Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids.

Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.

Temperature-sensitive control of protein activity by conditionally splicing inteins.

Conditional or temperature-sensitive (TS) alleles represent useful tools with which to investigate gene function. Indeed, much of our understanding of yeast has relied on temperature-sensitive mutations which, when available, also provide important insights into other model systems. However, the rarity of temperature-sensitive alleles and difficulty in identifying them has limited their use. Here we describe a system to generate temperature-sensitive alleles based on conditionally active inteins. We have identified temperature-sensitive splicing variants of the yeast Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein inserted within Gal4 and transferred these into Gal80. We show that Gal80-intein(TS) is able to efficiently provide temporal regulation of the Gal4/upstream activation sequence (UAS) system in a temperature-dependent manner in Drosophila melanogaster. Given the minimal host requirements necessary for temperature-sensitive intein splicing, this technique has the potential to allow the generation and use of conditionally active inteins in multiple host proteins and model systems, thereby widening the use of temperature-sensitive alleles for functional protein analysis.

Cloning and expression of Drosophila SOCS36E and its potential regulation by the JAK/STAT pathway.

The suppressor of cytokine signalling (SOCS) gene family was originally identified as an immediate early response to cytokine signalling and function as negative regulators of the Janus kinase (JAK)/signal tranducers and activators of transcription (STAT) signal transduction pathway [Krebs and Hilton, J. Cell Sci. 113 (2000) 2813; Starr and Hilton, Int. J. Biochem. Cell Biol. 30 (1998) 1081]. Although key components of the Drosophila JAK/STAT pathway have been identified [Brown et al., Curr. Biol. 11 (2001) 1700, reviewed in Zeidler et al., Oncogene 19 (2000) 2598], regulators of the pathway, and SOCS genes in particular, have not yet been characterised. Here we report the cloning of Drosophila SOCS36E and show its expression pattern during embryonic and imaginal disc development. SOCS36E is expressed in an essentially identical pattern to the Drosophila JAK/STAT pathway ligand unpaired (Upd). It is not expressed in upd mutant embryos and is upregulated in response to ectopic activation of the pathway during both embryonic and imaginal development.

Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand.

The characterisation of ligands that activate the JAK/STAT pathway has the potential to throw light onto a comparatively poorly understood aspect of this important signal transduction cascade. Here, we describe our analysis of the only invertebrate JAK/STAT pathway ligands identified to date, the Drosophila unpaired-like family. We show that upd2 is expressed in a pattern essentially identical to that of upd and demonstrate that the proteins encoded by this region activate JAK/STAT pathway signalling. Mutational analysis demonstrates a mutual semi-redundancy that can be visualised in multiple tissues known to require JAK/STAT signalling. In order to better characterise the in vivo function of these ligands, we developed a reporter based on a natural JAK/STAT pathway responsive enhancer and show that ectopic upd2 expression can effectively activate the JAK/STAT pathway. While both Upd and Upd2 are secreted JAK/STAT pathway agonists, tissue culture assays show that the signal-sequences of Upd and Upd2 confer distinct properties, with Upd associated primarily with the extracellular matrix and Upd2 secreted into the media. The differing biophysical characteristics identified for Upd-like molecules have implications for their function in vivo and adds another aspect to our understanding of cytokine signalling in Drosophila.