Secretory proteins are transported to the Golgi complex in vesicles that bud from the endoplasmic reticulum. The cytoplasmic coat protein complex II (COPII) is responsible for cargo sorting and vesicle morphogenesis. COPII was first described in Saccharomyces cerevisiae, but its basic function is conserved throughout all eukaryotes. Nevertheless, the COPII coat has adapted to the higher complexity of mammalian physiology, achieving more sophisticated levels of secretory regulation. In this review we cover aspects of mammalian COPII-mediated regulation of secretion, in particular related to the function of COPII paralogues, the spatial organization of cargo export and the role of accessory proteins.

Endosomal sorting complex required for transport-III (ESCRT-III) is a large complex built from related ESCRT-III proteins involved in multivesicular body biogenesis. Little is known about the structure and function of this complex. Here, we compare four human ESCRT-III proteins -hVps2-1/CHMP2a, hVps24/CHMP3, hVps20/ CHMP6, and hSnf7-1/CHMP4a -to each other, studying the effects of deleting predicted a-helical domains on their behavior in transfected cells. Surprisingly, removing $40 amino acids from the C-terminus of each protein unmasks a common ability to associate with endosomal membranes and assemble into large polymeric complexes. Expressing these truncated ESCRT-III proteins in cultured cells causes ubiquitinated cargo to accumulate on enlarged endosomes and inhibits viral budding, while expressing full-length proteins does not. hVps2-1/ CHMP2a lacking its C-terminal 42 amino acids further fails to bind to the AAA1 adenosine triphosphatase VPS4B/SKD1, indicating that C-terminal sequences are important for interaction of ESCRT-III proteins with VPS4. Overall, our study supports a model in which ESCRT-III proteins cycle between a default 'closed' state and an activated 'open' state under control of sequences at their C-terminus and associated factors.

The ESCRT machinery along with the AAA+ ATPase Vps4 drive membrane scission for trafficking into multivesicular bodies in the endocytic pathway and for the topologically related processes of viral budding and cytokinesis, but how they accomplish this remains unclear. Using deep-etch electron microscopy, we find that endogenous ESCRT-III filaments stabilized by depleting cells of Vps4 create uniform membrane-deforming conical spirals which are assemblies of specific ESCRT-III heteropolymers. To explore functional roles for ESCRT-III filaments, we examine HIV-1 Gag-mediated budding of virus-like particles and find that depleting Vps4 traps ESCRT-III filaments around nascent Gag assemblies. Interpolating between the observed structures suggests a new role for Vps4 in separating ESCRT-III from Gag or other cargo to allow centripetal growth of a neck constricting ESCRT-III spiral.DOI: http://dx.doi.org/10.7554/eLife.02184.001

The AAA؉ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/ hVps2-1, and CHMP3/hVps24 but not CHMP4A/hSnf7-1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with "MIT interacting motifs" (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.

The ESCRT (endosomal sorting complex required for transport) machinery comprises a set of protein complexes that regulate sorting and trafficking into multivesicular bodies en route to the lysosome. The physical mechanism responsible for generating lumenal vesicles in this pathway is unknown. Here we review recent studies suggesting that components of the ESCRT-III complex drive lumenal vesicle formation and consider possible mechanisms for this reaction.