Nuclear targeting of prothymosin alpha

Prothymosin alpha is a highly acidic protein which lacks an amino-terminal signal peptide, yet was once thought to be a precursor for thymosin alpha 1, a putative peptide hormone secreted by the thymus. Here, two lines of evidence are presented that strongly implicate prothymosin alpha as a nuclear protein: 1) in COS cells transfected with the human prothymosin alpha gene copious amounts of prothymosin alpha were present in sealed nuclei obtained by treating these cells with cytochalasin B and enucleating them centrifugally. 2) Constructs in which human prothymosin alpha nucleic acid sequences were fused in-frame either near the amino terminus of the beta-galactosidase gene in pCH110 or at the carboxyl terminus, when expressed in COS cells, resulted in nuclear localization of the fusion protein; indirect immunofluorescence in situ was used as the assay. The basic cluster of amino acids at the carboxyl terminus of prothymosin alpha, TKKQKT, has been identified as part of the nuclear targeting signal, whereas the basic cluster of amino acids situated within the thymosin alpha 1 sequence at the amino terminus failed to effect nuclear transport.     

Nuclear and mitotically enhanced epitope.

Salt-extracted proteins of taxol-stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle-dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re-accumulated at daughter nuclei and was concealed during G1 phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.     

Identification of amino acid sequence in the hinge region of human vitamin D receptor that transfers a cytosolic protein to the nucleus

The localization of human vitamin D receptor (VDR) in the absence of its ligand 1,25-dihydroxyvitamin D(3) was investigated using chimera proteins fused to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fusion proteins were transiently or stably introduced into COS7 cells, and the subcellular distribution of the fusion proteins was examined. GFP-tagged wild-type VDRs were located predominantly in nuclei but with a significant cytoplasmic presence, while GFP alone was equally distributed throughout the cells. 10(-8) M 1,25-dihydroxyvitamin D(3) promoted the nuclear import of VDR in a few hours. To identify the NLS, we constructed several mutated VDRs fused to GFP. Mutant VDRs that did not bind to DNA were also localized predominantly in nuclei, while the deletion of the hinge region resulted in the loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to translocate to nuclei. These results indicate that 1) VDR is located predominantly in nuclei with a significant presence in cytoplasm without the ligand and 2) an NLS consisting of 20 amino acids in the hinge region facilitates the transfer of VDR to the nucleus.     

The hydrophobic domain of cytochrome b5 is capable of anchoring beta-galactosidase in Escherichia coli membranes.

Cytochrome b5 is inserted posttranslationally into membranes in vivo and spontaneously into liposomes in vitro by a short carboxyl-terminal hydrophobic membrane-anchoring sequence. DNA corresponding to this hydrophobic sequence has been synthesized, and two gene fusions with the Escherichia coli enzyme beta-galactosidase have been constructed by locating the hydrophobic domain in one case at the EcoRI site near the C terminus and in the other at the normal C terminus of the enzyme. The latter fusion protein was enzymatically active, having approximately 50% of the specific activity of beta-galactosidase, and cells expressing this protein grew normally with lactose as the sole carbon source. Both fusion proteins were localized to the E. coli inner membrane, converting beta-galactosidase from a cytoplasmic enzyme to a membrane-associated enzyme. The hydrophobic domain of cytochrome b5 therefore contains the information required to target polypeptides containing this domain to the membrane. Use of the cytochrome b5 hydrophobic peptide, either alone or in conjunction with other localizing sequences such as signal sequences, provides a general procedure for associating proteins with membranes. Polypeptides bearing this hydrophobic peptide may have considerable use as pharmaceuticals when associated with liposomes or cellular membranes.     

Nuclear localization and cell cycle regulation of a murine protein tyrosine phosphatase.

