Nuclear localization of enzymatically active green fluorescent protein-CTP:phosphocholine cytidylyltransferase alpha fusion protein is independent of cell cycle conditions and cell types

To address the recent controversy about the subcellular localization of CTP:phosphocholine cytidylyltransferase alpha (CTalpha), this study was designed to visualize green fluorescent protein (GFP). CTalpha fusion proteins directly and continuously under different conditions of cell cycling and in various cell lines. The GFP. CTalpha fusion proteins were enzymatically active and capable of rescuing mutant cells with a temperature-sensitive CT. The expressed GFP.CTalpha fusion protein was localized to the nucleus in all cell lines and required the N-terminal nuclear targeting sequence. Serum depletion/replenishment did not cause shuttling of CTalpha between the nucleus and cytoplasm. Moreover, the subcellular localization of CTalpha was examined continuously through all stages of the cell cycle in synchronized cells. No shuttling of CTalpha between the nucleus and cytoplasm was observed at any stage of the cell cycle. Stimulation of cells with oleate had no effect on the localization of CTalpha. The GFP.CTalpha lacking the nuclear targeting sequence stayed exclusively in the cytoplasm. Regardless of their localization, the GFP.CTalpha fusion proteins were equally active for phosphatidylcholine synthesis and mutant rescue. We conclude that the nuclear localization of CTalpha is a biological event independent of cell cycle in most mammalian cells and is unrelated to activation of phosphatidylcholine synthesis.     

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.     

PNRC1核定位信号序列的鉴定

为鉴定富含脯氨酸核受体辅调节蛋白1(PNRC1)分子的核定位信号序列（nuclear localization signal sequence, NLS）,在生物信息学方法预测的基础上,先构建野生型PNRC1及删除预测NLS的PNRC1突变体的绿色荧光蛋白（GFP）重组表达载体,转染细胞后通过激光共聚焦显微镜观察PNRC1分子在删除预测NLS后细胞内的定位变化.然后,将预测的NLS编码序列直接连到GFP表达载体上,以及将预测的NLS加到胞浆蛋白上构建其GFP重组表达载体,转染细胞,观察预测的NLS能否把构建的重组体都带到细胞核内.结果显示,删除PNRC1中预测的NLS后,其定位从细胞核中变为主要定位在细胞浆中,而预测的NLS能把GFP或胞浆中的蛋白带到细胞核中.研究表明,预测的NLS为PNRC1分子真正的NLS.     

Protein transduction of an antioxidant enzyme: subcellular localization of superoxide dismutase fusion protein in cells

In protein therapy, it is important for exogenous protein to be delivered into the target subcellular localization. To transduce a therapeutic protein into its specific subcellular localization, we synthesized nuclear localization signal (NLS) and membrane translocation sequence signal (MTS) peptides and produced a genetic in-frame SOD fusion protein. The purified SOD fusion proteins were efficiently transduced into mammalian cells with enzymatic activities. Immunofluorescence and Western blot analysis revealed that the SOD fusion proteins successfully transduced into the nucleus and the cytosol in the cells. The viability of cells treated with paraquat was markedly increased by the transduced fusion proteins. Thus, our results suggest that these peptides should be useful for targeting the specific localization of therapeutic proteins in various human diseases.     

核定位信号分析示踪载体——pGST-EGFP的构建及功能鉴定

目的 构建谷胱甘肽转硫酶（GST）与EGFP相融合的新型蛋白质示踪载体--pGST-EGFP,以用于蛋白质细胞亚定位信号序列的深入分析.方法 以质粒pEGFP-N1为骨架,融合从pGEX-2TK载体中扩增的GST编码序列,构建成pGST-EGFP融合表达质粒;再插入人工合成的已知核定位蛋白SV40的核定位序列（NLS）,构建成pGST-EGFP-SV40 NLS作为阳性对照;另外,构建小分子量蛋白TNNI2在pGST-EGFP的融合表达质粒.将对照pEGFP-N1和各重组质粒分别用脂质体介导,瞬时转染HeLa细胞,荧光显微镜下观察蛋白的核定位情况.结果 单独表达的EGFP呈全细胞分布,而GST-EGFP融合蛋白只存在于细胞浆;SV40 NLS能将GST-EGFP融合蛋白带进细胞核.虽然TNNI2-EGFP融合蛋白的细胞亚定位呈现核内丰度更高的特点,但TNNI2-GST-EGFP融合蛋白仅限定于胞浆分布,提示TNNI2不能主动定位到细胞核中.结论 成功构建了蛋白质细胞亚定位示踪载体--pGST-EGFP.作为核定位信号分析系统,其对小分子蛋白细胞亚定位的示踪效果优于传统的pEGFP载体,更适用于科研工作中小分子量蛋白质核定位信号序列的研究.     

