Mutated fukutin-related protein (FKRP) localises as wild type in differentiated muscle cells

The mechanism of disease in forms of congenital and limb girdle muscular dystrophy linked to mutations in the gene encoding for Fukutin-related protein (FKRP) has previously been associated with the mis-localisation of FKRP from the Golgi apparatus. In the present report, we have transfected V5-tagged Fukutin-related protein expression constructs into differentiated C2C12 myotubes and the tibialis anterior of normal mice. The transfection of either wild type (WT) or several mutant constructs (P448L, C318Y, L276I) into myotubes consistently showed clear co-localisation with GM130, a Golgi marker. In contrast, whilst WT and the L276I localised to the Golgi of Cos-7 cells, the P448L and C318Y was mis-localised in the majority of these undifferentiated cells. The injection of the same constructs into the tibialis anterior of mice resulted in similar localisation of both the WT and all the mutants. Immunolabelling of FKRP in the muscle of MDC1C and LGMD2I patients was found to be indistinguishable from normal controls. Overall, these data suggest that retention in the endoplasmic reticulum of FKRP is not the main mechanism of disease but that this may instead relate to a disruption of the functional activity of this putative enzyme with its substrate(s) in the Golgi.     

Subcellular localization of the retinoblastoma protein

The subcellular localization of the retinoblastoma (RB) protein has been studied in primate cell lines by immunofluorescence staining using different monoclonal and polyclonal antibodies. The protein appeared as granules of heterogeneous size over the interphase nuclei. Computer assisted digital overlap analysis indicated that it was predominantly localized in euchromatic areas with low DNA density. The largest RB positive grains lined up on the heterochromatin/euchromatin boundary. During mitosis, the RB protein dissociated from the condensing chromosomes. It was dispersed throughout the cytoplasm during metaphase and anaphase, and it reassociated with the decondensing chromatin during telophase. A new monoclonal antibody, designated aRB1C1, was raised against a bacterial TrpE/human retinoblastoma protein. It specifically recognized the nonphosphorylated and differentially phosphorylated forms of the RB protein in immunoprecipitation experiments. A collection of RB expressing cell lines gave a positive staining reaction with the antibody, whereas the RB negative Weri-RB-27 retinoblastoma and OHS osteosarcoma cells failed to react. Wild-type RB complementary DNA was introduced into Weri-RB-27 by retrovirus mediated gene transfer. Similar experiments were performed with the DU145 prostatic carcinoma cell line that expresses a mutant RB protein. Reconstituted cells of both lines expressed the normal size RB protein and gave a positive immunofluorescence reaction with the aRB1C1 and other anti-RB antibodies. The new monoclonal antibody, however, showed cell type dependent differences of the staining pattern compared to other anti-RB antibodies, suggesting differentiation dependent accessibility to its epitope.     

Subcellular localization of an intermediate filament protein and its mRNA in glial cells.

Eucaryotic mRNAs are generally localized in the cell body, where most protein synthesis occurs. We have found that mRNAs encoding the glial intermediate filament protein are spatially distributed in the glial cell cytoplasm close to the location of the glial filaments. Whereas the glial filament protein mRNA was located predominantly in the distal process, actin mRNA was found almost exclusively in the apical portion of the glial cell. This pattern of mRNA localization might provide a mechanism for synthesis of proteins in specific subcellular compartments by mRNA translation locally.     

Subcellular localization of microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of apolipoprotein B-containing lipoproteins. Within the endoplasmic reticulum, it transfers lipid from the membrane to the forming lipoprotein. Recent evidence suggests that it may also function within the Golgi apparatus. To address this hypothesis, we developed a polyclonal antibody to MTP and used it in a series of studies on mouse liver and McArdle-RH7777 (McA) cells. Western blot analysis demonstrated the presence of MTP within mouse hepatic-Golgi apparatus-rich fractions. In addition, in vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within the Golgi fractions. Immunohistochemical studies with mouse liver demonstrated the presence of MTP within all hepatocytes, but not in nonparenchymal cells. The subcellular location of MTP in McA cells was investigated using confocal microscopy. MTP colocalized with the trans-Golgi network (TGN) 38 and Golgi SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) of 28 kDa (GS28), markers for the trans- and cis-Golgi apparatus, respectively. Morphometric analyses indicated that approximately 17% of the MTP signal colocalized with the TGN38, while 33% of the trans-Golgi marker colocalized with the MTP. Approximately 17% of the MTP signal colocalized with the GS28, whereas 53% of the cis-Golgi marker colocalized with the MTP. The results provide unequivocal evidence for the location of MTP within the Golgi apparatus, and further highlight the importance of this organelle in the assembly of lipoproteins.     

