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.
