCV protein expression on host cells, we established conditional expression of the full-length open reading frame (ORF) of an infectious cDNA clone of HCV (genotype 1a, H77 strain) in the nontransformed human hepatocyte line cell HH4 using the ecdysone receptor regulatory system. Treatment with the ecdysone analog ponasterone-A induced tightly regulated and dose-dependent full-length HCV ORF expression and properly processed HCV proteins. HCV Core, NS3, and NS5A colocalized in perinuclear regions and associated with the early endosomal protein EEA1. HCV ORF expression caused marked growth inhibition, increased intracellular reactive oxygen species, up-regulation of glutamate-l-cysteine ligase activity, increased glutathione level, and activation of nuclear factor kappaB. Although it was not directly cytotoxic, HCV ORF expression sensitized HH4 cells to Fas at certain concentrations but not to tumor necrosis factor-related apoptosis-inducing ligand. HCV ORF expression in HH4 cells up-regulated genes involved in innate immune response/inflammation and oxidative stress responses and down-regulated cell growth-related genes. Expression of HCV ORF in host cells may contribute to HCV pathogenesis by producing oxidative stress and increasing the expression of genes related to the innate immune response and inflammation.

Expression of G Protein-Coupled Receptor-30, a G Protein-Coupled Membrane Estrogen Receptor, in Oxytocin Neurons of the Rat Paraventricular and Supraoptic Nuclei

The regulatory actions of estrogens on magnocellular oxytocin (OT) neurons of the paraventricular and supraoptic nuclei are well documented. Although the expression and distribution of nuclear estrogen receptor-beta, but not estrogen receptor-alpha, in the OT neuron has been described, the nuclear receptors may not explain all aspects of estrogen function in the hypothalamic OT neuron. Recently a G protein-coupled receptor (GPR) for estrogens, GPR30, has been identified as a membrane-localized estrogen receptor in several cancer cell lines. In this study, we therefore investigated the expression and localization of GPR30 in magnocellular OT neurons to understand the mode of rapid estrogen actions within these neurons. Here we show that, in the paraventricular nucleus and supraoptic nucleus, GPR30 is expressed in magnocellular OT neurons at both mRNA and protein levels but is not expressed in vasopressin neurons. Specific markers for intracellular organelles and immunoelectron microscopy revealed that GPR30 was localized mainly in the Golgi apparatus of the neurons but could not be detected at the cell surface. In addition, the expression of GPR30 is also detected in the neurohypophysis. These results suggest that GPR30 may serve primarily as a nongenomic transducer of estrogen actions in the hypothalamo-neurohypophyseal system.

Differential Subcellular Localization of RIC-3 Isoforms and Their Role in Determining 5-HT3 Receptor Composition

RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT(3) receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse "slicks" and bright "halo" structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiled-coil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition.

Three-Dimensional Structure of the Human Cytomegalovirus Cytoplasmic Virion Assembly Complex Includes a Reoriented Secretory Apparatus

Human cytomegalovirus (HCMV) induces profound changes in infected cell morphology, including a large cytoplasmic inclusion that corresponds to the virion assembly complex (AC). In electron micrographs, the AC is a highly vacuolated part of the cytoplasm. Markers of cellular secretory organelles have been visualized at the outer edge of the AC, and we recently showed that a marker for early endosomes (i.e., early endosome antigen 1) localizes to the center of the AC. Here, we examined the relationship between the AC and components of the secretory apparatus, studied temporal aspects of the dramatic infection-induced cytoplasmic remodeling, examined the three-dimensional structure of the AC, and considered the implications of our observations for models of HCMV virion maturation and egress. We made three major observations. First, in addition to being relocated, the expression levels of some organelle markers change markedly during the period while the AC is developing. Second, based on three-dimensional reconstructions from z-series confocal microscopic images, the observed concentric rings of vesicles derived from the several compartments (Golgi bodies, the trans-Golgi network [TGN], and early endosomes) are arranged as nested cylinders of organelle-specific vesicles. Third, the membrane protein biosynthetic and exocytic pathways from the endoplasmic reticulum to the Golgi bodies, TGN, and early endosomes are in an unusual arrangement that nonetheless allows for a conventional order of biosynthesis and transport. Our model of AC structure suggests a mechanism by which the virus can regulate the order of tegument assembly.