A family of RS domain proteins with novel subcellular localization and trafficking

We report the sequence, conservation and cell biology of a novel protein, Psc1, which is expressed and regulated within the embryonic pluripotent cell population of the mouse. The Psc1 sequence includes an RS domain and an RNA recognition motif (RRM), and a sequential arrangement of protein motifs that has not been demonstrated for other RS domain proteins. This arrangement was conserved in a second mouse protein (BAC34721). The identification of Psc1 and BAC34721 homologues in vertebrates and related proteins, more widely throughout evolution, defines a new family of RS domain proteins termed acidic rich RS (ARRS) domain proteins. Psc1 incorporated into the nuclear speckles, but demonstrated novel aspects of subcellular distribution including localization to speckles proximal to the nuclear periphery and localization to punctate structures in the cytoplasm termed cytospeckles. Integration of Psc1 into cytospeckles was dependent on the RRM. Cytospeckles were dynamic within the cytoplasm and appeared to traffic into the nucleus. These observations suggest a novel role in RNA metabolism for ARRS proteins.     

Identification of a novel nuclear localization signal common to 69- and 82-kDa human choline acetyltransferase

We demonstrated previously that 69- and 82-kDa human choline acetyltransferase are localized predominantly to the cytoplasm and the nucleus, respectively. We have now identified a nuclear localization signal common to both forms of enzyme using confocal microscopy to study the subcellular compartmentalization of choline acetyltransferase tagged with green fluorescent protein in living HEK 293 cells. To identify functional nuclear localization and export signals, portions of full-length 69-kDa choline acetyltransferase were cloned into the vector peGFP-N1 and the cellular distribution patterns of the fusion proteins observed. Of the nine constructs studied, one yielded a protein with nuclear localization and another produced a protein with cytoplasmic localization. Mutation of the critical amino acids in this novel putative nuclear localization signal in the 69- and 82-kDa enzymes demonstrated that it is functional in both proteins. Moreover, 69-kDa choline acetyltransferase but not the 82-kDa enzyme is transported out of the nucleus by the leptomycin B-sensitive Crm-1 export pathway. By using bikaryon cells expressing both 82-kDa choline acetyltransferase and the nuclear protein heterogeneous nuclear ribonucleoprotein with green and red fluorescent tags, respectively, we found that the 82-kDa enzyme does not shuttle out of the nucleus in measurable amounts. These data suggest that 69-kDa choline acetyltransferase is a nucleocytoplasmic shuttling protein with a predominantly cytoplasmic localization determined by a functional nuclear localization signal and unidentified putative nuclear export signal. For 82-kDa choline acetyltransferase, the presence of the unique amino-terminal nuclear localization signal plus the newly identified nuclear localization signal may be involved in a process leading to predominantly nuclear accumulation of this enzyme, or alternatively, the two nuclear localization signals may be sufficient to overcome the force(s) driving nuclear export.     

