MVB (multivesicular body) formation occurs when the limiting membrane of an endosome invaginates into the intraluminal space and buds into the lumen, bringing with it a subset of transmembrane cargoes. Exvagination of the endosomal membrane from the cytosol is topologically similar to the budding of retroviral particles and cytokinesis, wherein membranes bud away from the cytoplasm, and the machinery responsible for MVB sorting has been implicated in these phenomena. The AAA (ATPase associated with various cellular activities) Vps4 (vacuolar protein sorting 4) performs a critical function in the MVB sorting pathway. Vps4 appears to dissociate the ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes during the course of MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. We have investigated the mechanisms by which ESCRT components stimulate the ATPase activity of Vps4. These studies support a model wherein Vps4 activity is subject to spatial and temporal regulation via distinct mechanisms during MVB sorting.

Introduction

The endosomal network co-ordinates protein sorting between the Golgi, plasma membrane and lysosome, thereby affecting protein composition within these subcellular compartments. MVBs (multivesicular bodies) are endosomal intermediates that arise when the limiting membrane of the endosome invaginates and buds into the endosomal lumen. Fusion of the MVB with the lysosome results in the delivery of the intraluminal vesicles to the hydrolytic lumen of the lysosome for degradation. Entry into this degradative pathway is highly regulated. Ubiquitin modification of endosomal proteins is the major signal for cargo inclusion into the MVB pathway. MVB sorting requires the function of the ESCRTs (endosomal sorting complexes required for transport), and cargo selection is thought to occur through ubiquitin-binding domains contained therein. ESCRTs can be broken into three complexes (I, II and III) conserved in most eukaryotes. Additional factors that are critical for the pathway include a set of adaptor proteins that are more divergent than the ESCRTs themselves [Hrs/Vps27, GGAs, Tom/Tollip, where Hrs is hepatocyte growth factor-regulated tyrosine kinase substrate, Vps is vacuolar protein sorting, GGA is Golgi-associated γ-adaptin ear homology domain Arf (ADP-ribosylation factor)-interacting protein, Tom is target of Myb1, and Tollip is Toll-inhibitory protein], deubiquitinating enzyme complexes (Doa4–Bro1 in yeast), and an AAA (ATPase associated with various cellular activities) [Vps4/SKD1 (suppressor of K+ transport defect 1)] and its modulators Ist1 and Vta1 (Vps20-associated 1)/SBP1/Lip5 (recently reviewed in [1,2]). Vps4 recruitment to the site of MVB formation occurs via interactions with the ESCRT-III family members [3,4]. The precise significance of these associations and the consequences of mechanical energy generated through Vps4 ATP hydrolysis remain unclear; however, one effect appears to be the removal of ESCRTs from the endosomal membrane. Additional speculation suggests that Vps4 ATP hydrolysis generates force for membrane deformation during intraluminal vesicle budding.

ESCRT-III assembly

ESCRT-III is unique among the ESCRTs in that the complex transiently assembles on the endosomal membrane; in contrast, ESCRT-I and -II exist as complexes in the cytoplasm that are transiently recruited to the site of MVB sorting [3,5,6]. Yeast possess six ESCRT-III subunits, of which four are essential for MVB function and are referred to as ‘core’ subunits (Vps20, Snf7, Vps2 and Vps24) and two are regulatory subunits (Did2 and Vps60) [3,7]. Mammals express 11 members of the ESCRT-III family, of which all but one [CHMP (charged multivesicular body protein) 7] are homologues of the yeast proteins [8]. Overexpression studies in mammalian cells have revealed that CHMP4 (homologue of yeast Snf7) can polymerize into filaments on the membrane and that these filaments are associated with membrane deformations consistent with the topology of invagination in MVB formation [9]. This observation has suggested that assembly of ESCRT-III itself may facilitate the membrane deformation permitting intraluminal vesicle formation, although disassembly by Vps4 also is required to complete the process.

Alignment of the ESCRT-III subunits reveals that they have highly similar charge composition and secondary structure, and structural studies of CHMP3 (homologue of yeast Vps24) have defined the five-helix core arrangement present throughout the family [10]. However, the C-termini are more divergent and have not been crystallized with the core. The conserved N-terminus has been implicated in membrane association and ESCRT-III oligomerization, whereas the C-termini (containing α6) seem to be more flexible and may be capable of adopting distinct closed and open conformations in the monomeric and oligomeric states [11]. This model has been supported by recent small-angle X-ray scattering analyses of CHMP3 that suggest ionic-dependent repositioning of the C-terminus [12]. The C-termini, along with α4 and α5 of the core, have also been implicated in mediating interactions with regulators of MVB sorting.

