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

Abstract

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...

A Beta-Stranded Motif Drives Capsid Protein Oligomers of the Parvovirus Minute Virus of Mice into the Nucleus for Viral Assembly

“…In contrast, VP-2 nuclear transport was halted by deletions created in any part of its sequence. While the activity of some NLSs is highly con- strained by a particular protein context (20,52), the extreme sensitivity of VP-2 nuclear transport to any deletion is unusual, an indication that this activity requires the correct cytoplasmic folding of the whole protein and hence its overall conformation. Indeed, wt and mutant VP-2 protein folding in the cytoplasm was verified by their ability to conform capsid epitopes in this compartment, while VP-1 protein appears unable to fold into similar structures (Fig.…”

Endogenous interleukin 1 alpha must be transported to the nucleus to exert its activity in human endothelial cells.

“…It is noteworthy that not only residues 79 to 85 but also the IL-la precursor can translocate n-Gal to the nucleus. Indeed, it has been reported that sequences distant from the NTS can affect its ability to function (13). Moreover, it has been shown that acidic fibroblast growth factor (aFGF) contains a putative NTS which is able to direct the expression of ,-Gal to the nucleus; however, this NTS is unable to target either aFGF itself or an aFGF-1-Gal fusion protein to the nucleus, suggesting that aFGF contains an additional sequence which prevents endogenously expressed aFGF from being translocated into the nucleus (50).…”

Nuclear and nucleolar targeting of human ribosomal protein S6.

“…The two identical NLSs KRRR of S6 represent a relatively strong signal because when this tetrapeptide is fused to 13-galactosidase it directs the hybrid protein exclusively into the nucleus (Figure 3). Efficiency of nuclear import can be influenced by the protein context of the NLS (Roberts et al, 1987;Nelson and Silver, 1989), by distant sequences (Gao and Knipe, 1992) …”