The heterotetrameric AP and F-COPI complexes help to define the cellular map of modern eukaryotes. To search for related machinery, we developed a structure-based bioinformatics tool, and identified the core subunits of TSET, a 'missing link' between the APs and COPI. Studies in Dictyostelium indicate that TSET is a heterohexamer, with two associated scaffolding proteins. TSET is non-essential in Dictyostelium, but may act in plasma membrane turnover, and is essentially identical to the recently described TPLATE complex, TPC. However, whereas TPC was reported to be plant-specific, we can identify a full or partial complex in every eukaryotic supergroup. An evolutionary path can be deduced from the earliest origins of the heterotetramer/scaffold coat to its multiple manifestations in modern organisms, including the mammalian muniscins, descendants of the TSET medium subunits. Thus, we have uncovered the machinery for an ancient and widespread pathway, which provides new insights into early eukaryotic evolution.DOI: http://dx.doi.org/10.7554/eLife.02866.001

A limitation to date of reverse transcriptase polymerase chain reactions (RT-PCRs) for the detection of small, round structured viruses (SRSVs) has been that they have detected only a narrow range of SRSVs due to the marked genomic diversity among strains. A total of 331 faecal samples collected from 136 separate incidents of gastroenteritis occurring in the UK between 1992 and 1994 were examined by RT-PCR employing a single primer pair (N1/E3). SRSV RNA was detected in samples from 93 of 101 (91%) incidents shown to be SRSV-associated by electron microscopy (EM) and in 5 of 35 (14%) SRSV-negative incidents. Amplification products were tested by Southern blot hybridisation with a pool of four digoxigenin (DIG)-labelled oligonucleotides derived from genomic sequence data of SRSV SPIEM types UK 1 to 4. Products from approximately 5% of amplified strains did not hybridise. The N1/E3 primer pair were shown to be SRSV-specific by their failure to amplify other faecal viruses including other human caliciviruses with typical calicivirus morphology. Hybridisation of PCR products with the individual oligonucleotides relating to SRSV SPIEM types UK 1-4 was investigated: 1 of 60 (1.7%) reacted with the UK1 probe, 2/60 (3.4%) reacted with the UK2 probe, 51/60 (85%) with the UK3 probe, and 27/60 (45%) reacted with the UK4 probe. All PCR products that hybridised with the UK4 probe hybridised with the UK3 probe; 6 (10%) failed to hybridise. Identification of this primer pair facilitates routine diagnosis of SRSV infection by RT-PCR and offers the potential for direct detection in food and environmental samples.

Abstract. Targeting of P-selectin to the regulated secretory organelles (RSOs) of phaeochromocytoma PC12 cells has been investigated. By expressing from cDNA a chimera composed of HRP and P-selectin, and then following HRP activity through subcellular fractionation, we have discovered that P-selectin contains signals that target HRP to the synaptic-like microvesicles (SLMV) as well as the dense-core granules (DCGs) of these cells. Mutagenesis of the chimera followed by transient expression in PC12 cells shows that at least two different sequences within the carboxy-terminal cytoplasmic tail of P-selectin are necessary, but that neither is sufficient for trafficking to the SLMV. One of these sequences is centred on the 10 amino acids of the membrane-proximal C1 exon that is also implicated in lysosomal targeting. The other sequence needed for trafficking to the SLMV includes the last four amino acids of the protein. The same series of mutations have a different effect on DCG targeting, showing that traffic to the two different RSOs depends on different features within the cytoplasmic domain of P-selectin. N'EUROENDOCRINE cells possess two kinds of organelles for regulated exocytosis; small synaptic or synaptic-like microvesicles (SLMVs) 1 and dense-core secretory granules (DCGs). The former contain only small molecules, the latter contain peptides and proteins as well. The ratio of these two regulated secretory organelles (RSOs) varies with cell type (35). Some proteins are found in the membranes of both organelles, reflecting the common requirements of exocytotic machinery (5,36). This raises the question of how such a biorganellar distribution is attained. The itinerary of such proteins must be complex, since the biogenesis of DCGs in the TGN is very different from SLMV formation, which probably occurs in some part of the endosomal system. Moreover, the relationship between proteins that become incorporated into the two different RSOs is unclear; for example, we do not know whether membrane proteins move from one organelle to the other via recycling, nor at what point the traffic bifurcates. In addition, very little is known at the present time about the signals involved in RSO targeting of membrane proteins.Please address all correspondence to Daniel F. Cutler, MRC Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, UCL, Gower Street, London WC1E 6BT, United Kingdom.1. Abbreviations used in this paper: BB, blotting buffer; DCG, dense-core granule; HB, homogenization buffer; PAM, peptidyl-amidating monooxygenase; RSO, regulated secretory organelle; SLMV, synaptic-like microvesicle; TfnR, transferrin receptor.Biogenesis of the SLMVs, and by implication that of small synaptic vesicles in neurons, is poorly understood. Most evidence suggests a central role for an endosomal compartment in SLMV origins. First, expression in nonneuronal cells usually leads to localization in endosomal compartments, often but not always those enriched in the transferrin receptor (4,14,25,28,29)....