Subcellular localization of acidic and basic PR proteins in tobacco mosaic virus-infected tobacco

Summary Infection of Samsum NN tobacco with tobacco mosaic virus (TMV) results in the induction of the synthesis of acidic and basic isoforms of many pathogenesis-related (PR) proteins. By immunogold-electronmicroscopy we have shown that PR proteins accumulate mainly in cells around the necrotic spots of TMV-induced lesions. The acidic chitinases, -(1,3)-glucanases and thaumatin-like proteins were found to accumulate in extracellular pocket-like vesicles while the basic chitinases were found in electron dense inclusion bodies in the vacuoles. These structures were not detectable in PR-containing leaves devoid of virus nor in healthy plants.     

Subcellular localization of hepatitis C viral proteins in mammalian cells

Summary.  We determined the subcellular localization of hepatitis C viral (HCV) proteins as a first step towards the understanding of the functions of these proteins in the mammalian cell (CHO-K1). We used fluorescence emitted from green fluorescent protein (GFP)-fused to the viral proteins to determine the subcellular localization of the viral proteins. We found that most of the viral proteins were excluded from the nucleus. Core exhibited a globular pattern near the nucleus. NS2 was concentrated in the perinuclear space. NS4A accumulated in the ER and the Golgi regions. NS3 was detected in the nucleus as well as the cytoplasm, when it was expressed by itself. However, NS3 became restricted to the cytoplasm, when it was produced together with NS4A. NS4B showed a spot-like pattern throughout the cytoplasm. NS5A and NS5B were distributed throughout the cytoplasm in a mesh-like pattern. These results can provide a basis for further investigations into the functions of the HCV proteins. Accepted August 29, 1998 Received May 25, 1998     

Subcellular localization of intracellular protein tyrosine phosphatases in T cells

A high protein tyrosine phosphatase (PTPase) activity is required to maintain circulating T lymphocytes in a resting phenotype, and to limit the initiation of T cell activation. We report that 15 of the currently known 24 intracellular PTPases are expressed in T cells, namely HePTP, TCPTP, SHP1, SHP2, PEP, PTP-PEST, PTP-MEG2, PTEN, PTPH1, PTP-MEG1, PTP36, PTP-BAS, LMPTP, PRL-1 and OV-1. Most were found in the cytosol and many were enriched at the plasma membrane. Only TCPTP and PTP-MEG2 had subcellular localizations that essentially excludes them from a direct role in early T cell antigen receptor signaling events. Overexpression of 6 of the PTPases reduced IL-2 gene activation, 3 of them thereby identified as novel candidates for negative regulators of TCR signaling. Our findings expand the repertoire of PTPases that should be considered for a regulatory role in T cell activation.     

Subcellular localization of sphingomyelin revealed by two toxin-based probes in mammalian cells

Sphingomyelin (SM) is an abundant phospholipid in cell membranes. However, owing to the lack of appropriate probes, the subcellular distribution of SM remains unclear. In this study, we examined the localization of SM in COS-1 cells (green monkey kidney cells) by using two SM probes, lysenin and equinatoxin-II (EqtII). Both toxins stained SM in the plasma membrane (PM), and the stains were abolished by sphingomyelin synthase 2 (SMS2) knockdown or sphingomyelinase (SMase) treatment. Simultaneous labeling by the two toxins showed that the PM has heterogeneous SM pools: a SM pool stained by only lysenin, a SM pool stained only by EqtII, and a SM pool stained by both toxins. In permeabilized cells, lysenin exclusively stained late endosomes (LEs) among intracellular organelles, whereas EqtII stained recycling endosomes (REs) in addition to LEs. The intracellular SM stains by EqtII were abolished by sphingomyelin synthase 1 (SMS1) knockdown, but not by SMS2 knockdown. These results indicate that lysenin and EqtII label different SM pools and that SMS2 and SMS1 are responsible for the synthesis of SM in the PM and endomembranes, respectively, in COS-1 cells. The use of the two SM-binding probes may provide more insights into various sphingomyelin-mediated processes in different topological domains.     

Neuraminidase activity of influenza virus-infected cells: localization and properties.

Infection of chick embryo cell (CEC) cultures with influenza virus results in the appearance of neuraminidase activity lost by the cells as a result of cultivation. Neuraminidase activity is associated mainly with the lysomal cell fraction. Different distribution of neuraminidase activity in lysosomes with various densities, different reaction to sodium ethylene diamine tetracetate (EDTA) and differenct optimal pH suggest that at early stages of viral infection the cell enzyme is activated and by the 7th hour of infection viral neuraminidase is synthesized.     