A multicolored set of in vivo organelle markers for co‐localization studies in Arabidopsis and other plants

Genome sequencing has resulted in the identification of a large number of uncharacterized genes with unknown functions. It is widely recognized that determination of the intracellular localization of the encoded proteins may aid in identifying their functions. To facilitate these localization experiments, we have generated a series of fluorescent organelle markers based on well-established targeting sequences that can be used for co-localization studies. In particular, this organelle marker set contains indicators for the endoplasmic reticulum, the Golgi apparatus, the tonoplast, peroxisomes, mitochondria, plastids and the plasma membrane. All markers were generated with four different fluorescent proteins (FP) (green, cyan, yellow or red FPs) in two different binary plasmids for kanamycin or glufosinate selection, respectively, to allow for flexible combinations. The labeled organelles displayed characteristic morphologies consistent with previous descriptions that could be used for their positive identification. Determination of the intracellular distribution of three previously uncharacterized proteins demonstrated the usefulness of the markers in testing predicted subcellular localizations. This organelle marker set should be a valuable resource for the plant community for such co-localization studies. In addition, the Arabidopsis organelle marker lines can also be employed in plant cell biology teaching labs to demonstrate the distribution and dynamics of these organelles.

Three-dimensional structure of yellow fluorescent protein zYFP538 from Zoanthus sp. at the resolution 1.8 Å

The three-dimensional structure of yellow fluorescent proteins zYFP538 (zFP538) from the button polyp Zoanthus sp. was determined at a resolution of 1.8 angstrom by X-ray analysis. The monomer of zYFP538 adopts a structure characteristic of the green fluorescent protein (GFP) family, a beta-barrel formed from 11 antiparallel beta segments and one internal alpha helix with a chromophore embedded into it. Like the TurboGFP, the beta-barrel of zYFP538 contains a water-filled pore leading to the chromophore Tyr67 residue, which presumably provides access of molecular oxygen necessary for the maturation process. The post-translational modification of the chromophore-forming triad Lys66-Tyr67-Gly68 results in a tricyclic structure consisting of a five-membered imidazolinone ring, a phenol ring of the Tyr67 residue, and an additional six-membered tetrahydropyridine ring. The chromophore formation is completed by cleavage of the protein backbone at the Calpha-N bond of Lys66. It was suggested that the energy conflict between the buried positive charge of the intact Lys66 side chain in the hydrophobic pocket formed by the Ile44, Leu46, Phe65, Leu204 and Leu219 side chains is the most probable trigger that induces the transformation of the bicyclic green form to the tricyclic yellow form. A stereochemical analysis of the contacting surfaces at the intratetramer interfaces helped reveal a group of conserved key residues responsible for the oligomerization. Along with others, these residues should be taken into account in designing monomeric forms suitable for practical application as markers of proteins and cell organelles.

Subcellular Localization and Physiological Significance of Intracellular Mannan-binding Protein

Mannan-binding protein (MBP) is a C-type mammalian lectin specific for mannose and N-acetylglucosamine. MBP is mainly synthesized in the liver and occurs naturally in two forms, serum MBP (S-MBP) and intracellular MBP (I-MBP). S-MBP activates complement in association with MBP-associated serine proteases via the lectin pathway. Despite our previous study (Mori, K., Kawasaki, T., and Yamashina, I. (1984) Arch. Biochem. Biophys. 232, 223-233), the subcellular localization of I-MBP and its functional implication have not been clarified yet. Here, as an extension of our previous studies, we have demonstrated that the expression of human MBP cDNA reproduces native MBP differentiation of S-MBP and I-MBP in human hepatoma cells. I-MBP shows distinct accumulation in cytoplasmic granules, and is predominantly localized in the endoplasmic reticulum (ER) and involved in COPII vesicle-mediated ER-to-Golgi transport. However, the subcellular localization of either a mutant (C236S/C244S) I-MBP, which lacks carbohydrate-binding activity, or the wild-type I-MBP in tunicamycin-treated cells shows an equally diffuse cytoplasmic distribution, suggesting that the unique accumulation of I-MBP in the ER and COPII vesicles is mediated by an N-glycan-lectin interaction. Furthermore, the binding of I-MBP with glycoprotein intermediates occurs in the ER, which is carbohydrate- and pH-dependent, and is affected by glucose-trimmed high-mannose-type oligosaccharides. These results strongly indicate that I-MBP may function as a cargo transport lectin facilitating ER-to-Golgi traffic in glycoprotein quality control.