ESCRT-III effector interactions

ESCRT-III is responsible for co-ordinating a number of activities required at a late stage of MVB sorting, including the recruitment of Vps4 and its regulators (Ist1, Vta1) as well as deubiquitinating enzymes such as Doa4, AMSH [associated molecule with the SH3 (Src homology 3) domain of STAM (signal-transducing adaptor molecule)] and UBPY (ubiquitin-specific protease Y) [3,7,1317]. The contributions of distinct ESCRT-III subunits to this process and the mechanisms enabling this specificity are becoming apparent. Snf7 and the human CHMP4 (Snf7 homologue) bind in a specific manner to the Bro1 domain proteins Bro1 and Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] respectively, with the Bro1–Snf7 interaction facilitating recruitment of the ubiquitin isopeptidase Doa4 [15,18,19]. CHMP1 (Did2), CHMP2 (Vps2) and CHMP3 (Vps24) can interact with two mammalian deubiquitinating enzymes, AMSH and UBPY, to facilitate their recruitment directly [17,20,21]. These interactions are mediated by three helix MIT (microtubule-interacting and transport) domains present within AMSH and UBPY. Vps4 harbours a MIT domain as well and binds to these same three ESCRT-III subunits [22,23]. Determination of the Vps4 MIT domain in complex with the C-termini of CHMP1 and Vps2 revealed that type 1 MIM (MIT-interaction motif) in α6 of Did2/CHMP1, Vps2/CHMP2 and Vps24/CHMP3 are recognized by the surface formed by the second and third helices of the MIT domain [24,25]. This MIM1 is similar to the Snf7 α6 sequence recognized by the Bro1 domain, except distinct spacing of hydrophobic residues discriminate between the two epitopes [18]. Vps4 also binds Vps20/CHMP6 via the MIT domain; however, this interaction occurs via a surface formed by the first and third MIT helices and recognizes a distinct MIM (MIM2) in the loop connecting CHMP6 α4 and α5 [26]. The ESCRT-III α4–α5 region has also been implicated in interactions between Did2 and the Vps4 inhibitor Ist1 and between Vps60/CHMP5 and the Vps4 activator Vta1/Lip5 [7,14,16]. The Vta1/Lip5 N-terminus mediating this association also interacts with the Did2/CHMP1 α6, and elucidation of the structure of the Vta1 N-terminus revealed an arrangement of dual MIT domains [7,26,27]. These observations highlight two elements (Bro1 domains and MIT domains) recognizing these ESCRT-III subunits and two regions of ESCRT-III proteins critical for interactions with effectors (the α6 amphipathic helix and the α4–α5 region). The conformational changes in ESCRT-III subunits between monomeric and oligomeric states probably have an impact on associations with effectors through repositioning α4–α5 and the C-terminal helix. The assembly of ESCRT-III oligomers is anticipated to present a multitude of distinct epitopes important for co-ordinating cargo deubiquitination and ESCRT disassembly.

