Primary structure of chicken muscle pyruvate kinase mRNA.

The major DNA-binding protein, or infected-cell protein 8 (ICP8), encoded by herpes simplex virus can localize to the cell nucleus independently of other viral proteins. To define the nuclear localization signals within ICP8, we performed several forms of mutagenesis on the cloned ICP8 gene. Deletion analysis of the ICP8 gene showed that several portions of ICP8 are involved in its nuclear localization. To determine whether these regions were independent localization signals, we introduced various portions of the ICP8 gene into a series of cassette plasmids which allowed expression of fusion proteins containing pyruvate kinase, normally a cytoplasmic protein, fused to various portions of ICP8. These results showed that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of targeting protein kinase into the nucleus. However, inclusion of certain additional regions of ICP8 into the fusion protein led to an inhibition of nuclear localization.Therefore, the carboxyl-terminal 28 residues of ICP8 can act independently as a nuclear localization signal, but certain conformational constraints or folding or assembly requirements in the remainder of the protein can affect the nuclear localization of the protein. Our results demonstrate that sequences distant from a nuclear localization signal can affect its ability to function. A set of fusion vectors has been isolated which should be of general use for making 5' or 3' fusions in any reading frame to rapidly map localization signals.Nuclear localization of proteins is believed to occur through at least two mechanisms: small proteins lacking nuclear localization signals may diffuse into the nucleus through nuclear pores, where they are then sequestered by binding to intranuclear components, whereas larger proteins containing nuclear localization signals are actively transported from the cytoplasm to the nucleus (2, 7, 27). The latter process has been shown to involve two distinct steps in vitro: binding to the nuclear pore and translocation across the nuclear membrane (31, 38).Amino acid sequences responsible for the selective nuclear uptake of many proteins have been identified. These studies involved either construction of deletion mutants to determine which regions of the proteins are not needed for the nuclear localization or the construction of fusion proteins to define which regions of the proteins can direct cytoplasmic proteins such as pyruvate kinase (PK) (20), ,-galactosidase (17, 27, 28, 43), or invertase (30) into the nucleus. The simian virus 40 (SV40) T antigen contains one of the bestdefined nuclear localization signals, which consists of a highly basic stretch of amino acids (Pro-Lys-Lys-Lys-ArgLys-Val) (19,20,22). When these residues were fused to a cytoplasmic protein, PK, they could direct the fusion protein into the nucleus (20). Similar experiments were also performed to identify the sequences within other nuclear proteins which are sufficient to direct cytoplasmic proteins to the nucleus (17,26,27,30,43). These studies have shown tha...

Section: Resultsmentioning

confidence: 99%

Distal protein sequences can affect the function of a nuclear localization signal.

Gao

Knipe

1992

“…Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process. (16) and rabbit anti-chicken muscle pyruvate kinase antibody (24) have been demonstrated previously. A synthetic peptide (Tyr-Cys-Thr-ProPro-Lys-Lys-Lys-Arg-Lys-Val-Tyr) homologous to the SV40 large-T-antigen nuclear location signal sequence was obtained from Genetic Design.…”

