Role of Rab1 in the maturation of transport intermediates exported from Endoplasmic Reticulum

PABLO MONETTA AND CECILIA ALVAREZ

Iguazú, Misiones. Argentinas

Congreso; SAIB; 2004

Recruitment of type I coat protein complex (COPI) is required for maturation of vesicles exported from the Endoplasmic Reticulum (ER) to the Golgi complex. COPI is recruited to membranes by a small GTPase of the ARF family. To become active, ARF must interact with GBF1, a guanine nucleotide exchange factor that stimulates the exchange of GDP for GTP. Previous works indicate that the small GTPase Rab1 modulates COPI recruitment; however, the molecular mechanism that links Rab1 activity and COPI recruitment is unknown. In this report, GBF1 was identified as a Rab1 effector protein. GST pull down assays show that GBF1 binds preferentially to Rab1-GTP. Additionally, doubleimmunofluo- rescence assays show that over-expression of the Rab1 GTP-restricted mutant (Rab1-Q67L) increases membrane association of COPI proteins and GBF1, preferentially at ER exit sites. Finally, in order to characterize in vivo the effect of the Rab1-Q67L on COPI dynamic, Fluorescence Recovery After Photobleaching (FRAP) was performed in GFP-e-COP expressing cells co-transfected with or without Rab1-Q67L. Fluorescence recovery was significantly slower in Rab1-Q67L expressing cells, indicating that COPI association from membranes is dependent on Rab1-GTP hydrolysis. Taken together our results suggest that Rab1 recruits GBF1 to membranes, which in turns activate ARF1, which then recruits COPI and allows COPII/COPI exchange on transport intermediates derived from the ER.