Function of the Vps4 oligomer

The mechanisms by which the assembly and disassembly of ESCRT-III permit membrane invagination remain unclear, but nucleotide hydrolysis by Vps4 plays a critical role in this process. Vps4 is a type 1 AAA with a single AAA domain per monomer. Type 2 AAAs (such as p97 and NSF) contain two AAA domains per subunit and exist as stable hexamers with one ring of AAA domains generating force through ATP hydrolysis, while the second ring stabilizes the hexameric form [28]. In contrast, type 1 AAAs, including Vps4, have been suggested to follow a cycle of ATP-stimulated oligomerization, oligomerization-stimulated ATP hydrolysis and subsequent dissociation. In the case of Vps4, this cycle has also been suggested to be co-ordinated with membrane recruitment. In this model, association with ESCRT-III facilitates Vps4 oligomerization, whereas subsequent ATP hydrolysis leads to ESCRT release along with Vps4 disassembly and membrane dissociation [4,29]. However, a number of studies suggest that this model is inaccurate. Vta1 has been demonstrated to stimulate Vps4 ATPase activity in part through promoting Vps4 oligomerization [30]; this suggests that the Vta1–Vps4 oligomer is stable throughout the ATPase cycle in vivo. Cryo-electron microscopy analysis of the Vps4 dodecamer has also revealed that the Vps4 oligomer exhibits 6-fold symmetry, suggesting that the upper and lower rings function differentially [31]. This asymmetry between rings is reminiscent of the type 2 AAAs and also suggests that the Vps4 oligomer may remain assembled throughout the ATP hydrolysis cycle even in the absence of Vta1. Although initial models proposed that Vps4 subunits hydrolyse ATP concomitantly, elegant studies with the bacterial AAA ClpX have demonstrated that the concerted hydrolysis does not occur and that ATP hydrolysis by even a single subunit can support substrate unfolding by the ClpX6 ring [32,33]. These observations suggest that the previous model of Vps4 function requires reassessment.

ESCRT-III activation of the Vps4 oligomer

The Vta1–Vps4 complex plays a critical role in co-ordinating disassembly of ESCRT-III, and MIT domains within both Vta1 and Vps4 contribute to this activity [4,7]. In an attempt to understand the significance of the interactions between ESCRT-III and Vta1–Vps4, we have focused on the ability of ESCRT-III subunits to modulate Vps4 ATPase activity. These studies have revealed at least two distinct mechanisms by which ESCRT-III family members can stimulate Vps4 [7]. Direct interaction of Vps2 or Did2 with the MIT domain of Vps4 results in stimulation of Vps4 ATPase activity. Additionally, Did2 and Vps60 can stimulate Vps4 ATPase activity indirectly via the MIT domains of Vta1. Vta1 itself can also stimulate Vps4 ATPase activity, indicating that Vps4 ATPase activity is regulated on a number of levels. These observations suggest a co-ordination of ESCRT-III assembly with its disassembly as stimulated by Vta1–Vps4. How this process facilitates membrane deformation to complete MVB sorting remains to be determined, but in vitro reconstitution of this process is being pursued to examine this question.

The formation of the MVB represents a topological conundrum as membrane must evaginate from the cytoplasm to form the intraluminal vesicle. The mechanisms by which the cytoplasmic ESCRT machinery accomplishes this task without being consumed by the process are as yet unclear. Formation of ESCRT-III has been implicated in promoting the membrane deformation, and ATP hydrolysis by Vps4 is required to complete vesicle budding and recycle the ESCRT-III subunits. The co-ordination of ESCRT-III assembly with disassembly appears critical for this process, and the mechanisms by which the Vta1–Vps4 complex is activated to promote ESCRT-III disassembly are becoming clear. Further dissection of how Vta1–Vps4 dissociates ESCRT-III to permit completion of intraluminal vesicle budding should resolve a critical question of MVB sorting.

ESCRTs: from Cell Biology to Pathogenesis: Biochemical Society Focused Meeting held at Robinson College, Cambridge, U.K., 26–28 August 2008. Organized and Edited by Katherine Bowers (University College London, U.K.), Juan Martin-Serrano (King's College London, U.K.) and Paul Whitley (Bath, U.K.).

Abbreviations

     
  • AAA

    ATPase associated with various cellular activities

  •  
  • AMSH

    associated molecule with the SH3 (Src homology 3) domain of STAM (signal-transducing adaptor molecule)

  •  
  • CHMP

    charged multivesicular body protein

  •  
  • ESCRT

    endosomal sorting complex required for transport

  •  
  • MIT

    microtubule-interacting and transport

  •  
  • MIM

    MIT-interaction motif

  •  
  • MVB

    multivesicular body

  •  
  • UBPY

    ubiquitin-specific protease Y

  •  
  • Vps

    vacuolar protein sorting

  •  
  • Vta1

    Vps20-associated 1

We thank Dr Markus Babst, Dr Bruce Horazdovsky, Dr Wesley Sundquist and Dr Zhaohui Xu for helpful discussions and Dr Darren Carney for critical evaluation of the manuscript.

Funding

This work was supported by a grant from the National Institutes of Health to D.J.K. [grant number GM73024] and a Predoctoral Fellowship from the American Heart Association to I.F.A. [grant number AHA07-155882].

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