MAPPING OF THE INTERMOLECULAR ASSOCIATION OF THE HUMAN T-CELL LEUKAEMIA LYMPHOTROPIC VIRUS TYPE-I P12(I) AND THE VACUOLAR H+-ATPASE 16 KDA SUBUNIT PROTEIN/

The p12(I) protein, a small hydrophobic protein encoded by the human T cell leukaemia/lymphotropic virus type I pX region, contains a prolin e-rich region located between two putative transmembrane (TM) domains. The p12(I) protein is associated with cellular endomembranes, and phy sically binds to the 16 kDa subunit of the vacuolar H+-ATPase proton p ump. To investigate the nature of the 16 kDa and p12(I) interaction an d to determine the oncogenic domain of p12(I), we constructed p12(I) m utant proteins in which various portions of the TM domains were delete d, as well as a p12(I) mutant containing a single amino acid substitut ion. These mutants were tested for binding to the 16 kDa subunit of th e vacuolar H+-ATPase in HeLa/Tat cells and for the capability to poten tiate transformation by bovine papillomavirus type 1 E5 oncoprotein in mouse C127 cells. The results indicated that both TM domains of the p 12(I) protein were dispensable for its interaction with the 16 kDa pro tein, whereas partial or complete deletion of the proline-rich region resulted in decreased or no binding of the p12(I) protein to the 16 kD a subunit. Immunofluorescence analysis of HeLa/Tat cells transfected w ith the p12(I) mutants showed that deletion of the proline-rich region did not alter the subcellular localization of these mutant p12(I) pro teins, suggesting direct involvement of the proline-rich domain in bin ding rather than the failure of these p12(I) mutants to reach the appr opriate cellular compartment. Mapping of 16 kDa subunit mutants in bin ding with the p12(I) protein suggested that molecular determinants loc ated between the second and third TM domain of the 16 kDa protein migh t be involved in this interaction. Finally, most of the p12(I) mutants lost the ability to potentiate transformation of C127 cells indicatin g that binding of p12(I) to the 16 kDa subunit does not directly corre late with oncogenicity.