Sec71 separates Golgi stacks in Drosophila S2 cells

Abstract

Golgi stacks are the basic structural units of the Golgi apparatus. Golgi stacks are separated from each other and scattered in the cytoplasm of Drosophila cells. Here, we report that the ARF-GEF inhibitor Brefeldin A (BFA) induces the formation of BFA bodies, which are aggregates of Golgi stacks, trans-Golgi networks, and recycling endosomes. Recycling endosomes are located in the centers of BFA bodies, while Golgi stacks surround them on their trans sides. Live imaging of S2 cells revealed that Golgi stacks repeatedly merged and separated on their trans sides, and BFA caused successive merger by inhibiting separation, forming BFA bodies. S2 cells carrying genome-edited BFA-resistant mutant Sec71M717L did not form BFA bodies at high concentrations of BFA; S2 cells carrying genome-edited BFA-hypersensitive mutant Sec71F713Y produced BFA bodies at low concentrations of BFA. These results indicate that Sec71 is the sole BFA target for BFA body formation and controls Golgi stack separation. Finally, we showed that impairment of Sec71 in fly photoreceptors induces BFA body formation with accumulation of both apical and basolateral cargos, resulting in inhibition of polarized transport.

The Golgi Apparatus and its Next-Door Neighbors

“…Recently, Fujii et al (2020b) showed in Drosophila cells that knockdown of Arf GEF Garz also induces dynamic relocalization of Golgi proteins to the ER, while leaving behind GM130, a component of cis -Golgi, in the cytoplasm, again suggesting the existence of a GECCO-like compartment in invertebrate cells (see Figure 4C ). It should be mentioned here that mammalian GM130 was also shown to stay in BFA-resistant structures together with ERGIC ( Nakamura et al, 1995 ).…”

“…BFA treatment of Drosophila cells also gave very interesting results ( Fujii et al, 2020b ) ( Figures 4B,C ). Drosophila has two Arf GEFs, called Sec71 (homolog of mammalian BIG and yeast Sec7, which functions in TGN) and Garz (homolog of mammalian GBF1 and yeast Gea1, which functions in cis -Golgi).…”

“…Very similar alignment is seen in the BFA bodies of Arabidopsis cells ( Langhans et al, 2011 ). On the other hand, when the other Arf GEF, Garz, is knocked down in Drosophila, most of the Golgi markers are absorbed into the ER ( Figure 4C ), like the case with mammalian cells ( Fujii et al, 2020b ). As GNL1, the Arf GEF working in the ER-Golgi traffic in Arabidopsis, is insensitive to BFA, the BFA body formation in Arabidopsis and Drosophila is most likely based on the inhibition of Arf activation only in the post-Golgi processes, leading to the aggregation of the TGN-RE system.…”

“…This finding manifests amazing similarity to the case of plant Golgi and TGN. Indeed, markers of TGN and RE show close localization and even partial overlap in Drosophila cells ( Fujii et al, 2020a ; Fujii et al, 2020b ).…”

“…In the BFA body, RE sits at the center, TGN follows next and Golgi cisternae align from trans to cis toward the periphery. (C) When the other Arf GEF of Drosophila Garz, which is insensitive to BFA, is knocked down, the Golgi main body is mostly absorbed into the ER, while leaving behind a cis component (GM130, red spots) ( Fujii et al, 2020b ).…”