Antonello D’Arrigo

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.

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 cysteine-rich 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 Zn2+. 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 GTPase-deficient 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.

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.

Hrs, an essential tyrosine kinase substrate, has been implicated in intracellular trafficking and signal transduction pathways. The protein contains several distinctive domains, including an N-terminal VHS domain, a phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding FYVE domain and two coiled-coil domains. Here we have investigated the roles of these domains in the subcellular localisation of Hrs. Hrs was found to colocalise extensively with EEA1, an established marker of early endosomes. While the membrane association of EEA1 was abolished in the presence of a dominant negative mutant of the endosomal GTPase Rab5, the localisation of Hrs to early endosomes was Rab5 independent. The VHS-domain was nonessential for the subcellular targeting of Hrs. In contrast, the FYVE domain as well as the second coiled-coil domain, which has been shown to bind to SNAP-25, were required for targeting of Hrs to early endosomes. A small construct consisting of only these two domains was correctly localised to early endosomes, whereas a point mutation (R183A) in the PtdIns(3)P-binding pocket of the FYVE domain inhibited the membrane targeting of Hrs. Thus, like EEA1, the endosomal targeting of Hrs is mediated by a PtdIns(3)P-binding FYVE domain in cooperation with an additional domain. We speculate that binding to PtdIns(3)P and a SNAP-25-related molecule may target Hrs specifically to early endosomes.