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.     

Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae.

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.     

A nuclear localization domain in the hnRNP A1 protein

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta- galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.     

Identification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal

The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands. To gain more insight into the regulation of PLAG1 function, we searched for PLAG1-interacting proteins. Using the yeast two-hybrid system, we identified karyopherin alpha2 as a PLAG1-interacting protein. Physical interaction between PLAG1 and karyopherin alpha2 was confirmed by an in vitro glutathione S-transferase pull-down assay. Karyopherin alpha2 escorts proteins into the nucleus via interaction with a nuclear localization sequence (NLS) composed of short stretches of basic amino acids. Two putative NLSs were identified in PLAG1. The predicted NLS1 (KRKR) was essential for physical interaction with karyopherin alpha2 in glutathione S-transferase pull-down assay, and its mutation resulted in decreased nuclear import of PLAG1. Moreover, NLS1 was able to drive the nuclear import of the cytoplasmic protein beta-galactosidase. In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin alpha2 binding nor in its nuclear import. The residual nuclear import of PLAG1 after mutation of the NLS1 was assigned to the zinc finger domain of PLAG1. These observations indicate that the nuclear import of PLAG1 is governed by its zinc finger domain and by NLS1, a karyopherin alpha2 recognition site.     

Context affects nuclear protein localization in Saccharomyces cerevisiae.

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.     

Yeast cytochrome b2 gene: isolation with antibody probes

An efficient technique was used to clone the gene for yeast cytochrome b2, (a nuclear encoded mitochondrial protein) using the expression vector, lambda gt11 (lac 5 nin 5 c1857 S100). This enables the insertion of yeast DNA into the beta-galactosidase structural gene (lacZ) and promotes synthesis of hybrid proteins. Screening of antigen producing clones in the lambda gt11 recombinant genomic library was achieved using antiserum against cytochrome b2 according to Young and Davis (1983) Two recombinants containing part of the gene coding for cytochrome b2 were isolated and characterized as follows: by their expression in Escherichia coli cells, examined by immuno-blotting with antibodies to pure cytochrome b2. by DNA sequence analysis. One recombinant carries a 3 Kb yeast DNA insert which contains the whole nucleotide sequence encoding cytochrome b2 and a few amino acids of the amino terminal presequence.     

Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei.

The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.     

The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.     

Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor

We have shown that hybrid proteins composed of the yeast repressible acid phosphatase (PHO5) and bacterial beta-galactosidase (lacZ) interfere with secretion of native acid phosphatase (Wolfe, P. B. (1988) J. Biol. Chem. 263, 6908-6915). We now report that PHO5-LacZ hybrid proteins have a more general effect on secretion and prevent translocation of several secreted proteins. Translocation of both the mating pheromone alpha-factor and the vacuolar protease carboxypeptidase Y is partially blocked when PHO5-LacZ hybrids are expressed. Cell fractionation and protease sensitivity indicate that alpha-factor and carboxypeptidase Y accumulate in precursor form on the cytoplasmic surface of the endoplasmic reticulum. Indirect immunofluorescence with antibody directed against beta-galactosidase supports the localization of hybrid proteins to the endoplasmic reticulum. Analysis of the hybrid protein phenotype in vivo and in vitro suggests that the hybrid proteins deplete a soluble factor required for efficient translocation across the endoplasmic reticulum. First, a decrease in the expression of a hybrid protein in vivo decreases its effect on translocation. Second, an in vitro translation/translocation reaction, prepared from a hybrid-bearing strain, is deficient in its ability to translocate prepro-alpha-factor across yeast microsomal membranes. This deficiency is complemented by addition of cytosol prepared from wild type cells. Finally, the hybrid protein phenotype is shown to be independent of the requirement for SSA gene products.     

The actin-related protein Act3p of Saccharomyces cerevisiae is located in the nucleus.

Actin-related proteins, a group of protein families that exhibit about 50% sequence identity among each other and to conventional actin, have been found in a variety of eukaryotic organisms. In the budding yeast Saccharomyces cerevisiae, genes for one conventional actin (ACT1) and for three actin-related proteins (ACT2, ACT3, and ACT5) are known. ACT3, which we recently discovered, is an essential gene coding for a polypeptide of 489 amino acids (Act3p), with a calculated molecular mass of 54.8 kDa. Besides its homology to conventional actin, Act3p possesses a domain exhibiting weak similarity to the chromosomal protein HMG-14 as well as a potential nuclear localization signal. An antiserum prepared against a specific segment of the ACT3 gene product recognizes a polypeptide band of approximately 55 kDa in yeast extract. Indirect immunofluorescence experiments with this antiserum revealed that Act3p is located in the nucleus. Nuclear staining was observed in all cells regardless of the stage of the cell cycle. Independently, immunoblotting experiments with subcellular fractions showed that Act3p is indeed highly enriched in the nuclear fraction. We suggest that Act3p is an essential constituent of yeast chromatin.     

