GTP‐stimulated membrane fission by the N‐BAR protein AMPH‐1

Abstract

Membrane‐enclosed transport carriers sort biological molecules between stations in the cell in a dynamic process that is fundamental to the physiology of eukaryotic organisms. While much is known about the formation and release of carriers from specific intracellular membranes, the mechanism of carrier formation from the recycling endosome, a compartment central to cellular signaling, remains to be resolved. In Caenorhabditis elegans, formation of transport carriers from the recycling endosome requires the dynamin‐like, Eps15‐homology domain (EHD) protein, RME‐1, functioning with the Bin/Amphiphysin/Rvs (N‐BAR) domain protein, AMPH‐1. Here we show, using a free‐solution single‐particle technique known as burst analysis spectroscopy (BAS), that AMPH‐1 alone creates small, tubular‐vesicular products from large, unilamellar vesicles by membrane fission. Membrane fission requires the amphipathic H0 helix of AMPH‐1 and is slowed in the presence of RME‐1. Unexpectedly, AMPH‐1‐induced membrane fission is stimulated in the presence of GTP. Furthermore, the GTP‐stimulated membrane fission activity seen for AMPH‐1 is recapitulated by the heterodimeric N‐BAR amphiphysin protein from yeast, Rvs161/167p, strongly suggesting that GTP‐stimulated membrane fission is a general property of this important class of N‐BAR proteins.

The phospho‐regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis

“…Amph1 is a modular protein, with an N‐terminal Bin/Amphiphysin/Rvs (N‐BAR) domain characterised by the presence of long stretches of amphiphilic α‐helices, and can either homodimerize or form heterodimers with another neuronal isoform, Amph2 (Ramjaun et al, 1999; Wigge et al, 1997). The dimeric amphipathic helices of the N‐BAR domain recruit Amph1 to highly‐curved membranes and exhibit fission‐inducing activity (Kustigian et al, 2023; Peter et al, 2004; Snead et al, 2019). The central region of Amph1 contains a clathrin‐ and adaptor‐protein complex 2 (AP2)‐binding (CLAP) domain (Slepnev et al, 2000), which facilitates cargo sorting at endocytic pits (Wang et al, 1995) and, when disrupted, inhibits SV endocytosis (Evergren et al, 2004; Jockusch et al, 2005).…”