Abstract
ABSTRACTSec7 protein is a guanine nucleotide exchange factor in the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. Aplysia Sec7 proteins (ApSec7s) play many roles in neurite outgrowth and synaptic facilitation in Aplysia neurons. However, the binding property of Aplysia ARF1 by ApSec7 isoforms has not been examined. In this study, we found that the cloned Aplysia ARF1 (ApARF1) protein only localized to the Golgi complex when it was expressed alone in HEK293T cells; however, if ApARF1 was co-expressed with plasma membrane-targeted ApSec7, it localized to both the plasma membrane and the Golgi complex via association with the Sec7 domain of ApSec7. Moreover, in HEK293T cells expressing both ApARF1 and another Sec7 isoform, ApSec7(VPKIS), the pleckstrin homology domain of ApSec7(VPKIS) associated with ApARF1, resulting in its localization to the Golgi complex. Overall, we propose a model in which ApSec7(VPKIS) activates ApARF1 in the Golgi complex, while ApSec7 recruits ApARF1 to the plasma membrane where it activates ApARF1/6 downstream signaling.
Highlights
ADP-ribosylation factors (ARFs) are key molecules regulating vesicle and organelle trafficking in cells (Donaldson & Jackson 2011)
The plasma membrane localization of ApARF6EGFP was enhanced by overexpression of mRFPApSec7-pleckstrin homology (PH) but not of mRFP-Aplysia Sec7 proteins (ApSec7s)-PH(VPKIS) (p < .001 compared to that of all other groups, one-way ANOVA; F = 14.7, Tukey’s post-hoc test) (Figure 3(d)). These results indicate that Aplysia ARF1 (ApARF1) can associate with the PH domain of ApSec7-PH(VPKIS) but not with the PH domain of ApSec7, whereas Aplysia ARF6 (ApARF6) binds to the PH domain of ApSec7 but not to that of ApSec7(VPKIS)
PI(3,4,5)P3 is indispensable for binding of the PH domain of ARNO/ cytohesin2 to active ARF6 (Santy & Casanova 2001)
Summary
ADP-ribosylation factors (ARFs) are key molecules regulating vesicle and organelle trafficking in cells (Donaldson & Jackson 2011). ARFs play key roles in various intracellular compartments (Tsuchiya et al 1991). ARF1 is localized to the Golgi complex and is an important mediator of the trafficking between the Golgi complex and endocytic compartments, whereas ARF6 is localized to the plasma membrane and is involved in neurite outgrowth, endocytosis, and trafficking between endosomes and the plasma membrane (D’Souza-Schorey & Chavrier 2006). It has been suggested that ARF1 is targeted to the plasma membrane where it exerts its functions (Maranda et al 2001; Li et al 2003; Cohen et al 2007). Accumulating evidence suggests that ARF1 plays a role in both the plasma membrane and Golgi complex
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