Subcellular localization of the Schlafen protein family

Although the first members of the Schlafen gene family were first described almost 10 years ago, the precise molecular/biochemical functions of the proteins they encode still remain largely unknown. Roles in cell growth, haematopoietic cell differentiation, and T cell development/maturation have, with some experimental support, been postulated, but none have been conclusively verified. Here, we have determined the subcellular localization of Schlafens 1, 2, 4, 5, 8, and 9, representing all three of the murine subgroups. We show that the proteins from subgroups I and II localize to the cytoplasm, while the longer forms in subgroup III localize exclusively to the nuclear compartment. We also demonstrate upregulation of Schlafen2 upon differentiation of haematopoietic cells and show this endogenous protein localizes to the cytoplasm. Thus, we propose the different subgroups of Schlafen proteins are likely to have functionally distinct roles, reflecting their differing localizations within the cell.     

Subcellular localization of MAK-V/Hunk protein kinase expressed in COS-1 cells

MAK-V/Hunk is a MARK/Par-1-related protein kinase, whose function is unknown. We studied the subcellular localization of MAK-V/Hunk in COS-1 cells by immunofluorescence. It has a nucleocytoplasmic distribution and is localized to the centrosome, as indicated by co-localization with gamma-tubulin. A putative kinase-deficient mutant, with a mutation in the invariant lysine residue in the catalytic domain, was not targeted to the nucleus or centrosome. These results suggest that the nuclear and centrosomal targeting of MAK-V/Hunk is specific, and is likely to be coupled to its catalytic activity.     

衣原体蛋白的亚细胞定位

沙眼衣原体(Chlamydia trachomatis,CT)是一种严格细胞内寄生、有独特发育周期的原核细胞型微生物.CT在宿主细胞浆内增殖,形成光镜可见的典型细胞内包涵体,包涵体为CT在宿主细胞内的生长繁殖提供屏障保护,同时也是CT与宿主细胞进行物质交换和信息传递的门户,CT不仅可从宿主细胞摄取营养物质,还可分泌效应蛋白进入宿主细胞质调节宿主细胞功能.CT基因组DNA序列和功能注释完成后,衣原体蛋白的亚细胞定位、结构和功能的研究已成为衣原体研究领域的热点之一[1-3].在CT与宿主细胞相互作用过程中,Inc蛋白、分泌蛋白等衣原体蛋白可能发挥着重要作用,鉴于蛋白质的亚细胞定位情况往往与其功能密切相关,衣原体蛋白在感染细胞中的定位认识成为其功能研究中的重要环节.     

Subcellular localization of protein kinase CK2

More than 46 years ago, Burnett and Kennedy first described protein kinase CK2 (formerly known as casein kinase 2) in liver extracts. Since then, protein kinase CK2 has been investigated in many organisms from yeast to man. It is now well established that protein kinase CK2 is a pleiotropic and ubiquitous serine or threonine kinase, which is highly conserved during evolution. A great number of studies deal with substrates of CK2, but the fact that over 160 substrates exist is more confusing than elucidatory. The holoenzyme is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits. There is now increasing evidence for individual functions of the subunits that are different from their functions in the holoenzyme. Furthermore, more and more studies describe interacting partners of the kinase that may be decisive in the regulation of this enzyme. A big step forward has been the determination of the crystal structure of the two subunits of protein kinase CK2. Now the interactions of the catalytic subunit of CK2 with ATP as well as GTP and the interaction between the regulatory subunits can be explained. However, cellular functions of protein kinase CK2 still remain unclear. In the present review we will focus our interest on the subcellular localization of protein kinase CK2. Protein kinase CK2 is found in many organisms and tissues and nearly every subcellular compartment. There is ample evidence that protein kinase CK2 has different functions in these compartments and that the subcellular localization of protein kinase CK2 is tightly regulated. Therefore studying the subcellular localization of protein kinase CK2 may be a key to its function.     

