Gene regulation by steroid hormones.

A short amino acid sequence able to specify nuclear location

The amino acid sequences of ten proteins with intracellular half-lives less than 2 hours contain one or more regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T). These PEST regions are generally, but not always, flanked by clusters containing several positively charged amino acids. Similar inspection of 35 proteins with intracellular half-lives between 20 and 220 hours revealed that only three contain a PEST region. On the basis of this information, it was anticipated that caseins, which contain several PEST sequences, would be rapidly degraded within eukaryotic cells. This expectation was confirmed by red blood cell-mediated microinjection of 125I-labeled caseins into HeLa cells where they exhibited half-lives of less than 2 hours. The rapid degradation of injected alpha- and beta-casein as well as the inverse correlation of PEST regions with intracellular stability indicate that the presence of these regions can result in the rapid intracellular degradation of the proteins containing them.

http://science.sciencemag.org/content/sci/234/4774/364.full.pdf

Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis

▪ Abstract The compartmentation of eukaryotic cells requires all nuclear proteins to be imported from the cytoplasm, whereas, for example, transfer RNAs, messenger RNAs, and ribosomes are made in the nucleus and need to be exported to the cytoplasm. Nuclear import and export proceed through nuclear pore complexes and can occur along a great number of distinct pathways, many of which are mediated by importin β-related nuclear transport receptors. These receptors shuttle between nucleus and cytoplasm, and they bind transport substrates either directly or via adapter molecules. They all cooperate with the RanGTPase system to regulate the interactions with their cargoes. Another focus of our review is nuclear export of messenger RNA, which apparently largely relies on export mediators distinct from importin β-related factors. We discuss mechanistic aspects and the energetics of transport receptor function and describe a number of pathways in detail.

Transport between the cell nucleus and the cytoplasm.

Nuclear targeting sequences--a consensus?

A point mutation in the simian virus 40 large-T gene, which was generated by mixed oligonucleotide mutagenesis and resulted in the conversion of Lys 128 to Thr, produced a large-T antigen that was detected in the cytoplasm but not the nucleus of cells Deletions within the surrounding sequence Lys-128Lys-Lys-Arg-Lys-Val-Glu also produce cytoplasmic large-T and define a region of the protein involved in nuclear location

Sequence requirements for nuclear location of simian virus 40 large-T antigen.

A short sequence of predominantly basic amino acids Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val from SV40 Large T is responsible for the normal nuclear location of the protein. Alteration of Lys-128 to each of six different residues other than Arg renders Large T cytoplasmic, whereas single amino acid changes in the surrounding region impair but do not prevent nuclear accumulation. When transposed to the amino terminus of cytoplasmic Large T species, or Escherichia coli beta-galactosidase or of chicken muscle pyruvate kinase, the sequence around Lys-128 of Large T is able to direct the recipient protein to the nucleus. This demonstrates that these amino acids can be sufficient for nuclear location and can act as a nuclear location signal. A computer search of over 2500 proteins reveals that some other nuclear proteins (for example, BK virus Large T, SV40 VP2 and adenovirus 72kDa DNA binding protein) contain very similar basic tracts, but so too do some presumed non-nuclear proteins (for example, poliovirus VP3). We suggest that the related sequence acts as the nuclear location signal in the other nuclear proteins but that the sequence does not function in all cases, perhaps because it is not accessible. A similar, but shorter or less basic sequence, was detected in a number of other nuclear proteins, for example, polyoma virus Large T, SV40 VP1 and several histones. However, such sequences were also found in many other proteins. Perhaps the shorter basic sequences can also act as nuclear location signals, but to be functional they need to be exposed (for example, at the amino terminus of the protein as in SV40 VP1) or to be present in multiple copies.

The nuclear location signal.

The ability of polyoma virus to transform cells results primarily from the action of one of the virus-coded early proteins, called middle-T antigen1–3. Middle-T has an associated tyrosine-specific protein kinase activity that can be measured in vitro and results in the phosphorylation of middle-T itself4–7. Almost all mutants so far tested that lack the ability to transform cells, also lack associated kinase activity2,4. Attempts to map within middle-T the tyrosine residue(s) that are phosphorylated in vitro suggest that a likely site of phosphorylation is tyrosine 315 (refs8–10 and unpublished results). The amino acid sequence preceding Tyr 315 includes a tract of six contiguous glutamic acid residues and bears some homology with that preceding the tyrosine phosphorylated in vivo in pp60v–src, the transforming protein of Rous sarcoma virus11, and with a region in the polypeptide hormone, gastrin, preceding a tyrosine that is sulphated12. Furthermore, although surprisingly large tracts of middle-T may be removed without affecting its transforming activity2,13–16, mutants that lack the sequences corresponding to amino acids 311–318 inclusive are transformation defective. Because the likely site of phosphorylation, the homology with pp60v–src and gastrin and the sequence apparently required for transformation all overlap, it has generally been accepted that this region of middle-T may form part of an essential region, possibly an active site on the protein17. Here we have used techniques of site-directed and site-specific mutagenesis to probe the sequence requirements in more detail. Contrary to expectation, the results obtained strongly suggest that Tyr 315 and conservation of the surrounding amino acid sequence are not essential for transformation.

Alexander F. Markham

Papers

Sequence requirements for nuclear location of simian virus 40 large-T antigen.

The nuclear location signal.

Transforming activity of polyoma virus middle-T antigen probed by site-directed mutagenesis.