Delayed Onset of Positive Feedback Activation of Rab5 by Rabex-5 and Rabaptin-5 in Endocytosis

Background: Rabex-5 is a guanine nucleotide exchange factor (GEF) that specifically activates Rab5, i.e., converting Rab5-GDP to Rab5-GTP, through two distinct pathways to promote endosome fusion and endocytosis. The direct pathway involves a pool of membrane-associated Rabex-5 that targets to the membrane via an early endosomal targeting (EET) domain. The indirect pathway, on the other hand, involves a cytosolic pool of Rabex-5/Rabaptin-5 complex. The complex is recruited to the membrane via Rabaptin-5 binding to Rab5-GTP, suggesting a positive feedback mechanism. The relationship of these two pathways for Rab5 activation in the cell is unclear. Methodology/Principal Findings: We dissect the relative contribution of each pathway to Rab5 activation via mathematical modeling and kinetic analysis in the cell. These studies show that the indirect pathway constitutes a positive feedback loop for converting Rab5-GDP to Rab5-GTP on the endosomal membrane and allows sensitive regulation of endosome fusion activity by the levels of Rab5 and Rabex-5 in the cell. The onset of this positive feedback effect, however, contains a threshold, which requires above endogenous levels of Rab5 or Rabex-5 in the cell. We term this novel phenomenon delayed response. The presence of the direct pathway reduces the delay by increasing the basal level of Rab5-GTP, thus facilitates the function of the Rabex-5/Rabaptin-5-mediated positive feedback loop. Conclusion: Our data support the mathematical model. With the model's guidance, the data reveal the affinity of Rabex-5/Rabaptin-5/Rab5-GTP interaction in the cell, which is quantitatively related to the Rabex-5 concentration for the onset of the indirect positive feedback pathway. The presence of the direct pathway and increased Rab5 concentration can reduce the Rabex-5 concentration required for the onset of the positive feedback loop. Thus the direct and indirect pathways cooperate in the regulation of early endosome fusion.