Abstract
Acute inhibition is a powerful technique to test proteins for direct roles and order their activities in a pathway, but as a general gene-based strategy, it is mostly unavailable in mammalian systems. As a consequence, the precise roles of proteins in membrane trafficking have been difficult to assess in vivo. Here we used a strategy based on a genetically encoded fluorescent protein that generates highly localized and damaging reactive oxygen species to rapidly inactivate exit from the endoplasmic reticulum (ER) during live-cell imaging and address the long-standing question of whether the integrity of the Golgi complex depends on constant input from the ER. Light-induced blockade of ER exit immediately perturbed Golgi membranes, and surprisingly, revealed that cis-Golgi-resident proteins continuously cycle to peripheral ER-Golgi intermediate compartment (ERGIC) membranes and depend on ER exit for their return to the Golgi. These experiments demonstrate that ER exit and extensive cycling of cis-Golgi components to the cell periphery sustain the mammalian Golgi complex.