Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.     

A new human cellular protein AUP1. III. The intracellular localization of AUP1 protein in different human and rat cell lines

Previously, we cloned a full-length cDNA of human Aup1 and showed that AUP1 may represent a new cellular target for the two adenovirus oncoproteins, E1A Ad5 and E4ORF3. In this study, we generated a polyclonal anti-AUP1 antibody and examined the subcellular localization of AUP1 in MCF7 cells, HeLa cells, H1299 cells, 293 cells, BRK1 cells and transfectants expressing adenoviruse E1 genes. Double staining of AUP1 and various markers for cytoplasmic structures showed that the pattern of AUP1 distribution in the cytoplasm was puctuate and diffuse and without any colocalization with Golgi apparatus or endoplasmic reticulum. Additional studies with ectopically expressed AUP1, fused with red fluorescent protein (RFP) in H1299 and McG7 human cell lines and BRK1 rat cell line, showed cytoplasmic localization of RFP-AUP1. Western blot analysis revealed that AUP1 was expressed at similar levels in all tested cell lines and had the same molecular weight as the rat protein (45 kDa). Taken together, these results suggest that AUP1 is a cytoplasmic protein that is expressed in all cell lines we examined.     

Involvement of the actin cytoskeleton in the regulation of serotonin transporter (SET) activity: possible mechanism underlying SET regulation by protein kinase C

Our previous report has revealed that PKC activation by 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the uptake activity of serotonin transporter (SET), via an indirect mechanism unknown, but not likely via direct phosphorylation of SET by PKC (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). To elucidate whether PKC can directly phosphorylate SET in vivo, FLAG-tagged SET (FLAG-SET) was expressed in COS-7 cells and the TPA-induced incorporation of (32)P into immunoprecipitated FLAG-SET was examined. PKC activation with TPA caused no phosphorylation of FLAG-SET expressed in COS-7 cells. On the other hand, morphological change associated with the disruption of filamentous actin (F-actin) was seen in TPA-treated COS-7 cells. Therefore, we studied the effects of cytochalasin D, an inhibitor of actin polymerization, on the uptake activity of the serotonin transporter (SET) to elucidate whether the actin cytoskeleton modulates the SET uptake activity. The treatment with cytochalasin D inhibited the uptake activity of both native and recombinant SET in a concentration-dependent manner. Eadie-Hofstee analysis revealed that cytochalasin D down-regulated the recombinant SET uptake activity by reducing the V(max), but not the K(m), mimicking the result observed in TPA-induced inhibition of SET activity (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). The cytochalasin D-induced inhibition of SET activity was partially, but significantly, reversed by jasplakinolide, a cell permeable stabilizer of F-actin, whereas TPA-induced inhibition of SET activity was not reversed by jasplakinolide. To elucidate whether the subcellular localization of SET was changed in response to cytochalasin D or TPA, we expressed the SET fused with the green fluorescent protein (SET-GFP) in COS-7 cells and observed the subcellular distribution of SET-GFP under a confocal laser scanning fluorescent microscope. Neither cytochalasin D nor TPA markedly changed the SET-GFP cellular localization, although these drugs caused morphological change in the GFP-transfected COS-7 cells. In addition, SET activity was not altered by the treatment with either colchicine, an inhibitor of microtubule polymerization, or taxol, a stabilizer of microtubule polymerization. These results suggest that the SET uptake activity was regulated by the state of the actin cytoskeleton and that TPA exerts its inhibitory action on SET activity, in part, via disruption of F-actin and subsequent morphological change in cells.     

Ultraspiracle promotes the nuclear localization of ecdysteroid receptor in mammalian cells

The heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (USP), both of which are members of the nuclear receptor superfamily, is considered to be the functional ecdysteroid receptor. Here we analyzed the subcellular distribution of EcR and USP fused to fluorescent proteins. The experiments were carried out in mammalian COS-7, CHO-K1 and HeLa cells to facilitate investigation of the subcellular trafficking of EcR and USP in the absence of endogenous expression of these two receptors. The distribution of USP tagged with a yellow fluorescent protein (YFP-USP) was almost exclusively nuclear in all cell types analyzed. The nuclear localization remained constant for at least 1 day after the first visible signs of expression. In contrast, the intracellular distribution of EcR tagged with a yellow fluorescent protein (YFP-EcR) varied and was dependent on time and cell type, although YFP-EcR alone was also able to partially translocate into the nuclear compartment. Coexpression of YFP-EcR with USP tagged with a cyan fluorescent protein (CFP-USP) resulted in exclusively nuclear localization of both proteins in all cell types analyzed. The USP-induced nuclear localization of YFP-EcR was stable for at least 20 hours. These experiments suggest that USP has a profound effect on the subcellular distribution of EcR.     

