A glutamine residue in the membrane-associating domain of the bovine papillomavirus type 1 E5 oncoprotein mediates its binding to a transmembrane component of the vacuolar H(+)-ATPase.

The 44-amino-acid E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. It is a highly hydrophobic polypeptide which dimerizes and localizes to the Golgi apparatus and endoplasmic reticulum membranes. Recent evidence suggests that E5 modulates the phosphorylation and internalization of the epidermal growth factor and colony-stimulating factor 1 receptors and constitutively activates platelet-derived growth factor receptors in C127 and FR3T3 cells. Although no direct interaction with these growth factor receptors has yet been identified, the E5 oncoprotein has been shown recently to interact with the hydrophobic 16-kDa component of the vacuolar H(+)-ATPase (16K protein) [D. J. Goldstein, M. E. Finbow, T. Andresson, P. McLean, K. Smith, V. Bubb, and R. Schlegel, Nature (London) 352:347-349, 1991]. In the current study, we have further analyzed the E5-16K protein complex by fast protein liquid chromatography and shown that each E5 dimer appears to bind two 16K proteins. In order to define the specific amino acid residues of E5 which participate in this binding, mutated E5 epitope fusion proteins were analyzed for their ability to coprecipitate 16K protein. Transformation-defective mutants containing amino acid substitutions within the short hydrophilic carboxyl-terminal domain retained the ability to associate with the 16K protein. However, E5 mutants lacking the glutamine residue in the hydrophobic domain were markedly inhibited in 16K protein binding. Most interestingly, the placement of a glutamine in several random hydrophobic sequences facilitated 16K protein binding, defining this residue as a potential binding site for the 16K protein component of the proton pump and exemplifying the critical role of hydrophilic amino acids for mediating specific interactions between transmembrane proteins.