Regulating imports

The yeast transport factor Kap95p (importin-β in mammals) binds cytoplasmic cargo and transports it through the nuclear pore complex. This active transport is regulated by nuclear RanGTP, which disassembles import complexes by binding to importin, making nuclear import irreversible. Importin is a flexible molecule comprised of 19 HEAT repeats that form a helicoid structure. A previous structure of a truncated importin-β in complex with RanGppNHp showed interactions between the N-terminal arch (HEAT repeats 1–11) of importin-β. These involved primarily the switch II loop and a patch of basic residues on Ran. Now, crystallographic studies by Stewart and colleagues on the full-length Kap95p–RanGTP complex reveal interactions between the RanGTP switch I loop, an area that undergoes large conformational change dependent upon its nucleotide-bound state, and the C-terminal arch (HEAT repeats 12–19) of Kap95p. Comparison with other importin-β–cargo complexes shows that RanGTP produces substantial conformational changes in Kap95p by causing minor changes in orientation between the 19 HEAT repeat elements. Mutational analysis indicates that the interaction between the switch I loop and the C-terminal arch is critical for RanGTP's ability to dissociate cargo from Kap95p. The authors propose that RanGTP employs an unzipping mechanism to actively displace cargo by first binding to the N-terminal arch of Kap95p and then its C-terminal arch. By binding to both the N- and C-terminal halves of Kap95p, they suggest RanGTP locks Kap95p in an inflexible conformation that is unable to bind cargo. (Nature advance online publication, 1 May 2005 10.1038/nature03578) MM

Fine-tuning adaptation

Adapting to the environment requires that an organism sense and respond to external signals such as changes in nutrient supply, temperature and level of oxidative stress. At the cellular level, the response begins with detection of stimuli at the cell surface and activation of signaling cascades that control gene expression. For example, TIF-IA, a transcription factor in the nucleolus that modulates the activity of polymerase I (Pol I), is phosphorylated at multiple sites in response to different environmental conditions. When nutrients are available, TIF-IA is modified at serine residues by the mTOR and MAPK-dependent signaling cascades and activated for transcription initiation. Mayer and colleagues show that when cells experience oxidative or ribotoxic stress, TIF-IA is phosphorylated at Thr200 by the c-Jun N-terminal protein kinase 2 (JNK2) and inactivated. TIF-IA, through its interactions with other transcription factors, targets Pol I to ribosomal DNA promoters. The authors show that phosphorylation of Thr200 prevents TIF-IA from interacting with Pol I thus preventing the formation of the transcription initiation complex at these promoters. This subsequently affects ribosome biogenesis and hence protein synthesis in nutrient starved cells. Interestingly, Thr200 phosphorylation also causes TIF-IA to move from the nucleolus to the nucleoplasm where it is sequestered from Pol I. Previous data had shown that retention in the nucleolus was controlled by mTOR hyperphosphorylation at Ser199. So it seems that reversible phosphorylation at two neighboring sites, Ser199 and Thr200, can modulate the activity and affect the localization of TIF-IA. How this exquisite fine-tuning is achieved by the simple addition of a phosphoryl group to a side chain will require further study. (Genes Dev. 19, 933–941, 2005) EJ

Keeping mold out

Plants have several mechanisms to resist the pathogens such as viruses, bacteria and fungi. One of these mechanisms is called the hypersensitive response (HR), which involves the formation of localized necrotic lesions near infected sites to restrict the spread of pathogens. In tomato, HR against the leaf mold fungus Cladosporium fulvum is mediated by the host Cf-2 gene product that senses the fungal avirulence protein (Avr2) secreted during infection. Also required in this process is a secreted tomato cysteine protease Rcr3. How these components function to trigger HR is not well understood. To characterize the role of Avr2 and Rcr3 in this surveillance mechanism, de Wit and colleagues tested whether Avr2 directly targets Rcr3. They show that Avr2 can compete with E64, which irreversibly modifies and inhibits Rcr3, and that this activity does not require any other plant factors. These observations suggest that Avr2 also inhibits Rcr3 and that Avr2 and Rcr3 physically interact. Co-immunoprecipitation data confirm this latter suggestion. They further demonstrate that the Avr2–Rcr3 complex is required for the initiation of HR. Taken together, these observations support the 'guard' hypothesis of how the Cf-2 protein functions in HR: Avr2 targets a host factor to facilitate growth of the leaf mold, and Cf-2 monitors the status of the host target. Further studies will be necessary to understand whether Cf-2 recognizes Avr2–Rcr3 and how recognition leads to HR. (Science, published online 21 April 2005 10.1126/science1111404) HPF

Gap filling

DNA base excision repair (BER) is a multistep process that fixes damage to and loss of DNA bases. BER is initiated when a DNA glycosylase recognizes and cleaves the N-glycosylic bond that links the base to the phosphodiester backbone. This hydrolytic reaction generates a so-called abasic site. Next the AP endonuclease cleaves the DNA backbone and removes some neighboring nucleotides. Finally, the gap is filled and sealed by DNA polymerase I and DNA ligase. DNA lesions can impede DNA- and RNA-polymerase leading to replication or transcription defects or to error-prone bypass synthesis. Therefore, for BER to be faithful, recognition and hydrolysis of the damaged base must be tightly coupled to excision of the AP site and restoration of the information and integrity of the DNA. Previous work showed that human thymine-DNA glycosylase (TDG) displays product inhibition, that is, it remains tightly bound to the AP site and that modification of TDG with small ubiquitin-like modifiers (SUMOs) induces its dissociation. However the details of how this occurred remained unclear. Steinacher and Schar now show that upon interaction with DNA, the N-terminal domain of TDG undergoes a large conformational change that facilitates its tight association with the substrate/product DNA. They further demonstrate that SUMOylation of the C-terminal domain alters this conformation of the N terminus in a way that facilitates dissociation of TDG from the product AP site. This allows the subsequent handover of the repair intermediate to the downstream enzymes of BER. (Curr. Biol. 15, 616–623, 2005) BK

Research highlights written by Hwa-ping Feng, Evelyn Jabri,Boyana Konforti and Michelle Montoya.