Alteration of poly(ADP-ribose) glycohydrolase nucleocytoplasmic shuttling characteristics upon cleavage by apoptotic proteases

Poly(ADP-ribosyl)ation is an important post-translational modification which mostly affects nuclear proteins. The major roles of poly(ADP-ribose) synthesis are assigned to DNA damage signalling during base excision repair, apoptosis and excitotoxicity. The transient nature and modulation of poly(ADP-ribose) levels depend mainly on the activity of poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG), the key catabolic enzyme of poly(ADP-ribose). Given the fact that PARG substrate, poly(ADP-ribose), is found almost exclusively in the nucleus and that PARG is mainly localized in the cytoplasm, we wanted to have a closer look at PARG subcellular localization in order to better understand the mechanism by which PARG regulates intracellular poly(ADP-ribose) levels. We examined the subcellular distribution of PARG and of its two enzymatically active C-terminal apoptotic fragments both biochemically and by fluorescence microscopy. Green fluorescent protein (GFP) fusion proteins were constructed for PARG (GFP-PARG), its 74 kDa (GFP-74) and 85 kDa (GFP-85) apoptotic fragments and transiently expressed in COS-7 cells. Localization experiments reveal that all three fusion proteins localize predominantly to the cytoplasm and that a fraction also co-localizes with the Golgi marker FTCD. Moreover, leptomycin B, a drug that specifically inhibits nuclear export signal (NES)-dependent nuclear export, induces a redistribution of GFP-PARG from the cytoplasm to the nucleus and this nuclear accumulation is even more pronounced for the GFP-74 and GFP-85 apoptotic fragments. This observation confirms our hypothesis for the presence of important regions in the PARG sequence that would allow the protein to engage in CRM1-dependent nuclear export. Moreover, the altered nuclear import kinetics found for the apoptotic fragments highlights the importance of PARG N-terminal sequence in modulating PARG nucleocytoplasmic trafficking properties.     

A unique C-terminal repeat domain maintains the cytosolic localization of the placenta-specific tristetraprolin family member ZFP36L3

Members of the tristetraprolin family of CCCH tandem zinc finger proteins bind to AU-rich elements in certain cellular mRNAs, leading to their deadenylation and destabilization. Studies in knock-out mice demonstrated roles for three of the family members, tristetraprolin, ZFP36L1, and ZFP36L2, in inflammation, chorioallantoic fusion, and early embryonic development, respectively. However, little is known about a recently discovered placenta-specific tristetraprolin family member, ZFP36L3. Tristetraprolin, ZFP36L1, and ZFP36L2 have been shown to shuttle between the nucleus and cytoplasm, using typical hydrophobic amino acid-rich nuclear export sequences, and nuclear localization sequences located within the tandem zinc finger domain. In contrast, we previously showed that green fluorescent protein-labeled ZFP36L3, expressed in HEK 293 cells, remained cytosolic, even in the presence of the nuclear export blocker leptomycin B. We show here that the conserved tandem zinc finger domain contains an active nuclear localization signal. However, the sequence corresponding to the nuclear export signal in the other family members was nonfunctional, and thus did not contribute to the cytosolic localization. The unique C-terminal repeat domain could override the activity of the nuclear localization sequence, preventing the import of ZFP36L3 into the nucleus. Immunostaining of mouse placenta demonstrated that ZFP36L3 was located only in the cytoplasm of trophoblast cells. Thus, in contrast to the other mammalian members of this protein family, ZFP36L3 is a "full-time" cytosolic protein, rather than a nucleocytoplasmic shuttling protein. The significance of this difference in subcellular localization to the physiology of placental trophoblast cells, where ZFP36L3 is selectively expressed, remains to be determined.     

A conserved motif controls nuclear localization of Drosophila Muscleblind

Human Muscleblind-like proteins are alternative splicing regulators that are functionally altered in the RNA-mediated disease myotonic dystrophy. There are different Muscleblind protein isoforms in Drosophila and we previously determined that these have different subcellular localizations in the COS-M6 cell line. Here, we describe the conservation of the sequence motif KRAEK in isoforms C and E and propose a specific function for this motif. Different Muscleblind isoforms localize to the peri-plasma membrane (MblA), cytoplasm (MblB), or show no preference for the nuclear or cytoplasmic compartment (MblC and MblD) in Drosophila S2 cells transiently transfected with Musclebind expression plasmids. Mutation of the KRAEK motif reduces MblC nuclear localization, whereas fusion of a single KRAEK motif to the heterologous protein β-galactosidase is sufficient to target the reporter protein to the nucleus of S2 cells. This motif is not exclusive to Muscleblind proteins and is detected in several other protein types. Taken together, these results suggest that the KRAEK motif regulates nuclear translocation of Muscleblind and may constitute a new class of nuclear localization signal.     