The Role of the Calcium Sensing Receptor in Regulating Intracellular Calcium Handling in Human Epidermal Keratinocytes

Calcium is critical for controlling the balance of proliferation and differentiation in epidermal keratinocytes. We previously reported that the calcium sensing receptor (CaR) is required for mediating Ca2+ signaling and extracellular Ca2+ (Ca2+(o))-induced differentiation. In this study, we investigated the mechanism by which CaR regulates intracellular Ca2+ (Ca2+(i)) and its role in differentiation. Membrane fractionation, fluorescence immunolocalization, and co-immunoprecipitation studies were performed to assess potential interactions between CaR and other regulators of Ca2+ stores and channels. We found that the glycosylated form of CaR forms a complex with phospholipase C gamma1, IP3 receptor (IP3R), and the Golgi Ca2+-ATPase, secretory pathway Ca2+-ATPase 1, in the trans-Golgi. Inactivation of the endogenous CaR gene by adenoviral expression of a CaR antisense cDNA inhibited Ca2+(i) response to Ca2+(o), decreased Ca2+(i) stores, decreased Ca2+(o)-induced differentiation, but augmented store-operated channel activity and Ca2+ uptake by intracellular organelles. Our results indicate that CaR regulates keratinocyte differentiation in part by modulating Ca2+(i) stores via interactions with Ca2+ pumps and channels that regulate those stores.

Enigmatic Brefeldin A

The fungal macrocyclic lactone brefeldin A (BFA) has been a useful tool in studying protein trafficking in the secretory and endocytic pathways in plant cells. The development of various GFP-tagged organelle markers expressed in transgenic plant cells has allowed dynamic study of organelles in response to BFA in living cells. Several organelles including the endoplasmic reticulum (ER), the Golgi apparatus and endosomal compartment have been shown to have visible morphological changes in response to BFA treatment, resulting in the formation of BFA-induced aggregated compartments or ER-Golgi hybrids in various plant cells. Using transgenic tobacco BY-2 cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi apparatus or prevacuolar compartment (PVC), we have recently demonstrated that Golgi and PVC organelles have different sensitivity to BFA, where BFA at recoverable high concentrations (50 to 100 μg/ml) also induced PVC or multivesicular body (MVB) to form aggregates in plant cells. We have thus extended the BFA action to plant PVCs/MVBs, which will serve as a useful tool for studying PVC-mediated protein sorting and PVC biogenesis.

Can Population Genetic Structure Be Predicted from Life‐History Traits?

Population genetic structure is a key parameter in evolutionary biology. Earlier comparative studies have shown that genetic structure depends on species ecological attributes and life-history traits, but species phylogenetic relatedness had not been accounted for. Here we reevaluate the relationships between genetic structure and species traits in seed plants. Each species is characterized by a set of life-history and ecological features as well as by its geographic range size, its heterozygote deficit, and its genetic structure at nuclear and organelle markers to distinguish between pollen- and seed-mediated gene flow. We use both a conventional regression approach and a method that controls for phylogenetic relationships. Once phylogenetic conservatism and covariation among traits are taken into account, genetic structure is shown to be related with only a few synthetic traits, such as mating system for nuclear markers and seed dispersal mode or geographic range size for organelle markers. Along with other studies on invasiveness or rarity, our work illustrates the fact that predicting the fate of species across a broad taxonomic assemblage on the basis of simple traits is rarely possible, a testimony of the highly contingent nature of evolution.