mentioning

confidence: 99%

Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

Markland¹,

Smith²,

Roberts³

1987

An in vitro nuclear translocation system is described in which isolated rat liver nuclei were incubated in a defined buffered medium containing radiolabeled or fluorescently labeled exogenous proteins. The nuclei were rapidly recovered, extracted, and analyzed for the presence of associated radiolabeled or fluorescently labeled proteins. The isolated nuclei exhibited the same specificity for protein uptake as seen previously in vivo, accumulating simian virus 40 wild-type large-T antigen and p53 while excluding a cytoplasmic variant of large-T antigen (dlO) and bovine serum albumin. The rapid nuclear accumulation of wild-type large-T antigen was shown to be selective and dependent upon the recognition of a wild-type nuclear location signal, ATP and temperature dependent, and unidirectional. Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process.A nuclear location signal sequence has been identified in simian virus 40 (SV40) large-T antigen (18). The sequence Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu is both necessary and sufficient for the nuclear localization of large-T antigen variants as well as otherwise cytoplasmic proteins (19). Synthetic peptides homologous to the SV40 large-T antigen nuclear signal can also target proteins of various sizes to the nucleus (15,22).Several known nuclear proteins contain basic amino acid sequences homologous to the SV40 Iarge-T antigen prototypic signal sequence (36). In some cases, it has been shown that these sequences constitute the putative nuclear location signals of their respective proteins (32, 39). The large-T antigen of polyomavirus contains two basic sequences which contribute to the nuclear accumulation of this protein (30). Recently, it has been demonstrated that the efficacy of a nuclear location signal is dependent upon the context of the protein within which it is present (33).Little is known about how nuclear location signals function. The SV40 large-T antigen signal permits the rapid nuclear entry of proteins which would otherwise be too large to accumulate in the nucleus by passive diffusion (15,19,22). This process exhibits a high degree of specificity, since a single point mutation at codon 128 of the signal sequence can completely abolish this phenomenon (5,18,21). This suggests that a highly efficient and selective cellular apparatus must be responsible for the recognition and nuclear translocation of proteins containing nuclear location signals.The development of a cell-free system to study the signaldependent nuclear accumulation of proteins would facilitate the characterization of nuclear signal-mediated translocation of proteins across the nuclear envelope and the identification of the translocation apparatus itself. The inclusion of homogeneous preparations of isolated nuclei in such a system would enhance the likelihood that it would reproducibly exhibit nuclear transport characteristics similar to those observed previously in vi...

“…Residue numbers reported correspond to the sequence of the Prague C strain of Rous sarcoma virus (40). The amino acids derived from the chicken Ml PK (residues 120 to 529) are the same in all cases (26). The nucleotide sequence encoding PK was derived from plasmid RL142PK1OX, RL18PK8X, or RL18PK12X as required to maintain an open reading frame.…”

mentioning

confidence: 99%

The src protein contains multiple domains for specific attachment to membranes.

Kaplan¹,

Varmus²,

Bishop³

1990

107

The proteins encoded by the oncogene v-src and its cellular counterpart c-src (designated generically here as ppWsrc) are tightly associated with both plasma membranes and intracellular membranes. This association is due in part to the amino-terminal myristylation of pp6OsrC, but several lines of evidence suggest that amino-terminal portions of the protein itself are also involved. We now report that pp60src contains at least three domains which, in conjunction with myristylation, are capable of mediating attachment to membranes and determining subcellular localization. We identified these domains by fusing various portions of pp6fsrc to pyruvate kinase, which is normally a cytoplasmic protein. Amino acids 1 to 14 of pp6Osc are sufficient to mediate both myristylation and the attachment of pyruvate kinase to cytoplasmic granules. In contrast, amino acids 38 to 111 mediate association with the plasma membrane and perinuclear membranes, whereas amino acids 204 to 259 mediate association primarily with perinuclear membranes. We conclude that pp60Osc contains independent domains that target the protein to distinctive subcellular locations and thus may facilitate diverse biological functions of the protein.The products of the retroviral oncogene v-src and its cellular counterpart c-src (designated generically here as pp6Osrc) are protein tyrosine kinases that are associated with membranes. Association of pp60 src with membranes is essential for transformation by v-src (19). In various cell types, the viral and cellular src proteins are associated with plasma membranes (7,8,21,44), with perinuclear membranes (36), with secretory organelles in both chromaffin cells and platelets (30, 33), and with growth cones in developing neurons (27,41). Little is known about how pp6Osrc is specifically targeted to these subcellular locations.The amino terminus of pp6Osrc is covalently coupled to a 14-carbon fatty acid, myristic acid (4, 39). The myristyl moiety promotes the association of pp6Osrc with membranes (2, 11). On the other hand, not all myristylated src proteins are associated with membranes (3, 11), and some nonmyristylated src proteins are membrane associated, albeit weakly (12,20). These results suggest that pp6Osrc contains amino acid sequences which, in conjunction with amino-terminal myristylation, mediate the association of the protein with membranes.Previous work has implicated the amino-terminal 10 kilodaltons (kDa) of pp6Osrc in attachment to membranes (22,35