Nuclear transport of plant potyviral proteins.

We have used immunoblotting, immunocytochemical, and gene fusion methods to examine the differential subcellular partitioning of tobacco etch potyvirus proteins that are potentially associated with RNA replication. From the earliest timepoints at which viral proteins could be detected, proteins Nla (49-kilodalton proteinase) and Nlb (58-kilodalton polymerase) were localized primarily in the nucleus, whereas the 71-kilodalton cylindrical inclusion protein was identified in the cytoplasm. The Nla and Nlb coding regions were fused to the beta-glucuronidase (GUS) sequence in a plant expression vector, resulting in synthesis of chimeric proteins in transfected protoplasts and in transgenic plants. In situ localization of GUS activity revealed nuclear localization of the GUS-Nla and GUS-Nlb fusion proteins and cytoplasmic localization of nonfused GUS. These results indicate that both Nla and Nlb contain nuclear targeting signals, and that they may serve as useful models for studies of plant cell nuclear transport. A discussion of the general utility of the nuclear transport system described here, as well as the role of nuclear translocation of potyviral proteins, is presented.     

Matricellular protein SPARC is translocated to the nuclei of immortalized murine lens epithelial cells

The matricellular glycoprotein, secreted protein acidic and rich in cysteine (SPARC), has complex biological activities and is important for lens epithelial cell function and regulation of cataract formation. To understand how SPARC influences lens epithelial cell activity and homeostasis, we have studied the subcellular distribution of SPARC in murine lens epithelial cells in vitro. We demonstrate that endogenous SPARC is located in the cytoplasm of either quiescent or dividing lens epithelial cells in culture. However, cytoplasmic SPARC was translocated into the nuclei of immortalized lens epithelial cells upon a significant reduction of intracellular SPARC in these cells. Recombinant human (rh) SPARC added to the culture media was quickly and efficiently internalized into the cytosol of SPARC-null lens epithelial cells. Moreover, cytoplasmic rhSPARC was also translocated into the nucleus after exogenous rhSPARC was removed from the culture media. The translocation of SPARC into the nucleus was therefore triggered by the reduction of SPARC protein normally available to the cells. A mouse SPARC-EGFP chimeric fusion protein (70 kDa) was expressed in lens epithelial cells and 293-EBNA cells, and was observed both in the cytoplasm and culture medium, but not in the nucleus. SPARC does not appear to have a strong nuclear localization sequence. Alternatively, SPARC might pass through the nuclear pore complex by passive diffusion. SPARC therefore functions not only as an extracellular protein but also potentially as an intracellular protein to influence cellular activities and homeostasis.     

Nuclear localization of enhanced green fluorescent protein homomultimers

The green fluorescent protein (GFP) and its variants are used in many studies to determine the subcellular localization of other proteins by analyzing fusion proteins. The main problem for nuclear localization studies is the fact that, to some extent, GFP translocates to the nucleus on its own. Because the nuclear import could be due to unspecific diffusion of the relatively small GFP through the nuclear pores, we analyzed the localization of multimers of a GFP variant, the enhanced GFP (EGFP). By detecting the fluorescence of the expressed proteins in gels after nonreducing SDS-PAGE, we demonstrate the integrity of the expressed proteins. Nevertheless, even EGFP homotetramers and homohexamers are found in the nuclei of the five analyzed mammalian cell lines. The use of fusion constructs of small proteins with multimeric EGFP alone, therefore, is not adequate to prove nuclear import processes. Fusion to tetrameric EGFP in combination with a careful quantification of the fluorescence intensities in the nucleus and cytoplasm might be sufficient in many cases to identify a significant difference between the fusion protein and tetrameric EGFP alone to deduce a nuclear localization signal.     