Subcellular localization of fumarase in mammalian cells and tissues

Fumarase, a mitochondrial matrix protein, is previously indicated to be present in substantial amounts in the cytosol as well. However, recent studies show that newly synthesized human fumarase is efficiently imported into mitochondria with no detectable amount in the cytosol. To clarify its subcellular localization, the subcellular distribution of fumarase in mammalian cells/tissues was examined by a number of different methods. Cell fractionation using either a mitochondria fraction kit or extraction with low concentrations of digitonin, detected no fumarase in a 100,000 g supernatant fraction. Immunoflourescence labeling with an affinity-purified antibody to fumarase and an antibody to the mitochondrial Hsp60 protein showed identical labeling pattern with labeling seen mainly in mitochondria. Detailed studies were performed using high-resolution immunogold electron microscopy to determine the subcellular localization of fumarase in rat tissues, embedded in LR White resin. In thin sections from kidney, liver, heart, adrenal gland and anterior pituitary, strong and specific labeling due to fumarase antibody was only detected in mitochondria. However, in the pancreatic acinar cells, in addition to mitochondria, highly significant labeling was also observed in the zymogen granules and endoplasmic reticulum. The observed labeling in all cases was completely abolished upon omission of the primary antibody indicating that it was specific. In a western blot of purified zymogen granules, a fumarase-antibody cross-reactive protein of the same molecular mass as seen in the mitochondria was present. These results provide evidence that fumarase in mammalian cells/tissues is mainly localized in mitochondria and significant amounts of this protein are not present in the cytosol. However, these studies also reveal that in certain tissues, in addition to mitochondria, this protein is also present at specific extramitochondrial sites. Although the cellular function of fumarase at these extramitochondrial locations is not known, the appearance/localization of fumarase outside mitochondria may help explain how mutations in this mitochondrial protein can give rise to a number of different types of cancers.     

Subcellular localization specified by protein acylation and phosphorylation

Modification of proteins by both lipophilic and hydrophilic moieties is widely documented. Here we present recent insights into how protein targeting is influenced by protein modification, with particular emphasis on dynamic regulation by fatty acylation and phosphorylation of proteins.     

Subcellular localization of phospholipase C isoforms in vascular smooth muscle

The phospholipase C (PLC) isoform most important during agonist-activated IP(3) production in vascular smooth muscle is still unknown. When PLC activity in rat tail artery homogenate was determined, this activity was shown to be inhibited by an antibody directed against PLCbeta2. Antibodies directed against the gamma1, beta1, beta3 and delta1 isoforms of PLC failed to inhibit PLC activity in this tissue. Both PLCbeta2 and PLCgamma1 were isolated from rat tail artery by DEAE column chromatography and PLCbeta2 activity was shown to be 3-fold greater than PLCgamma1 activity. When rat tail artery was treated with norepinephrine (10 mM), PLCbeta2 was shown to translocate from cytosol to membranes. When subcellular fractions of rat tail artery were isolated by sucrose density gradient centrifugation, including nuclei, plasma membrane, and cytosol, PLCbeta2 was detected in the plasma membrane and the cytosol but not in the nuclei. PLCdelta1 and PLCgamma1 were found only in cytosol. This evidence is consistent with the model wherein an agonist such as norepinephrine can activate smooth muscle contraction via interaction with a plasma membrane receptor which can easily interact with a plasma membrane-associated isoform of PLC, such as PLCbeta2.     

Subcellular localization and secretion of activity-dependent neuroprotective protein in astrocytes

Activity-dependent neuroprotective protein (ADNP, approximately 123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using alpha-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by approximately 1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.     

Subcellular localization of hyaluronate synthetase in oligodendroglioma cells

In order to provide some insight into the mechanism of hyaluronate synthesis, the subcellular localization of the synthetase system for hyaluronate was determined in eukaryotic cells. The mouse oligodendroglioma cell line G26-24, which produces copious amounts of hyaluronate in culture, was chosen as a system for these studies. Protease treatment and homogenization of cells followed by hyaluronate synthetase assay suggested that nucleotide-binding sites and trypsin-sensitive synthetase sites were not exposed at the outer membrane surface. Protease treatment following homogenization did result in decreased activity. Membrane fragments, prepared by gentle homogenization in iso- and hypotonic buffers, were subjected to differential centrifugation followed by several continuous and discontinuous sucrose equilibrium and velocity gradient systems. Hyaluronate synthetase activity co-fractionated with a plasma membrane marker in all systems, including those in which Golgi markers were separable. Treatment of intact cells in culture with several hyaluronidases resulted in a marked stimulation of cell-free synthetase activity. The stimulated activity was also found exclusively in plasma membrane-enriched fractions.     

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.     

Subcellular localization of the human proto-oncogene protein DEK

Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/arginine-repeat proteins. However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro. This could indicate that the DEK protein resides on cellular chromatin. To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis. Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase. Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK co-sedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo. Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin. We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle. In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture.     

Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells

The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.