Photodynamic therapy of malignant glioma with hypericin: Comprehensive in vitro study in human glioblastoma cell lines

The poor prognosis of patients suffering from malignant glioma requires further efforts. Photodynamic therapy (PDT) might be a therapeutic option to increase surgical radicality. Hypericin (HY) exhibit high phototoxicity to malignant cells and accumulates to a higher extent in glioblastoma cells as compared to neurons. Therefore, the impact of various experimental parameters on cytotoxicity, intracellular accumulation and phototoxicity of HY was quantitatively assessed in the three human glioblastoma cell lines U373 MG, LN229 and T98G. Additionally, intracellular location of HY was studied with fluorescence microscopic techniques. For all three cell lines, no cytotoxicity was found for incubation concentrations up to 5 microM. For short-time incubation (2 h), maximum HY fluorescence was achieved at an incubation concentration of about 5 microM. However, uptake kinetics of HY was dependent on its incubation concentration. Moreover, increase in HY fluorescence was negligible at 4 degrees C, which strongly indicates that the compound is taken up by an energy-dependent process. HY exhibited high phototoxicity (at 595 nm) in all three cell lines with ID50-values ranging from 0.15 J/cm(2) to 0.22 J/cm(2), but sensitivity decreased in the order U373 MG > LN229 > T98G. However, assessment of phototoxicity at different wavelengths revealed that highest cell inactivation was achieved at 600 nm. Fluorescence microscopy showed that HY fluorescence arose predominantly from the perinuclear region and the nuclear membrane. Fluorescence pattern of HY was significantly different from those observed for organelle markers staining lysosomes or mitochondria. Location of HY in the plasma membrane was proven by total internal reflection fluorescence microscopy. Thus, the present study demonstrates that glioblastoma cells can be effectively inactivated by HY-PDT after short-time incubation and exposure to low light doses. These results obtained in cell culture are encouraging and justify further evaluation HY-PDT for the treatment of malignant glioma in animal experiments.

Overexpression of Rat Long Chain Acyl-CoA Synthetase 1 Alters Fatty Acid Metabolism in Rat Primary Hepatocytes

Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 mum [1-(14)C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, beta-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and beta-oxidation. The increase in [1,2,3-(3)H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [(14)C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [(14)C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.

Identification and Characterization of a Lysosomal Membrane Protein ofDictyostelium discoideumwith Monoclonal Antibodies

Lysosomes are responsible for the degradation of macromolecules derived from the cell exterior by endocytosis, or from within the cell by autophagy. While our knowledge of the biosynthesis and targeting of lysosomal hydrolases is considerable, much less is known about the lysosomal membrane itself. To identify the lysosomal membrane proteins that mediate these functions, we have isolated lysosomes from amebae and injected them into mice to produce monoclonal antibodies (MAbs). We produced nine MAbs against Dictyostelium lysosomes from the batches of fused cells. Among them, three MAbs were specific to lysosomal membrane and gave a strong signal, and thus used in this study. The MAbs specifically reacted with a single protein band of 27 kd and stained a lysosome-like structure by immunofluorescence microscopy. To identify the antigen that the MAbs recognize, we processed differential centrifugation with whole-cell extract of Dictyostelium and traced p27 protein by activity assay of organelle marker enzyme. We showed that p27 is one component of the lysosomal system on the basis of comigration with a lysosomal marker enzyme. We also demonstrated that the 27-kd lysosomal protein is a tightly bound integral membrane protein by using a phase separation method of Triton X-114.

Dynamic Response of Prevacuolar Compartments to Brefeldin A in Plant Cells

Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) in the secretory pathway. Using transgenic tobacco (Nicotiana tabacum) Bright-Yellow-2 (BY-2) cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi or PVCs, we have recently demonstrated that PVCs are mobile multivesicular bodies defined by vacuolar sorting receptor proteins. Here, we demonstrate that Golgi and PVCs have different sensitivity in response to brefeldin A (BFA) treatment in living tobacco BY-2 cells. BFA at low concentrations (5-10 microg mL(-1)) induced YFP-marked Golgi stacks to form both endoplasmic reticulum-Golgi hybrid structures and BFA-induced aggregates, but had little effect on YFP-marked PVCs in transgenic BY-2 cells at both confocal and immunogold electron microscopy levels. However, BFA at high concentrations (50-100 microg mL(-1)) caused both YFP-marked Golgi stacks and PVCs to form aggregates in a dose- and time-dependent manner. Normal Golgi or PVC signals can be recovered upon removal of BFA from the culture media. Confocal immunofluorescence and immunogold electron microscopy studies with specific organelle markers further demonstrate that the PVC aggregates are distinct, but physically associated, with Golgi aggregates in BFA-treated cells and that PVCs might lose their internal vesicle structures at high BFA concentration. In addition, vacuolar sorting receptor-marked PVCs in root-tip cells of tobacco, pea (Pisum sativum), mung bean (Vigna radiata), and Arabidopsis (Arabidopsis thaliana) upon BFA treatment are also induced to form similar aggregates. Thus, we have demonstrated that the effects of BFA are not limited to endoplasmic reticulum and Golgi, but extend to PVC in the endomembrane system, which might provide a quick tool for distinguishing Golgi from PVC for its identification and characterization, as well as a possible new tool in studying PVC-mediated protein traffic in plant cells.

