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.


RESULTS
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.


CONCLUSIONS
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.
Zhu
Huaiping
H
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
Qian
Hong
H
Li
Guangpu
G
D000465:Algorithms; D000818:Animals; D002478:Cells, Cultured; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D004789:Enzyme Activation; D025461:Feedback, Physiological; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008954:Models, Biological; D011485:Protein Binding; D015398:Signal Transduction; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; C510870:RabGEF1 protein, mouse; C486276:Rabep1 protein, mouse; D033921:Vesicular Transport Proteins; D006153:Guanosine Diphosphate; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D020696:rab5 GTP-Binding Proteins