Abstract
ER membrane contact sites (MCSs) define the position where early and late endosomes undergo constriction and fission. ER tubules are recruited to the divide between cargo sorting domains on endosomes that will then constrict and undergo fission. Our aim was to purify ER factors that regulate MCS formation and fission in animal cells. Based on the identity of the candidate proteins, we hope to be able to test hypotheses about the mechanisms used by the ER to drive organelle constriction and division at MCSs. Our strategy to identify MCS proteins was to target a promiscuous biotin ligase (BirA) to ER‐associated fission sites. BirA has a modification reach to approximately 30nm, which is ideal for bridging membrane contacts (which have a typical spacer distance of ~5–15nm). Our hybrid construct localizes as predicted and biotinylates ER proteins at MCSs in mammalian cells that we could purify and then identify by mass spec. This strategy has identified ER proteins that regulate ER‐associated endosome fission.
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