Effects of a highly basic region of human immunodeficiency virus Tat protein on nucleolar localization.

Human immunodeficiency virus type 1 encodes a positive trans-activator protein, Tat, which is located predominantly in the cell nucleolus. To study the role of the basic region of Tat in nucleolar localization, we constructed fusion genes encoding serially deleted segments of Tat joined to the amino-terminal end of the Escherichia coli beta-galactosidase molecule. We show that the basic region of Tat was sufficient for nuclear localization but not for nucleolar localization. Addition of three amino acids (59, 60, and 61) of the Tat sequence at the C-terminal end of the basic region was necessary for the chimeric beta-galactosidase to localize in the nucleus as well as in the nucleolus. We demonstrate that a short amino acid sequence (G-48 RKKRRQRRRA HQ N-61), when fused to the amino terminus of beta-galactosidase, can act as a nucleolar localization signal.     

Degradation of a nuclear-localized protein in mammalian COS cells, using Escherichia coli beta-galactosidase as a model protein.

To investigate the mechanism of degradation of proteins localized in the nucleus, we constructed genes encoding modified Escherichia coli beta-galactosidases and expressed them in mammalian COS cells. When the beta-galactosidase with a nuclear localization signal from SV 40 T antigen was expressed in COS cells, the beta-galactosidase polypeptide was localized in the nuclei and was stable for at least 4 h. When 16 amino acid residues were deleted from the C-terminal end, the beta-galactosidase polypeptide was also observed in the nuclei but it was degraded rapidly, with a half-life of 1.6 h. When the nuclear localizing signal was replaced with a mutant sequence, which lacks nuclear targeting activity, the beta-galactosidase polypeptides were present throughout the cells rather than in the nuclei. The beta-galactosidase polypeptide with the complete C terminus was stable and the cytoplasmic truncated polypeptide was degraded at the same rate as the nuclear C terminus truncated polypeptide. The beta-galactosidase polypeptides with the complete C terminus were present as a tetramer as reported previously and had beta-galactosidase activity, but the C terminus truncated polypeptides were present as monomer and had no enzyme activity, indicating that C terminus truncated beta-galactosidase is malfolded. Together, the results suggest that a nuclear-localized malfolded protein is degraded as rapidly as a cytoplasmic malfolded protein.     

A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional- lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.     

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.     

Identification and functional analysis of the nuclear localization signals of ribosomal protein L25 from Saccharomyces cerevisiae

The regions of the large subunit ribosomal protein L25 from Saccharomyces cerevisiae responsible for nuclear localization of the protein were identified by constructing fusion genes encoding various segments of L25 linked to the amino terminus of beta-galactosidase. Indirect immunofluorescence of yeast cells expressing the fusions demonstrated that amino acid residues 1 to 17 as well as 18 to 41 of L25 promote import of the reporter protein into the nucleus. Both nuclear localization signal (NLS) sequences appear to consist of two distinct functional parts: one showed relatively weak nuclear targeting activity, whereas the other considerably enhances this activity but does not promote nuclear import by itself. Microinjection of in vitro prepared intact and N-terminally truncated L25 into Xenopus laevis oocytes demonstrated that the region containing the two NLS sequences is indeed required for efficient nuclear localization of the ribosomal protein. This conclusion was confirmed by complementation experiments using a yeast strain that conditionally expresses wild-type L25. The latter experiments also indicated that amino acid residues 1 to 41 of L25 are required for full functional activity of yeast 60 S ribosomal subunits. Yeast cells expressing forms of L25 that lack this region are viable, but show impaired growth and a highly abnormal cell morphology.     

Engineering nuclear localization signals in modular protein vehicles for gene therapy

Amino acids from 126 to 135 of the SV40 virus T antigen act as efficient nuclear localization signal during infection but also when fused to recombinant proteins. This peptide has been inserted into two alternative acceptor sites of a modified Escherichia coli beta-galactosidase which also displays a DNA-binding domain, a cell-binding motif for integrin alpha(v)beta(3) targeting and cell internalization, and a cryptic nuclear targeting signal naturally present in the bacterial enzyme. In cultured cells, the presence of the SV40 peptide enhances the expression of a delivered DNA up to 30-fold. However, the DNA expression levels are largely depending on the chosen insertion site for the SV40 segment concomitant to the structural impact of peptide accommodation on the protein vehicle. The structural stability of the hybrid protein, apparently critical for efficient gene transfer, is discussed in the context of modular protein engineering to develop non-viral vectors for gene therapy.