Cytoplasmic, nuclear, and golgi localization of RGS proteins. Evidence for N-terminal and RGS domain sequences as intracellular targeting motifs

RGS proteins comprise a family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. Biochemical studies suggest that members of this protein family act as GTPase-activating proteins for certain Galpha subunits, thereby accelerating the turn-off mechanism of Galpha and terminating signaling by both Galpha and Gbetagamma subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several RGS proteins in COS-7 cells expressing RGS-green fluorescent protein (GFP) fusion proteins and in cells expressing RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse neuroblastoma cells, and RGS10 exhibited nuclear localization in human glioma cells. Deletion or alanine substitution of an N-terminal leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other proteins, promoted nuclear localization of these proteins in COS-7 cells. In agreement with this observation, treatment of mouse neuroblastoma cells with leptomycin B to inhibit nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of RGS proteins localized in the nucleus, suggesting that nuclear localization of RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the cysteine motif promoted nuclear localization of RGSZ. None of the RGS proteins examined were localized at the plasma membrane. These results demonstrate that RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. These findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned.     

M-LP,Mpv17-like protein,has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene

M-LP (Mpv17-like protein) has been identified as a new protein that has high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early onset glomerulosclerosis. In this study, we verified the peroxisomal localization of M-LP by performing dual-color confocal analysis of COS-7 cells cotransfected with green fluorescent protein-tagged M-LP and DsRED2-PTS1, a red fluorescent peroxisomal marker. To characterize the peroxisomal membrane targeting signal, we examined the intracellular localizations of several green fluorescent protein-tagged deletion mutants and demonstrated that, of the three transmembrane segments predicted, the first near the NH(2) terminus and NH(2)-terminal half of the following loop region, which is abundant in positively charged amino acids, were necessary and sufficient for peroxisomal targeting. To elucidate the function of M-LP, we examined the activities of several enzymes involved in reactive oxygen species metabolism in COS-7 cells and found that transfection with M-LP increased the superoxide dismutase activity significantly. Quantitative real-time PCR analysis revealed that the manganese SOD (SOD2) mRNA level of COS-7 cells transfected with M-LP was elevated. These results indicate that M-LP participates in reactive oxygen species metabolism.     

Cell cycle-dependent subcellular localization of exchange factor directly activated by cAMP

Epac belongs to a new family of proteins that can directly mediate the action of the intracellular second messenger cAMP by activating a downstream small GTPase Rap1. The Epac/Rap1 pathway represents a novel cAMP-signaling cascade that is independent of the cAMP-dependent protein kinase (PKA). In this study, we have used fluorescence microscopy to probe the intracellular targeting of Epac during different stages of the cell division cycle and the structural features that are important for Epac localization. Our results suggest Epac, endogenous or expressed as a green fluorescent protein fusion protein, is mainly localized to the nuclear membrane and mitochondria during interphase in COS-7 cells. Deletion mutagenesis analysis reveals that whereas the DEP domain is responsible for membrane association, the mitochondrial-targeting sequence is located at the N terminus. Although Epac predominantly exhibits perinuclear localization in interphase, the subcellular localization of Epac is cell cycle-dependent. Epac disassociates from the nuclear membrane and localizes to the mitotic spindle and centrosomes in metaphase. At the end of the cell cycle, Epac is observed to reassociate with the nuclear envelope and concentrate around the contractile ring. Furthermore, overexpression of Epac in COS-7 cells leads to an increase in multinuclear cell populations. These results suggest that Epac may play an important role in mitosis.     

