Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-a

The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser(112) in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.

Domains of the TGN: Coats, tethers and G proteins

The trans-Golgi network is the major sorting compartment of the secretory pathway for protein, lipid and membrane traffic. There is a constant flow of membrane and cargo to and from this compartment. Evidence is emerging that the trans-Golgi network has multiple biochemically and functionally distinct subdomains, each of which contributes to the combined sorting and transport requirements of this dynamic compartment. The recruitment of distinct arrays of protein complexes to trans-Golgi network membranes is likely to produce the diversity of structure and biochemistry observed amongst subdomains that serve to generate different carriers or maintain resident trans-Golgi network components. This review discusses how these subdomains may be formed and examines the molecular players involved, including G proteins, clathrin adaptors and golgin tethers. Diversity within these protein families is highlighted and shown to be critical for the functionality of the trans-Golgi network, as a mediator of protein sorting and membrane transport, and for the maintenance of Golgi structure.

Television satire, decmocracy and the decay of public language: John Clarke's verbal caricature

This paper examines the contributions of John Clarke to the field of political satire through his interviews with straight-man Bryan Dawe on ABC TV’s The 7.30 Report. Clarke’s work represents one of the last vestiges of what was once a vigorous satiric tradition in TV comedy, specifically the practice of political caricature. There was The Mavis Bramston Show in the 1960s and The Naked Vicar Show in the 1970s, while The Gillies Report in the 1980s was probably the best example of sustained political caricature in television comedy. Even in later sketch-based shows such as Fast Forward and The Late Show in the early 1990s, political caricature was a significant component of the material, whereas it seems to have all but disappeared from current television comedy. The paper investigates the disappearance of this type of comedy from Australian television screens and also discusses why the longevity, consistency, not to mention accuracy, of Clarke’s satire is so important in the current political climate. Clarke’s political caricature is almost entirely language-based, expertly parodying the spin-doctored rhetoric of our elected representatives and business leaders. This leads to a secondary focus of the paper, which is a discussion of Clarke’s unique form of satire in the context of what an historian (and former satirist) identifies as ‘the decay of public language’.