MPTP is a murine homolog of the human T-cell protein tyrosine phosphatase (PTPase) and the rat PTP-S enzyme. Enzymatic activity of this ubiquitously expressed protein was demonstrated in immunoprecipitates from NIH 3T3 cells and in recombinant protein overexpressed in bacteria. Expression of beta-galactosidase-MPTP MPTP chimeric proteins in COS1 cells identified a nuclear localization signal at the carboxyl terminus of the MPTP that was sufficient to direct beta-galactosidase as well as a tagged version of the MPTP to the nucleus. Deletion analysis of amino acids within the nuclear targeting signal showed that this sequence does not conform to the bipartite type of nuclear localization signals. Furthermore, it was shown that the steady-state levels of MPTP RNA fluctuate in a cell cycle-specific manner. On the basis of these experiments, we discuss the possible function of MPTP in the cell cycle and other nuclear processes.     

Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus: an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport.

Large amounts of pp65 (UL83) of human cytomegalovirus are translocated to the cell nucleus during the first minutes after uptake of the tegument protein from infecting viral particles. Two stretches of basic amino acids which resembled nuclear localization signals (NLS) of both the simian virus 40 type and the bipartite type were found in the primary structure of pp65. Deletion of these sequences significantly impaired nuclear localization of the truncated proteins after transient expression. The results indicated that both elements contributed to the nuclear localization of the protein. When fused to the bacterial beta-galactosidase, only one of the two basic elements was sufficient to mediate nuclear translocation. This element consisted of two clusters of basic amino acids (boxes C and D), which were separated by a short spacer sequence. In contrast to other bipartite NLS of animal cells, both basic boxes C and D functioned independently in nuclear transport, thus resembling simian virus 40-type NLS. Yet, complete translocation of beta-galactosidase was only found in the bipartite configuration. When both boxes C and D were fused, thereby deleting the intervening sequences, the nuclear transport of beta-galactosidase was reduced to levels seen with constructs in which only one of the boxes was present. Appropriate spacing, therefore, was important but not absolutely required. This was in contrast with results for other bipartite NLS, in which spacer deletions led to complete cytoplasmic retention. The presented results demonstrate that efficient nuclear transport of pp65 is mediated by one dominant NLS and additional targeting sequences. The major NLS of pp65 is an unusual signal sequence composed of two weak NLS which function together as one strong bipartite nuclear targeting signal.     

Nuclear localization of the PEP protein tyrosine phosphatase.

PEP is an intracellular protein tyrosine phosphatase expressed primarily by cells of hematopoietic origin that can be divided structurally into a catalytic domain and a large carboxy-terminal domain. The carboxy-terminal domain is enriched in proline, glutamic acid, serine, and threonine residues (PEST sequences) and contains a nonperfect tandem repeat sequence enriched in proline residues and a carboxy terminus enriched in basic amino acids. Here we show that PEP is diffusely expressed in lymphoid tissues, consistent with expression by many different cell types. Analysis of the PEP protein identifies a nuclear localization sequence within the extreme carboxy terminus. Transfer of 18 amino acids from the carboxy terminus of PEP to beta-galactosidase conferred nuclear localization, indicating that this sequence was sufficient for nuclear localization. Proteins enriched in PEST sequences are often rapidly degraded. However, pulse-chase analysis indicates that PEP has a half-life of greater than 5 h.     

Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence

Uncleaved signal-anchor sequences of membrane proteins inserted into the endoplasmic reticulum initiate the translocation of either the amino-terminal or the carboxyl-terminal polypeptide segment across the bilayer. Which topology is acquired is not determined by the apolar segment of the signal but rather by the hydrophilic sequences flanking it. To study the role of charged residues in determining the membrane topology, the insertion of mutants of the asialoglycoprotein receptor H1, a single-spanning protein with a cytoplasmic amino terminus, was analyzed in transfected COS-7 cells. When the charged amino acids flanking the hydrophobic signal were mutated to residues of opposite charge, half the polypeptides inserted with the inverted orientation. When, in addition, the amino-terminal domain of the mutant protein was truncated, approximately 90% of the polypeptides acquired the inverted topology. The transmembrane orientation appears to be primarily determined by the charges flanking the signal sequence but is modulated by the domains to be translocated.     

Analysis of protein-DNA and protein-protein interactions of centromere protein B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle.

