Slp4-a and MyRIP acting via Rab27A control the probability of WPB exocytosis independently of WPB pool size. (A) Rab27A cycles between the cytosol and the WPB membrane, a process thought to be dependent on GDP-dissociation inhibitor (GDI; rectangular insert, cellular components are not drawn to scale). Rab cycling distributes Rab27A approximately evenly between all WPBs within the cell. Because the cellular content of Rab27A remains constant in culture, increases in WPB pool size (gray gradient) over a “Physiologic range” (beige bar) result in a decrease in the amount of Rab27A on individual WPBs (purple gradient). The probability of WPB release (P_r, dashed line) remains constant over this “Physiologic range” of WPB pool sizes, indicating that the control of WPB exocytosis cannot solely depend on the absolute amount of WPB-Rab27A. As long as the cellular content of Rab27A (and its effectors) remains constant, the steady-state fractional occupancy of Rab27A by its effectors will also be constant, irrespective of the absolute Rab27A concentration on individual WPBs. This raises the possibility that the fractional occupancy of Rab27A by its effectors, rather than just the absolute amount of these molecules, sets the P_r. Substantial depletion (by KD, extreme right of panel A) or high overexpression (OE, extreme left in panel A; data not shown) of Rab27A results in a reduction in P_r. (B) WPB exocytosis is determined by the balance of occupancy of WPB-Rab27A by negative (MyRIP that binds only GTP-Rab27A) and positive (Slp4-a that binds both GTP- and GDP-Rab27A) effectors. Manipulations that alter either effector concentration or cellular Rab27A levels can perturb the fractional occupancy of WPB-Rab27A by its effectors, resulting in changes (small vertical arrows) in the probability of WPB exocytosis.