The SNARE protein family of Leishmania major

BackgroundLeishmania major is a protozoan parasite with a highly polarised cell shape that depends upon endocytosis and exocytosis from a single area of the plasma membrane, the flagellar pocket. SNAREs (soluble N-ethylmaleimide-sensitive factor adaptor proteins receptors) are key components of the intracellular vesicle-mediated transports that take place in all eukaryotic cells. They are membrane-bound proteins that facilitate the docking and fusion of vesicles with organelles. The recent availability of the genome sequence of L. major has allowed us to assess the complement of SNAREs in the parasite and to investigate their location in comparison with metazoans.ResultsBioinformatic searches of the L. major genome revealed a total of 27 SNARE domain-containing proteins that could be classified in structural groups by phylogenetic analysis. 25 of these possessed the expected features of functional SNAREs, whereas the other two could represent kinetoplastid-specific proteins that might act as regulators of the SNARE complexes. Other differences of Leishmania SNAREs were the absence of double SNARE domain-containing and of the brevin classes of these proteins. Members of the Qa group of Leishmania SNAREs showed differential expressions profiles in the two main parasite forms whereas their GFP-tagging and in vivo expression revealed localisations in the Golgi, late endosome/lysosome and near the flagellar pocket.ConclusionThe early-branching eukaryote L. major apparently possess a SNARE repertoire that equals in number the one of metazoans such as Drosophila, showing that the machinery for vesicle fusion is well conserved throughout the eukaryotes. However, the analysis revealed the absence of certain types of SNAREs found in metazoans and yeast, while suggesting the presence of original SNAREs as well as others with unusual localisation. This study also presented the intracellular localisation of the L. major SNAREs from the Qa group and reveals that these proteins could be useful as organelle markers in this parasitic protozoon.

African swine fever virus causes microtubule-dependent dispersal of the trans-golgi network and slows delivery of membrane protein to the plasma membrane.

Viral interference with secretory cargo is a common mechanism for pathogen immune evasion. Selective down regulation of critical immune system molecules such as major histocompatibility complex (MHC) proteins enables pathogens to mask themselves from their host. African swine fever virus (ASFV) disrupts the trans-Golgi network (TGN) by altering the localization of TGN46, an organelle marker for the distal secretory pathway. Reorganization of membrane transport components may provide a mechanism whereby ASFV can disrupt the correct secretion and/or cell surface expression of host proteins. In the study reported here, we used the tsO45 temperature-sensitive mutant of the G protein of vesicular stomatitis virus to show that ASFV significantly reduces the rate at which the protein is delivered to the plasma membrane. This is linked to a general reorganization of the secretory pathway during infection and a specific, microtubule-dependent disruption of structural components of the TGN. Golgin p230 and TGN46 are separated into distinct vesicles, whereupon TGN46 is depleted. These data suggest that disruption of the TGN by ASFV can slow membrane traffic during viral infection. This may be functionally important because infection of macrophages with virulent isolates of ASFV increased the expression of MHC class I genes, but there was no parallel increase in MHC class I molecule delivery to the plasma membrane.