We previously reported that centromere protein B (CENP-B) forms a stable complex (designated complex A) containing two alphoid DNAs in vitro. Domains in the CENP-B polypeptide involved in the formation of complex A were determined in the present study with truncated derivatives expressed in Escherichia coli and in rabbit reticulocyte lysates. It was revealed by gel mobility shift analyses that polypeptides containing the NH2-terminal DNA-binding domain bind a DNA molecule as a monomer, while dimerizing at a novel hydrophobic domain in the COOH-terminal region of 59 amino acid residues. This polypeptide dimerization activity at the COOH-terminal region was also confirmed with the two-hybrid system in Saccharomyces cerevisiae cells. The results thus proved that CENP-B polypeptides form a homodimer at the COOH-terminal hydrophobic domain, each binding a DNA strand at their NH2-terminal domains. The dimerization and DNA-binding domains fall into two of the three completely conserved sequences found in human and mouse CENP-B, and complex A-forming activity was also detected in nuclear extracts of mouse cells. Metaphase-specific phosphorylation of CENP-B was also detected, but this had no effect on its complex A-forming activity. On the basis of the present results, we propose that CENP-B plays an important role in the assembly of specific centromere structures by forming unique DNA-protein complexes at the sites of CENP-B boxes on the centromeric repetitive DNA both in interphase nuclei and on mitotic chromosomes.     

A structural model of human erythrocyte protein 4.1

Limited proteolysis and specific chemical cleavage methods have enabled a detailed structural characterization of human erythrocyte protein 4.1. This protein is composed of two chemically very similar polypeptide chains (a and b) with apparent molecular masses of 80,000 and 78,000 daltons. Cleavage of protein 4.1 at cysteine residues by 2-nitro-5-thiocyanobenzoic acid produces a series of doublets which differ by approximately 2,000 daltons and have identical peptide maps. Alignment of these peptides by mapping analysis has localized 4 cysteine residues within a 17,000-dalton segment on both a and b polypeptides. Mild chymotryptic treatment at 0 degrees C cleaves protein 4.1 primarily in three central locations and generates two families of unrelated peptides. Analysis of these fragments in two-dimensional gels and by peptide mapping reveals an unusual polarity in protein 4.1 structure in that each polypeptide chain contains two segments, one relatively acidic the other basic, that are segregated at opposite ends of the molecule. The basic region is digested into a cysteine-rich 30,000-dalton domain which resists further breakdown while the acidic region is readily degraded into smaller fragments. The peptides derived from the acidic region all appear as doublets suggesting that protein 4.1 a and b polypeptides differ close to the terminus of the acidic end. Similar phosphorylation sites occur on both polypeptides within a segment some 24,000-34,000 daltons from the acidic terminus.     

Construction and properties of bifunctionally active membrane-bound fusion proteins. Escherichia coli proline carrier linked with beta-galactosidase

For construction of bifunctionally active membrane-bound fusion proteins, we designed plasmids encoding fusion proteins in which the carboxyl terminus of Escherichia coli proline carrier was joined to the amino terminus of E. coli beta-galactosidase directly or with a collagen linker inserted between the two. The expressions of these fusion proteins complemented deficiencies in both proline transport and beta-galactosidase activity in E. coli cells. The fusion proteins were stable and mostly localized in the cytoplasmic membrane. The proline transport activities of the fusion proteins were kinetically similar to that of the wild type proline carrier. The beta-galactosidase moiety of the collagen-linked fusion protein was liberated from membrane vesicles by collagenase treatment. The Km value of released beta-galactosidase for o-nitrophenyl beta-D-galactopyranoside hydrolysis was similar to that of membrane-bound beta-galactosidase in the fusion protein. These results indicated that the fusion proteins are bifunctionally active and exhibit normal proline transport and beta-galactosidase activities. The crypticity of the beta-galactosidase activity associated with the fusion proteins indicated that the carboxyl terminus of the proline carrier was located on the cytoplasmic side of the membrane.     

Ventralization of the Drosophila embryo by deletion of extracellular leucine-rich repeats in the Toll protein.