Parkin is associated with cellular vesicles

We recently identified a novel gene, parkin, as a pathogenic gene for autosomal recessive juvenile parkinsonism. Parkin encodes a 52-kDa protein with a ubiquitin-like domain and two RING-finger motifs. To provide a insight into the function of parkin, we have examined its intracellular distribution in cultured cells. We found that parkin was localized in the trans-Golgi network and the secretory vesicles in U-373MG or SH-SY5Y cells by immunocytochemical analyses. In the subsequent subcellular fractionation studies of rat brain, we showed that parkin was copurified with the synaptic vesicles (SVs) when we used low ionic conditions throughout the procedure. An immunoelectromicroscopic analysis indicated that parkin was present on the SV membrane. Parkin was readily released from SVs into the soluble phase by increasing ionic strength at neutral pH, but not by a non-ionic detergent. To elucidate its responsible region for membrane association, we transfected with green fluorescent protein-tagged deletion mutants of parkin into COS-1 cells followed by subcellular fractionation. We demonstrated the ability of parkin to bind to the membranes through a broad region except for the ubiquitin-like domain. The significance of SV localization of parkin is discussed.     

Stabilization of FtsH-unfolded protein complex by binding of ATP and blocking of protease

Mutations in small heterodimer partner (SHP) and hepatocyte nuclear factor 4alpha (HNF4alpha) are associated with mild obesity and diabetes mellitus, respectively. Both receptors work together to determine the normal pancreatic beta-cell function. We examined their subcellular localization and interaction in living cells by tagging them with yellow and cyan variants of green fluorescent protein (GFP) variants. Expressed SHP resided only in the cytoplasm in COS-7 cells which lacks HNF4alpha, but predominantly in the nucleus in insulinoma cells (MIN6). HNF4alpha was localized exclusively in the nuclei of both cells, coexpressed with HNF4alpha in COS-7 cells, redistributed in the nucleus, depending on the amount of HNF4alpha. We found fluorescence resonance energy transfer between GFP-tagged SHP and HNF4alpha, indicating a specific close association between them in the nucleus. The results strongly suggest that SHP exists primarily in the cytoplasm and is translocated into the nucleus on interacting with its nuclear receptor partner HNF4alpha.     

Targeting of the AE2 anion exchanger to the Golgi apparatus is cell type-dependent and correlates with the expression of Ank(195), a Golgi membrane skeletal protein

Sodium-independent anion exchangers (AE1-4) show remarkable variability in their tissue-specific expression and subcellular localization. Currently, isoform-specific targeting mechanisms are considered to be responsible for this variable localization. Here, we report that targeting can also be cell type-specific. We show that the full-length AE2 protein and its green fluorescent protein- or DsRed-tagged variants localize predominantly either to the Golgi apparatus in COS-7 cells, or to the plasma membrane in HeLa cells. This alternative targeting did not seem to result from either translational or post-translational differences, but rather from differential expression of at least one of the Golgi membrane skeletal proteins, ankyrin(195) (Ank(195)), between the two cell types. Comparative studies with several different cell lines revealed that the Golgi localization of the AE2 protein correlated strictly with the expression of Ank(195) in the cells. The two Golgi-associated proteins also co-localized well and similarly resisted detergent extraction in the cold, whereas the plasma membrane-localized AE2 in Ank(195)-deficient cells was mostly detergent-soluble. Collectively, our results suggest that Ank(195) expression is a key determinant for the variable and cell type-dependent localization of the AE2 protein in the Golgi apparatus in mammalian cells.     

Proteolytic activity assayed by subcellular localization switching of a substrate

An approach to assay proteolytic activity in vivo by altering the subcellular localization of a labelled substrate was demonstrated. The assay included a protein shuttling between different cellular compartments and a site-specific recombinant protease. The shuttle protein used was the human immunodeficiency virus type 1 (HIV-1) Rev protein tandemly fused to the enhanced green fluorescent protein (EGFP) and the red fluorescent protein (RFP), while the protease was the site-specific protease VP24 from the herpes simplex virus type 1 (HSV-1). The fluorescent proteins in the Rev fusion protein were separated by a cleavage site specific for the VP24 protease. When co-expressed in COS-7 cells proteolysis was observed by fluorescence microscopy as a shift from a predominantly cytoplasmic localization of the fusion protein RevEGFP to a nuclear localization while the RFP part of the fusion protein remained in the cytoplasm. The cleavage of the fusion protein by VP24 was confirmed by Western blot analysis. The activity of VP24, when tagged N-terminally by the Myc-epitope, was found to be comparable to VP24. These results demonstrates that the activity and localization of a recombinantly expressed protease can be assessed by protease-mediated cleavage of fusion proteins containing a specific protease cleavage site.     

