The Context

Maintaining the nucleus as a distinct biochemical compartment requires a mechanism by which proteins are segregated between the nucleus and the cytoplasm. Studies in the 1970s established that small molecules diffuse rapidly across the nuclear envelope, but that most proteins are unable to do so. It therefore appeared likely that nuclear proteins are specifically recognized and selectively imported to the nucleus from their sites of synthesis on cytoplasmic ribosomes.

Earlier experiments of Günter Blobel and his colleagues had established that proteins are targeted to the endoplasmic reticulum by signal sequences consisting of short stretches of amino acids (see Chapter 9). In this 1984 paper, Alan Smith and his colleagues extended this principle to the targeting of nuclear proteins by identifying a short amino acid sequence that serves as a nuclear localization signal.

The Experiments

The viral protein SV40 T antigen was used as a model for studies of nuclear localization in animal cells. T antigen is a 94-kd protein that is required for SV40 DNA replication and is normally localized to the nucleus of SV40-infected cells. Previous studies in both Alan Smith's laboratory and in the laboratory of Janet Butel (Lanford and Butel, 1984, Cell 37: 801–813) had shown that mutation of Lys-128 to either Thr or Asn prevented the normal nuclear accumulation of T antigen in both rodent and monkey cells. Rather than being transported to the nucleus, these mutant T antigens remained in the cytoplasm, suggesting that Lys-128 was part of a nuclear localization signal. Kalderon and colleagues tested this hypothesis using two distinct experimental approaches.

First, they determined the effects of different deletions on the subcellular localization of T antigen. Mutant T antigens bearing deletions that eliminated amino acids either between residues 1 and 126 or between residue 136 and the C terminus were found to accumulate normally in the nucleus. In contrast, a mutant with a deletion of amino acids 127 to 132 remained in the cytoplasm. Thus, the amino acid sequence extending from residue 127 to 132 appeared to be responsible for nuclear localization of T antigen.

To determine whether this amino acid sequence was able to target other proteins to the nucleus, the investigators constructed chimeras in which the T antigen amino acid sequence was fused to proteins that were normally cytoplasmic. These experiments established that the addition of T antigen amino acids 126 to 132 to either β-galactosidase or pyruvate kinase is sufficient to specify the nuclear accumulation of these otherwise cytoplasmic proteins (see figure). This short amino acid sequence of SV40 T antigen thus functions as a nuclear localization signal, which is both necessary and sufficient to target proteins for nuclear import.

The Impact

As Kalderon and colleagues suggested in their 1984 paper, the nuclear localization signal of SV40 T antigen has proved to “represent a prototype of similar sequences in other nuclear proteins.” By targeting proteins for nuclear import, these signals are key to establishing the biochemical identity of the nucleus and maintaining the fundamental division of eukaryotic cells into nuclear and cytoplasmic compartments. Nuclear localization signals are now known to be recognized by cytoplasmic receptors that transport their substrate proteins to the nuclear pore complex. Although the mechanism of transport through the nuclear pore complex remains to be elucidated, the identification of nuclear localization signals was a key advance in understanding nuclear protein import.