COPI vesicles accumulating in the presence of a GTP restricted arf1 mutantare depleted of anterograde and retrograde cargo

Microinjection of the slowly hydrolyzable GTP analogue GTP gamma S or the e ctopic expression of a GTP restricted mutant of the small GTPase arf1 (arf1 [Q71L]) leads to the rapid accumulation of COPI coated vesicles and buds in living cells. This effect is blocked at 15 degrees C and by microinjection of antibodies against beta-COP, Anterograde and retrograde membrane protei n transport markers, which have been previously shown to be incorporated in to COPI vesicles between the endoplasmic reticulum and Golgi complex, are d epleted from the GTP gamma S or arf1[Q71L] induced COPI coated vesicles and buds. In contrast, in control cells 30 to 60% of the COPI carriers co-loca lize with these markers. These in vivo data corroborate recent in vitro wor k, suggesting that GTP gamma S and arf1[Q71L] interfere with the sorting of membrane proteins into Golgi derived COPI vesicles, and provide the first in vivo evidence for a role of GTP hydrolysis by arf1 in the sorting of car go into COPI coated vesicles and buds.