Nature © Macmillan Publishers Ltd 1998 8 F Fi ig gu ur re e 1 1 The endosomal localization of EEA1-CT and its Hrs FYVE-finger hybrid depends on PI(3)K activity and an intact FYVE finger. BHK21 cells were co-transfected with C215S (k, l) and then incubated for 20 minutes at 37 ᑻC in the absence (a, b, e-h, k, l) or presence (c, d and i, j) of 20 nM wortmannin. The cells were permeabilized with 0.05% saponin to wash out cytosolic protein, then fixed and finally stained with anti-Rab5 (a, c, e, g, i, k) or anti-Myc-epitope (b, d, f, h, j, l) antibodies and examined by confocal immunofluorescence microscopy 5 . a shows the same cell as b, c shows the same cell as d, and so on. Arrows indicate examples of co-localization of Rab5 with epitope-tagged protein. Scale bar, 10 Ȗm. Nature

EEA1, a 162-kDa autoantigen associated with subacute cutaneous systemic lupus erythematosus, is a coiled-coil protein localized to early endosomes and cytosol. At its C terminus, the protein contains a cysteinerich motif, which is shared with Vps27, Fab1, and Vac1, yeast proteins implicated in membrane traffic (Mu, F. T., Callaghan, J. M., Steele-Mortimer, O., Stenmark, H., Parton, R. G., Campbell, P. L., McCluskey, J., Yeo, J. P., Tock, E. P., and Toh, B. H. (1995) J. Biol. Chem. 270, 13503-13511). Here we show that this motif constitutes a genuine zinc binding domain, which we term the FYVE finger (based on the first letters of four proteins containing this motif). Profile-based data base searches identified the FYVE finger in 11 distinct proteins. The FYVE finger-containing C terminus of EEA1 was found to bind 2 mol equivalents of Zn 2؉. Mutations of conserved histidine and cysteine residues in the FYVE motif independently reduced zinc binding to 1 mol equivalent. Confocal immunofluorescence microscopy of transfected HEp2 cells revealed that the C-terminal part (residues 1277-1411) of EEA1 colocalizes extensively with a GTPasedeficient mutant of the early endosomal GTPase Rab5, while deletion of the FYVE finger or mutations that interfere with zinc binding cause a cytosolic localization. These results implicate the FYVE finger in the specific localization of EEA1 to endosomes.Endocytosis involves the cellular uptake of extracellular compounds by the invagination and pinching off of defined areas of the plasma membrane. The so formed endocytic vesicles fuse with early endosomes, from where the endocytosed material can be relocated to a number of alternative destinations (1). The sorting function of the early endosome compartment has been extensively studied (2), but still our knowledge about the molecular basis for endocytosis and endosome sorting is fragmentary. Only few molecules have so far been found specifically associated with the early endosome compartment. One of them is Rab5, a GTPase regulating homotypic fusion between early endosomes (3), and, presumably, the heterotypic fusion between endocytic vesicles and early endosomes (4). Recently, autoimmune sera from some patients with subacute systemic lupus erythematosus were found to react with a 162-kDa peripheral membrane protein specifically localized to early endosomes (5). This autoantigen, called early endosome antigen 1 (EEA1) 1 , comprises extensive coiled-coil regions, and at its N and C termini it contains sequence motifs reminiscent of zinc fingers, protein structures originally found in nucleic acid binding proteins (6, 7). The C-terminal zinc-finger-like domain is particularly interesting, as it is conserved among several non-nuclear proteins, some of which are involved in intracellular trafficking (5,8). In this report we have focused on this domain, which we have now found in 11 different proteins. We show that it binds two Zn 2ϩ ions and plays a major role in the intracellular localization of EEA1. EXPERIMENTAL PROCEDURES Materials-Hydrox...

DNA mismatch recognition and binding in human cells has been thought to be mediated by the hMSH2 protein. Here it is shown that the mismatch-binding factor consists of two distinct proteins, the 100-kilodalton hMSH2 and a 160-kilodalton polypeptide, GTBP (for G/T binding protein). Sequence analysis identified GTBP as a new member of the MutS homolog family. Both proteins are required for mismatch-specific binding, a result consistent with the finding that tumor-derived cell lines devoid of either protein are also devoid of mismatch-binding activity.

The molecular defects responsible for tumor cell hypermutability in humans have not yet been fully identified. Here the gene encoding a G/T mismatch-binding protein (GTBP) was localized to within 1 megabase of the related hMSH2 gene on chromosome 2 and was found to be inactivated in three hypermutable cell lines. Unlike cells defective in other mismatch repair genes, which display widespread alterations in mononucleotide, dinucleotide, and other simple repeated sequences, the GTBP-deficient cells showed alterations primarily in mononucleotide tracts. These results suggest that GTBP is important for maintaining the integrity of the human genome and document molecular defects accounting for variation in mutator phenotype.

Objective: Necrotising enterocolitis (NEC) remains one of the primary causes of morbidity and mortality in neonates and alternative strategies are needed. Stem cells have become a therapeutic option for other intestinal diseases, which share some features with NEC. We tested the hypothesis that amniotic fluid stem (AFS) cells exerted a beneficial effect in a neonatal rat model of NEC. Design: Rats intraperitoneally injected with AFS cells and their controls (bone marrow mesenchymal stem cells, myoblast) were analysed for survival, behaviour, bowel imaging (MRI scan), histology, bowel absorption and motility, immunofluorescence for AFS cell detection, degree of gut inflammation (myeloperoxidase and malondialdehyde), and enterocyte apoptosis and proliferation. Results: AFS cells integrated in the bowel wall and improved rat survival and clinical conditions, decreased NEC incidence and macroscopic gut damage, improved intestinal function, decreased bowel inflammation, increased enterocyte proliferation and reduced apoptosis. The beneficial effect was achieved via modulation of stromal cells expressing cyclooxygenase 2 in the lamina propria, as shown by survival studies using selective and non-selective cyclooxygenase 2 inhibitors. Interestingly, AFS cells differentially expressed genes of the Wnt/β-catenin pathway, which regulate intestinal epithelial stem cell function and cell migration and growth factors known to maintain gut epithelial integrity and reduce mucosal injury. Conclusions: We demonstrated here for the first time that AFS cells injected in an established model of NEC improve survival, clinical status, gut structure and function. Understanding the mechanism of this effect may help us to develop new cellular or pharmacological therapies for infants with NEC

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The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the transGolgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate in trans-Golgi network to endosome as well as in endocytic membrane traffic.

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