Nuclear localization of angiotensinogen in astrocytes

In the brain, angiotensinogen (AGT) is primarily expressed in astrocytes; brain ANG II derived from locally produced AGT has been shown to influence blood pressure. To better understand the molecular basis of AGT expression in the brain, we identified a human astrocytoma cell line, CCF-STTG1, that expresses endogenous AGT mRNA and produces AGT protein. Studies examining CCF-STTG1 cell AGT after N- and O-glycosidase suggest that AGT may not be posttranslationally modified by glycosylation in these cells as it is in plasma. Small amounts of AGT (5% of HepG2) were detected in the culture medium, suggesting a low rate of AGT secretion. Immunocytochemical examination of AGT in CCF-STTG1 cells revealed mainly nuclear localization. Although this has not been previously reported, it is consistent with nuclear localization of other serpin family members. To examine this further, we generated a fusion protein consisting of green fluorescent protein (GFP) and human AGT and examined subcellular localization by confocal microscopy after confirming expression of the fusion protein by Western blot. In CCF-STTG1 cells, a control GFP construct lacking AGT was mainly localized in the cytoplasm, whereas the GFP-AGT fusion protein was primarily localized in the nucleus. To map the location of a potential nuclear localization signal, overlapping 500-bp fragments of human AGT cDNA were fused in frame downstream of GFP. Although four of the fusion proteins exhibited either perinuclear or cytoplasmic localization, one fusion protein encoding the COOH terminus of AGT was localized in the nucleus. Importantly, nuclear localization of human AGT was confirmed in primary cultures of glial cells isolated from transgenic mice expressing the human AGT under the control of its own endogenous promoter. Our results suggest that AGT may have a novel intracellular role in the brain apart from its predicted endocrine function.     

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein-CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61-ECFP-CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.     

Identification of a bipartite nuclear localization sequence necessary for nuclear import of 5-lipoxygenase.

5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. This enzyme can reside either in the cytoplasm or the nucleus; its subcellular distribution is influenced by extracellular factors, and its nuclear import correlates with changes in leukotriene synthetic capacity. To identify sequences responsible for the nuclear import of 5-lipoxygenase, we transfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors encoding various 5-lipoxygenase constructs fused to green fluorescent protein. Overexpression of wild type 5-lipoxygenase with or without fusion to green fluorescent protein resulted in a predominantly intranuclear pattern of fluorescence, similar to the distribution of native 5-lipoxygenase in primary alveolar macrophages. Within the 5-lipoxygenase protein is a sequence (Arg(638)-Lys(655)) that closely resembles a bipartite nuclear localization signal. Studies using deletion mutants indicated that this region was necessary for nuclear import of 5-lipoxygenase. Analysis of mutants containing specific amino acid substitutions within this sequence confirmed that it was this sequence that was necessary for nuclear import of 5-lipoxygenase and that a specific arginine residue was critical for this function. As nuclear import of 5-lipoxygenase may regulate leukotriene production, natural or induced mutations in this bipartite nuclear localization sequence may also be important in affecting leukotriene synthesis.     

The Nucleo-cytoplasmic actin-binding protein CapG lacks a nuclear export sequence present in structurally related proteins

Despite thorough structure-function analyses, it remains unclear how CapG, a ubiquitous F-actin barbed end capping protein that controls actin microfilament turnover in cells, is able to reside in the nucleus and cytoplasm, whereas structurally related actin-binding proteins are predominantly cytoplasmic. Here we report the molecular basis for the different subcellular localization of CapG, severin, and fragminP. Green fluorescent protein-tagged fragminP and severin accumulate in the nucleus upon treatment of transfected cells with the CRM1 inhibitor leptomycin B. We identified a nuclear export sequence in severin and fragminP, which is absent in CapG. Deletion of amino acids Met(1)-Leu(27) resulted in nuclear accumulation of severin and fragminP. Tagging this sequence to CapG triggered nuclear export, whereas mutation of single leucine residues (Leu(17), Leu(21), and Leu(27)) in the export sequence inhibited nuclear export. Based on these findings, a nuclear export signal was identified in myopodin, a muscle-specific actin-binding protein, and the Bloom syndrome protein, a RecQ-like helicase. Deletion of the myopodin nuclear export sequence blocked invasion into collagen type I of C2C12 cells transiently overexpressing myopodin. Our findings explain regulated subcellular targeting of distinct classes of actin-binding proteins.