These were prepared as previously described with slight modification. Cells were collected at 2000 g, washed twice with KPi containing 0.1 mM PMSF, the pellets were suspended in the same buffer, the mixed with an equal volume of acid-washed glass beads. Cell suspensions were vortexed (4×15 s cycles), cell debris was removed by centrifugation at 1300 g for 10 min at 4 ⁰C, and the total protein concentration in the supernatants (i.e., the soluble protein extracts) was determined by the Bradford assay bovine serum albumin as a standard