Distinguishing mitochondrial inner membrane orientation of dual specific phosphatase 18 and 21.

Dual specificity phosphatase (DSP) 18 and 21 are members of a poorly understood subfamily of protein tyrosine phosphatases (PTP) that are unique in their ability to dephosphorylate both phosphotyrosine and phosphoserine/threonine residues in vitro. Because of the difficulty in identifying substrate specificity, determining subcellular localization can help to resolve biological function of these phosphatases. DSP18 and DSP21 are targeted to mitochondria by internal localization signals. Surprisingly, DSP18 and DSP21 are both peripherally associated with the mitochondrial inner membrane, however, DSP18 is oriented toward the intermembrane space while DSP21 is facing the matrix compartment. This chapter describes methodology for purification of recombinant protein and demonstration of phosphatase activity, for mitochondrial purification and subfractionation of mitochondria to determine submitochondrial localization and for determining membrane orientation and strength of membrane association.