Subcellular localization of the Huntington's disease gene product in cell lines by immunofluorescence and biochemical subcellular fractionation

Huntington's disease is a progressive neurodegenerative disorder, which is caused by expansion of a polymorphic (CAG)n repeat in the coding region of the Huntington's disease gene. The function of huntingtin has not been elucidated so far. Accordingly, detailed subcellular localization studies remain useful. In an immunohistochemical study, we have reported huntingtin to be present in the cytoplasm of cells in the majority of the tissues studied. In addition, we detected a signal in the nucleus of cells in some tissues, including neuronal cells. We have further extended these studies in various mammalian cell lines, using a panel of (affinity-purified) polyclonal huntingtin antibodies in immunofluorescence, confocal laser scanning microscopy and biochemical subcellular fractionation studies. In mouse embryonic fibroblasts, human skin fibroblasts and in mouse neuroblastoma cells huntingtin was present in the cytoplasm. All five antibodies, directed against different parts of huntingtin, also showed a signal in the nucleus. This signal could be competed by the original antigen. The localization of huntingtin in both cytoplasm and nucleus, was confirmed by biochemical subcellular fractionation studies. However, in most other studies, a nuclear location for huntingtin has not been found. Our results suggest, however, that besides its function(s) in the cytoplasm, a nuclear function of huntingtin at some stages of differentiation or in some phases of the cell cycle may not be excluded.      

Subcellular localization and trafficking of polycystins

Polycystin-2 is a member of the transient receptor potential (TRP) family of ion channels that is mutated in autosomal dominant polycystic kidney disease. Although its function as a non-selective cation channel has been demonstrated in several model systems, the precise subcellular localization of polycystin-2 (TRPP2) in tubular epithelial cells has remained controversial. Recent evidence suggests that the subcellular localization of TRPP2 is regulated by multiple protein interactions. This review will summarize our current knowledge about polycystin trafficking and highlight the experimental data that supports a compartment-specific function of cystogenic proteins.     

Subcellular localization of YKL-40 in normal and malignant epithelial cells of the breast

YKL-40 is a new prognostic biomarker in cancer. The biological function is only poorly understood. This study aimed at determining the subcellular localization of YKL-40, using immunogold labeling, in normal epithelial cells and in malignant tumor cells of the breast by immunoelectron microscopy. YKL-40 protein expression was redistributed in carcinoma versus normal glandular tissue of the breast. A reduced expression of YKL-40 in relation to intermediate filaments and desmosomes was found in tumor cells. Changes in YKL-40 expression suggest that the function of YKL-40 in cells of epithelial origin may be related to cell motility and cell-cell adhesion, features associated with invasion and migration potential of tumor cells.     

Subcellular localization of the yeast proteome

Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass approximately 5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability--a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu.     

Subcellular localization of WD40 repeat 1 protein in PC12 rat pheochromocytoma cells

The dynamics of actin filament protein is crucial for various physiological processes of the cells. Among the proteins correlating with actin dynamics, a novel 67-kDa WD40 repeat protein 1 (WDR1) was the vertebrate homologue of actin-interacting protein 1 (Aip1). Even though previous studies have provided the clues on the function of WDR1 in specific organs under pathological conditions, the exact subcellular localization of WDR1 is not known. Therefore, in the present study, we undertook to determine the distribution of WDR1 within PC12 pheochromocytoma cells (PC12 cells) using light and electron microscopic techniques. Double immunocytochemistry clearly showed that WDR1 immunoreactivities (IRs) were co-localized with anti-actin antibody, suggesting the involvement of WDR1 in actin dynamics. WDR1 immunoreactivities (IRs) in PC12 cells showed different distribution patterns as nerve growth factor (NGF) concentrations varied. During active proliferation, the distribution of WDR1 IRs seemed to be similar to those found in cortical actin patches, whereas WDR1 IR was observed in cytoplasmic actin cables after PC12 cells were induced to differentiate by treating with NGF. Though further studies are necessary to determine the function of WDR1, the current data represents a first step towards the in vitro study of WDR1 protein.     

Subcellular localization of histamine in neonatal rat brain

Conclusion Indeed, this idea is in agreement with (a) data on the turnover rate of HT in neonatal rat brain as compared to adult brain and mast cells [11]; (b) recent histological data demonstrating the presence of mast cells in rat brain [12]; and (c) the in vivo effect of compound 48/80 on the levels of HT in P₁ of neonatal rat brain [13].     

Subcellular localization of hexokinase in the rat cortex

The distribution of hexokinase, inactivated and activated by Triton X-100, in subcellular fractions of the rat cortex was investigated. Latent hexokinase, inactivated and activated by the detergent Triton X-100 in the mitochondria is confirmed by the parallel distribution of cytochrome oxidase and hexokinase activities. Specific hexokinase activity of the external mitochondrial membrane was almost twice as high as that of the internal membrane. The existence of a special molecular form of latent hexokinase is discussed.We are grateful to technical assistants R. Arnold and I. Scheel for their help. The investigation was financially supported by the Ministry of Science and Technology of the GDR.     

Subcellular localization of heparanase in human neutrophils.

The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel filtration analysis of 35S-labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECM-derived, heparan sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.