Membrane fission by protein crowding

Abstract

Membrane fission, which facilitates compartmentalization of biological processes into discrete, membrane-bound volumes, is essential for cellular life. Proteins with specific structural features including constricting rings, helical scaffolds, and hydrophobic membrane insertions are thought to be the primary drivers of fission. In contrast, here we report a mechanism of fission that is independent of protein structure-steric pressure among membrane-bound proteins. In particular, random collisions among crowded proteins generate substantial pressure, which if unbalanced on the opposite membrane surface can dramatically increase membrane curvature, leading to fission. Using the endocytic protein epsin1 N-terminal homology domain (ENTH), previously thought to drive fission by hydrophobic insertion, our results show that membrane coverage correlates equally with fission regardless of the hydrophobicity of insertions. Specifically, combining FRET-based measurements of membrane coverage with multiple, independent measurements of membrane vesiculation revealed that fission became spontaneous as steric pressure increased. Further, fission efficiency remained equally potent when helices were replaced by synthetic membrane-binding motifs. These data challenge the view that hydrophobic insertions drive membrane fission, suggesting instead that the role of insertions is to anchor proteins strongly to membrane surfaces, amplifying steric pressure. In line with these conclusions, even green fluorescent protein (GFP) was able to drive fission efficiently when bound to the membrane at high coverage. Our conclusions are further strengthened by the finding that intrinsically disordered proteins, which have large hydrodynamic radii yet lack a defined structure, drove fission with substantially greater potency than smaller, structured proteins.

A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission

“…In contrast, recent work from our group found that protein crowding can drive membrane fission independent of protein structural or assembly properties (Snead et al, 2017). Specifically, molecular crowding among proteins attached to membrane surfaces at high density generates steric pressure that can bend membranes and ultimately overcome the energetic barrier to membrane fission (Busch et al, 2015; Snead et al, 2017; Stachowiak et al, 2012).…”

“…In contrast, recent work from our group found that protein crowding can drive membrane fission independent of protein structural or assembly properties (Snead et al, 2017). Specifically, molecular crowding among proteins attached to membrane surfaces at high density generates steric pressure that can bend membranes and ultimately overcome the energetic barrier to membrane fission (Busch et al, 2015; Snead et al, 2017; Stachowiak et al, 2012). In support of this mechanism, we found that bulky, intrinsically disordered proteins, which occupy significantly larger membrane footprints in comparison to well-folded proteins of similar mass (Hofmann et al, 2012), can be potent drivers of membrane fission.…”

“…1B) and tethered vesicle experiments (Fig. 6) reveal that full-length Epsin 1 drives membrane fission more efficiently in comparison to the isolated ENTH domain (Snead et al, 2017). …”

“…This assay builds upon earlier work from the Stamou group (Hatzakis et al, 2009; Kunding, Mortensen, Christensen, & Stamou, 2008; Lohr, Kunding, Bhatia, & Stamou, 2009), which utilized immobilized vesicles to measure the sensitivity of proteins to membrane curvature. However, our procedure, first reported in a recent study (Snead et al, 2017), is the first to use this experimental approach for quantifying the vesicle diameter distribution created during membrane fission. We validated our procedure by comparing it to traditional negative stain TEM of vesicles.…”

Peripheral Protein Unfolding Drives Membrane Bending

“…13 A recent report suggests lateral pressure from protein crowding can stretch and bend the membrane and lead to membrane fission. 14 Additionally, theoretical analysis has shown that crowded membrane-bound proteins generate substantial pressure at membrane surfaces. 15 These observations suggest crowding of proteins locally confined on the membrane surface, such as a region of membrane phase separation, may function as a general mechanism of membrane bending regardless of the specific protein structures involved.…”