Subcellular Redistribution of the Serotonin Transporter by Secretory Carrier Membrane Protein 2

The serotonin transporter (SERT) belongs to the SLC6 family of sodium- and chloride-dependent neurotransmitter transporters responsible for uptake of amino acids and biogenic amines from extracellular spaces. Their activities and subcellular distributions are regulated by various cellular mechanisms, including interactions with other proteins. Using the yeast two-hybrid approach we screened a human brain cDNA library and identified secretory carrier membrane protein 2 (SCAMP2) as a novel SERT-interacting protein. GST-pulldown assays confirmed the physical interaction between SCAMP2 and the N-terminal domain of SERT. In addition, SERT was found to form a complex with SCAMP2 as demonstrated by co-immunoprecipitation from a heterologous expression system and from rat brain homogenate. Co-expression of SERT and SCAMP2 in mammalian cells results in the subcellular redistribution of SERT with a decrease in cell surface SERT and a concomitant reduction in 5-HT uptake activity. Using confocal microscopy we show that in neuronal cells endogenous SERT co-localizes with SCAMP2 in discrete structures also containing the lipid raft marker flotillin-1 and the SNARE protein syntaxin 1A. In contrast, SERT immunoreactivity is clearly segregated from transferrin receptor-containing endosomes. A single amino acid mutation, cysteine-201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, abolished SCAMP2-mediated down-regulation of SERT, although this mutation had no effect on the physical interaction between SERT and SCAMP2. Taken together, our results suggest that SCAMP2 plays an important role in the regulation of the subcellular distribution of SERT.

Fate of E-cadherin in Early RPE Cultures: Transient Accumulation of Truncated Peptides at Nonjunctional Sites

E-cadherin is known to accumulate variably and slowly at junctions of cultured human RPE cells. The intent of this investigation was to determine what limits E-cadherin protein accumulation in RPE cells by analyzing cultures at early postplating intervals when junctions of the dominant cadherin (N-cadherin) are first forming. RPE cell lines hTERT-RPE1 and ARPE-19 and RPE cultures established from human donors were analyzed within 48 hours after plating for E-cadherin gene and protein expression (by RT-PCR and Western blotting, respectively) and for protein distribution (by immunofluorescence and immuno-electron microscopy), including codistribution with markers for organelles. Cell surface localization was analyzed by biotinylation and trypsin cleavage of extracellular cadherin domains. The E-cadherin gene was constitutively expressed by RPE cultures, but the protein did not accumulate substantially in early RPE cultures. Instead small amounts of newly synthesized E-cadherin were detectable only transiently, peaking within a few hours after plating, at which time the protein was in the form of peptides of variable size rather the predicted 120-kDa molecular mass. Immunoreactive E-cadherin peptides did not traffic to the cell surface and localize to junctions. Rather they codistributed with several organelles including the endoplasmic reticulum (ER; but not the Golgi), sites of protein degradation (proteasomes, lysosomes, and autophagosomes) and unusual compartments (centrosomes and apposed to subdomains of the mitochondrial network). The results suggest that in RPE cells posttranscriptional mechanisms involving altered protein processing and rapid turnover exist to limit E-cadherin accumulation. The consequence may be to limit E-cadherin-specific inductive properties in the RPE, a cell type in which N-cadherin is the normal dominant cadherin.

The hereditary spastic paraplegia protein spartin localises to mitochondria

Hereditary spastic paraplegia describes a diverse group of disorders characterized by progressive paraparesis primarily affecting lower limbs. In Troyer syndrome, an autosomal recessive form of hereditary spastic paraplegia, patients have dysarthria, distal amyotrophy, developmental delay and short stature in addition to spastic paraparesis. It is caused by a frameshift mutation (1110delA) in SPG20 leading to premature truncation of spartin, a protein with no known function. The objective of this study was to determine the subcellular localization of spartin and investigate the effect of the 1110delA mutation. We observed cytoplasmic expression of spartin in all transfected cell lines. Using superimposed organelle markers or immunocytochemistry staining, we established that spartin localizes to mitochondria and that this localization is dependent on sequences in the C-terminal region. Mutant spartin containing the 1110delA mutation has lost mitochondrial localization. Immunocytochemistry staining using anti-alpha-tubulin antibody provided evidence for partial co-localization of spartin with microtubules. Analysis of fluorescence resonance energy transfer indicated that sequences in the amino terminal are important in mediating microtubule interaction. This study provides the first evidence of spartin subcellular localization and identifies it as the third mitochondrial protein implicated in hereditary spastic paraplegia. Our results suggest that Troyer syndrome may be due to defective microtubule-mediated trafficking of mitochondria and/or mitochondrial dysfunction.

Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins

BackgroundTrisomy of human chromosome 21 (Chr21) results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins.ResultsWe chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb) to MCM3AP (46.6 Mb), with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized.ConclusionThe cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.