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The p121 protein, a small hydrophobic protein encoded by the human T cell leukaemia/lymphotropic virus type I pX region, contains a proline-rich region located between two putative transmembrane (TM) domains. The p121 protein is associated with cellular endomembranes, and physically binds to the 16 kDa subunit of the vacuolar H+-ATPase proton pump. To investigate the nature of the 16 kDa and p121 interaction and to determine the oncogenic domain of p121, we constructed p121 mutant proteins in which various portions of the TM domains were deleted, as well as a p121 mutant containing a single amino acid substitution. These mutants were tested for binding to the 16 kDa subunit of the vacuolar H+-ATPase in HeLa/Tat cells and for the capability to potentiate transformation by bovine papillomavirus type 1 E5 oncoprotein in mouse C127 cells. The results indicated that both TM domains of the p121 protein were dispensable for its interaction with the 16 kDa protein, whereas partial or complete deletion of the proline-rich region resulted in decreased or no binding of the p121 protein to the 16 kDa subunit. Immunofluorescence analysis of HeLa/Tat cells transfected with the p121 mutants showed that deletion of the proline-rich region did not alter the subcellular localization of these mutant p121 proteins, suggesting direct involvement of the proline-rich domain in binding rather than the failure of these p121 mutants to reach the appropriate cellular compartment. Mapping of 16 kDa subunit mutants in binding with the p121 protein suggested that molecular determinants located between the second and third TM domain of the 16 kDa protein might be involved in this interaction. Finally, most of the p121 mutants lost the ability to potentiate transformation of C127 cells indicating that binding of p121 to the 16 kDa subunit does not directly correlate with oncogenicity.
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