Dorsoventral polarity of the Drosophila embryo is established by a signal transduction pathway in which the maternal transmembrane protein Toll appears to function as the receptor for a ventrally localized extracellular ligand. Certain dominant Toll alleles encode proteins that behave as partially ligand-independent receptors, causing embryos containing these proteins to become ventralized. In extracts of embryos derived from mothers carrying these dominant alleles, we detected a polypeptide of approximately 35 kDa in addition to full-length Toll polypeptides with antibodies to Toll. Our biochemical analyses suggest that the smaller polypeptide is a truncated form of Toll lacking extracellular domain sequences. To assay the biological activity of such a shortened form of Toll, we synthesized RNA encoding a mutant polypeptide lacking the leucine-rich repeats that comprise most of Toll's extracellular domain and injected this RNA into embryos. The truncated Toll protein elicited the most ventral cell fate independently of the wild-type Toll protein and its ligand. These results support the view that Toll is a receptor whose extracellular domain regulates the intrinsic signaling activity of its cytoplasmic domain.     

Visualizing the microtubule-associated protein tau in the nucleus

Although tau is mainly known as an axonal microtubule-associated protein,many studies indicate that it is not restricted to this subcellular compartment.Assessing tau’s subcellular distribution,however,is not trivial as is evident from transgenic mouse studies.When human tau is over-expressed,it can be immunohistochemically localized to axons and the somatodendritic domain,modeling what is found in neurodegenerative diseases such as Alzheimer’s disease.Yet,in wild-type mice,despite its abundance,tau is difficult to visualize even in the axon.It is even more challenging to detect this protein in the nucleus,where tau has been proposed to protect DNA from damage.To establish a framework for future studies into tau’s nuclear functions,we compared several methods to visualize endogenous nuclear tau in cell lines and mouse brain.While depending on the fixation and permeabilization protocol,we were able to detect nuclear tau in SH-SY5Y human neuroblastoma cells,we failed to do so in N2a murine neuroblastoma cells.As a second method we used subcellular fractionation of mouse tissue and found that in the nucleus tau is mainly present in a hypophosphorylated form.When either full-length or truncated human tau was expressed,both accumulated in the cytoplasm,but were also found in the nuclear fraction.Because subcellular fractionation methods have their limitations,we finally isolated nuclei to probe for nuclear tau and found that the nuclei were free of cytoplasmic contamination.Together our analysis identifies several protocols for detecting tau in the nucleus where it is found in a less phosphorylated form.     

Association of a cytoplasmic dynein-like protein recognizable by anti-MAP 1C with the mammalian mitotic spindle

A rabbit antibody to bovine brain MAP 1C was prepared. The antibody stained the mitotic spindle of PtK2 cells by immunofluorescence. On immunoblots of PtK2 cell extract the antibody reacted with polypeptides of molecular weights greater than 350 and 80 KD that resemble the subunit proteins of bovine brain MAP 1C. An additional 135 KD polypeptide in the extract was also stained. These results indicate that a cytoplasmic dynein recognizable by the anti-MAP 1C antibody is localized in the mitotic spindle.     

Characterization of monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both myosin-II and low molecular mass nuclear proteins

We characterized nine monoclonal antibodies that bind to the heavy chain of Acanthamoeba myosin-IA. Eight of these antibodies bind to myosin-IB and eight cross-react with Acanthamoeba myosin-II. All but one of the antibodies bind to a 30-kD chymotryptic peptide of myosin-IA that derives from the COOH terminus of the molecule, and to tryptic peptides as small as 17 kD, hence these epitopes are clustered closely together on the heavy chain. None of the antibodies prevent heavy chain phosphorylation by myosin-I heavy chain kinase. One antibody inhibits the K+-EDTA ATPase activity and three antibodies inhibit the actin- activated Mg++-ATPase activity of myosin-I under the set of conditions that we tested. When fluorescent antibody staining of both whole cells and isolated nuclei is done, several of these monoclonal antibodies react strongly with nuclei. These antibodies also stain the cytoplasmic matrix, especially the cortex near the plasma membrane. All nine of the monoclonal antibodies bind to polypeptides of 30-34 kD that are highly enriched in nuclei isolated from Acanthamoeba. There is no myosin-I in the isolated nuclei, so the 30-34-kD polypeptides, not myosin-I, are responsible for the nuclear staining.     

