We describe an assay for in Wivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-donain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When a C-te l fragment of ubiqultin (Cub) is expressed as a fusion to a reporter protein, the fusion is cleaved only If an N-terminal frgment of ubiquin (Nub) is ao expressed in the same cell. This reconstitution of native ubiquitin from Its rag ts, detectable by the in vivo cleavage assay, is not observed with a mutat ly altered Nub. However, if Cub and the altered Nub are each linked to polypeptides that interact in vivo, the cleavage of the fusion cotaining Cub is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in mvo protein interactions. This method, termed USPS (ubiquitin-based splitprotein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.
The specific and covalent labeling of fusion proteins with synthetic molecules opens up new ways to study protein function in the living cell. Here we present a novel method that allows for the specific and exclusive extracellular labeling of proteins on the surfaces of live cells with a large variety of synthetic molecules including fluorophores, protein ligands, or quantum dots. The approach is based on the specific labeling of fusion proteins of acyl carrier protein with synthetic molecules through post-translational modification catalyzed by phosphopantetheine transferase. The specificity and versatility of the labeling should allow it to become an important tool for studying and manipulating cell surface proteins and for complementing existing approaches in cell surface engineering.
Protein I is a hetero‐tetramer which contains two copies each of p11 and p36. p36 (calpactin I, lipocortin II) is a major substrate of retrovirally encoded tyrosine protein kinases, while p11 modulates several Ca2+‐induced properties also displayed by p36 alone. Here we have characterized the p11 binding site on p36 by fluorescence spectroscopy using porcine p36 labelled at cysteine 8 with the fluorophore Prodan (6‐proprionyl‐2‐dimethylamino‐naphthalene). We have used peptides of differing length from the amino‐terminal domain of p36 to restrict the major binding site to the first 12 residues. Noticeable binding is still observed with a peptide containing only the first nine residues. Interestingly the N‐terminal acetyl group of p36 forms a functional part of the p11 binding site. CD studies indicate that the binding region can form an alpha‐helix, which seems to have amphiphatic properties when projected on a helical wheel. This structural element is also known for a calmodulin binding protein. Thus the question is raised whether other p11/calmodulin‐related proteins interact with their target proteins via a similar mechanism. We also discuss how p11 could modulate p36 associated properties.
The split-Ubiquitin (split-Ub) technique was used to map the molecular environment of a membrane protein in vivo. C ub , the C-terminal half of Ub, was attached to Sec63p, and N ub , the N-terminal half of Ub, was attached to a selection of differently localized proteins of the yeast Saccharomyces cerevisiae. The efficiency of the N ub and C ub reassembly to the quasi-native Ub reflects the proximity between Sec63-C ub and the N ub -labeled proteins. By using a modified Ura3p as the reporter that is released from C ub , the local concentration between Sec63-C ub -RUra3p and the different N ub -constructs could be translated into the growth rate of yeast cells on media lacking uracil. We show that Sec63p interacts with Sec62p and Sec61p in vivo. Ssh1p is more distant to Sec63p than its close sequence homologue Sec61p. Employing N ub -and C ub -labeled versions of Ste14p, an enzyme of the protein isoprenylation pathway, we conclude that Ste14p is a membrane protein of the ER. Using Sec63p as a reference, a gradient of local concentrations of different t-and v-SNARES could be visualized in the living cell. The RUra3p reporter should further allow the selection of new binding partners of Sec63p and the selection of molecules or cellular conditions that interfere with the binding between Sec63p and one of its known partners.
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