Characterization of the nuclear localization and nuclear export signals of bovine herpesvirus 1 VP22

The bovine herpesvirus 1 (BHV-1) tegument protein VP22 is predominantly localized in the nucleus after viral infection. To analyze subcellular localization in the absence of other viral proteins, a plasmid expressing BHV-1 VP22 fused to enhanced yellow fluorescent protein (EYFP) was constructed. The transient expression of VP22 fused to EYFP in COS-7 cells confirmed the predominant nuclear localization of VP22. Analysis of the amino acid sequence of VP22 revealed that it does not have a classical nuclear localization signal (NLS). However, by constructing a series of deletion derivatives, we mapped the nuclear targeting domain of BHV-1 VP22 to amino acids (aa) 121 to 139. Furthermore, a 4-aa motif, 130PRPR133, was able to direct EYFP and an EYFP dimer (dEYFP) or trimer (tEYFP) predominantly into the nucleus, whereas a deletion or mutation of this arginine-rich motif abrogated the nuclear localization property of VP22. Thus, 130PRPR133 is a functional nonclassical NLS. Since we observed that the C-terminal 68 aa of VP22 mediated the cytoplasmic localization of EYFP, an analysis was performed on these C-terminal amino acid sequences, and a leucine-rich motif, 204LDRMLKSAAIRIL216, was detected. Replacement of the leucines in this putative nuclear export signal (NES) with neutral amino acids resulted in an exclusive nuclear localization of VP22. Furthermore, this motif was able to localize EYFP and dEYFP in the cytoplasm, and the nuclear export function of this NES could be blocked by leptomycin B. This demonstrates that this leucine-rich motif is a functional NES. These data represent the first identification of a functional NLS and NES in a herpesvirus VP22 homologue.     

SDF-1α基因与绿色荧光蛋白融合载体的构建及亚细胞定位

目的构建SDF-1α基因与绿色荧光蛋白的融合蛋白表达载体,进而观察SDF-1α基因编码蛋白在细胞内的定位情况。方法用EcoRI内切酶从pMD-T18一SDF-1α重组载体中酶切分离SDF-1α基因的完整ORF,构建pEGFP-C1-SDF-1α的融合表达载体,脂质体转染COS-7细胞,并在荧光显微镜下观察表达的融合蛋白。结果SDF-1α基因在COS-7细胞中高效表达,激光共聚焦的结果显示,SDF-1α基因定位在细胞质内。结论成功构建了pEGFP-C1-SDF-1α的融合表达载体,SDF-1α基因主要在细胞质中表达。     

Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.     

Exofacial epitope-tagged glucose transporter chimeras reveal COOH- terminal sequences governing cellular localization

The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti- HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH-terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transporters as important determinants of cellular localization in COS-7 and CHO cells.     

Visualization of G protein betagamma dimers using bimolecular fluorescence complementation demonstrates roles for both beta and gamma in subcellular targeting

To investigate the role of subcellular localization in regulating the specificity of G protein betagamma signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize betagamma dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to beta and a carboxyl-terminal yellow fluorescent protein fragment to gamma. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on betagamma assembly in that it was not obtained using beta2 and gamma1, which do not form a functional dimer. In addition to assembly, BiFC betagamma complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent beta and gamma subunits and by their abilities to potentiate activation of adenylyl cyclase by alpha(s) in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different beta subunits with three different gamma subunits were compared. BiFC betagamma complexes containing either beta1 or beta2 localized to the plasma membrane, whereas those containing beta5 accumulated in the cytosol or on intracellular membranes. These results indicate that the beta subunit can direct trafficking of the gamma subunit. Taken together with previous observations, these results show that the G protein alpha, beta, and gamma subunits all play roles in targeting each other. This method of specifically visualizing betagamma dimers will have many applications in sorting out roles for particular betagamma complexes in a wide variety of cell types.