Shortened cytoplasmic domain affects intracellular transport but not nuclear localization of a viral glycoprotein

Herpes simplex virus (HSV) buds from the inner nuclear membrane of the infected cells. The glycoprotein gB-1 of HSV contains a stretch of 69 hydrophobic amino acids near the COOH terminus and a 109-amino acid cytoplasmic domain. By oligonucleotide-directed mutagenesis, five gB-1 mutants were constructed which either lack a cytoplasmic tail or contained 3, 6, 22, or 43 amino acids in the cytoplasmic tail. When expressed in COS cells all of the mutant glycoproteins were synthesized but the rate of intracellular transport and the appearance at the cell surface of the mutant gB-1 protein lacking the cytoplasmic tail or containing 3 and 6 amino acids in the cytoplasmic domain was drastically reduced. The wild-type gB-1 as well as all of the mutants in the cytoplasmic tail were, however, located on the nuclear envelope. These results suggest that the cytoplasmic domain of the glycoprotein gB may play a role in intracellular transport but not in the nuclear localization.     

Putative terminase subunits of herpes simplex virus 1 form a complex in the cytoplasm and interact with portal protein in the nucleus

Herpes simplex virus (HSV) terminase is an essential component of the molecular motor that translocates DNA through the portal vertex in the capsid during DNA packaging. The HSV terminase is believed to consist of the UL15, UL28, and UL33 gene products (pUL15, pUL28, and pUL33, respectively), whereas the HSV type 1 portal vertex is encoded by UL6. Immunoprecipitation reactions revealed that pUL15, pUL28, and pUL33 interact in cytoplasmic and nuclear lysates. Deletion of a canonical nuclear localization signal (NLS) from pUL15 generated a dominant-negative protein that, when expressed in an engineered cell line, decreased the replication of wild-type virus up to 80-fold. When engineered into the genome of recombinant HSV, this mutation did not interfere with the coimmunoprecipitation of pUL15, pUL28, and pUL33 from cytoplasmic lysates of infected cells but prevented viral replication, most nuclear import of both pUL15 and pUL28, and coimmunoprecipitation of pUL15, pUL28, and pUL33 from nuclear lysates. When the pUL15/pUL28 interaction was reduced in infected cells by the truncation of the C terminus of pUL28, pUL28 remained in the cytoplasm. Whether putative terminase components localized in the nucleus or cytoplasm, pUL6 localized in infected cell nuclei, as viewed by indirect immunofluorescence. The finding that the portal and terminase do eventually interact was supported by the observation that pUL6 coimmunoprecipitated strongly with pUL15 and weakly with pUL28 from extracts of infected cells in 1.0 M NaCl. These data are consistent with the hypothesis that the pUL15/pUL28/pUL33 complex forms in the cytoplasm and that an NLS in pUL15 is used to import the complex into the nucleus where at least pUL15 and pUL28 interact with the portal to mediate DNA packaging.     

Isolation of a novel 190 kDa protein from tobacco BY-2 cells: possible involvement in the interaction between actin filaments and microtubules

Interaction between actin filaments (AFs) and microtubules (MTs) has been reported in various plant cells, and the presence of a factor(s) connecting these two cytoskeletal networks has been suggested, but its molecular entity has not been elucidated yet. We obtained a fraction containing MT-binding polypeptides, which induced bundling of AFs and of MTs. A 190 kDa polypeptide which associated with AFs was selectively isolated from the fraction. This polypeptide was thought to have an ability to bind to both AFs and MTs. We raised a monoclonal antibody against the 190 kDa polypeptide. Immunostaining demonstrated the association of the 190 kDa polypeptide with AF bundles and with MT bundles formed in vitro. Immunocytochemical studies throughout the cell cycle revealed that the 190 kDa polypeptide was localized in the nucleus before nuclear envelope breakdown, and in the spindle and the phragmoplast during cell division. After the re-formation of the nuclear envelope, the 190 kDa polypeptide was sequestered to the daughter nuclei. Using the antibody, we succeeded in cloning a cDNA encoding the 190 kDa polypeptide.