A Screen for Membrane Fission Catalysts Identifies the ATPase EHD1

Abstract

Membrane fission manifests during cell division, synaptic transmission, vesicular transport, and organelle biogenesis, yet identifying proteins that catalyze fission remains a challenge. Using a facile and robust assay system of supported membrane tubes in a microscopic screen that directly monitors membrane tube scission, we detect robust GTP- and ATP-dependent as well as nucleotide-independent fission activity in the brain cytosol. Using previously established interacting partner proteins as bait for pulldowns, we attribute the GTP-dependent fission activity to dynamin. Biochemical fractionation followed by mass spectrometric analyses identifies the Eps15-homology domain-containing protein1 (EHD1) as a novel ATP-dependent membrane fission catalyst. Together, our approach establishes an experimental workflow for the discovery of novel membrane fission catalysts.

Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission

“…It is of interest that acute siRNA knockdown of EHD4 induces much larger endosome size than the more chronic EHD4 knock-out in the CRISPR/Cas9 NIH3T3 cells, suggesting that compensation may be occurring in the latter cells. Overall, these data are consistent with the role for EHD1 in vesiculation of tubular and vesicular recycling endosomes [19,20,22,23,45] and further support the notion that both EHD1 and EHD4 regulate endosomal fission, as their depletion leads to enlarged SE in cells.…”

“…Whereas previous studies have addressed the localization of EHD4 to SE [32,33], these studies predated the concept of EHD1 as a major endosomal fission protein [19,22,23,45,74]. Moreover, while previous studies demonstrated that EHD4 was in part recruited to SE [32], those studies did not quantify the degree of recruitment, and in our current study we observe significantly impaired recruitment of EHD1 to SE.…”

“…Given the role of EHD1 in endosomal fission [19][20][21][22][23][24][25][26] and our previous study suggesting that EHD4 regulates endosomal size [32], we hypothesized that EHD4 might coordinate endosome fission and size with EHD1. To test this idea, we took untreated cells, mock-shRNA transfected cells, or cells subjected to EHD4-shRNA knock-down and examined EEA1-labeled SE after immunostaining (Fig 2A-2F).…”

“…Over the past two decades, the Eps15 Homology Domain protein 1 (EHD1) has emerged as a major regulator of endocytic recycling [17,18]. Recent studies have demonstrated that in vitro, EHD1 is capable of membrane fission, whereas in cells it localizes to SE and recycling endosomes, and induces ATP-catalyzed membrane fission [19][20][21][22][23][24][25][26]. We have recently demonstrated that EHD1 undergoes recruitment to endosomal membranes upon induction of receptor-mediated endocytosis, where it interacts with SNX17 and promotes endosomal fission [27].…”

Cellular functions and intrinsic attributes of the ATP‐binding Eps15 homology domain‐containing proteins

“…This in turn expands the bulged region on the tube causing intervening regions to thin down and ultimately undergo scission (Figure 6b). Importantly, EHD1 both in a purified state as well as in a complex mixture of brain cytosol displayed a dramatic capacity to catalyze fission 23,81 . Such reciprocal shape change in membrane tubes arises from a conservation of membrane area in systems with finite reservoir.…”