Bro1 stimulates Vps4 activity to promote Intralumenal Vesicle Formation during Multivesicular Body biogenesis

Abstract

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) form intralumenal vesicles (ILVs) during the conversion of endosomes to multivesicular bodies (MVBs). The assembly and disassembly of an ESCRT-III polymer facilitates membrane remodeling and scission during this process. The ESCRT-III-associated protein Bro1 (the yeast homolog of mammalian proteins ALIX and HD-PTP) promotes ESCRT-III assembly and inhibits disassembly of ESCRT-III filaments by impeding Vps4, a AAA-ATPase that dismantles ESCRT-III polymers. Here we show that the evolutionarily conserved “V domain” of Bro1-family proteins directly stimulate Vps4 ATPase activity and this activity is enhanced by interaction with ubiquitin. Surprisingly, a carboxyl-terminal fragment of Bro1 containing the V domain supports ILV formation but not sorting of cargo into ILVs, revealing that these two processes can be uncoupled. These studies implicate Bro1 as a factor coordinating cargo sorting with direct regulation of Vps4 to modulate ESCRT-III driven ILV formation during MVB biogenesis.

Interactions of ubiquitin and CHMP5 with the V domain of HD-PTP reveals role for regulation of Vps4 ATPase

“…In addition, the V domains from HD-PTP and Bro1 also mediate Ub-enhanced Vps4 stimulation as shown in this study (Fig. 8) and previous studies (Tseng et al, 2021). These interactions provide a number of potential molecular mechanisms that could specify targets and timing of Vps4 activity to not only ensure proper ILV scission, but also ensure that cargo is retained in these ILVs and can undergo a proper degree of deubiquitination and allow HD-PTP/Bro1 to escape incorporation into ILVs.…”

“…Similarly, HD-PTP binds the MIT domains of yeast and mammalian Vps4 isoforms (Fig. 8 A, B) and has been shown to also stimulate Vps4 activity in vitro (Tseng et al, 2021). Our structural studies that defined Ub-binding sites on the V HDPTP allows us to show Ub binding via HD-PTP enhances its ability stimulate Vps4 ATPase activity.…”

“…This supports the idea that we have identified the major binding sites for Ub and highlights a conserved feature in the function of the V domains in general. Previous work defined mutations in V Bro1 that still bound the MIT domain of Vps4 but lacked the ability to stimulate Vps4 activity by Ub, and these alleles in vivo had major defects in cargo sorting (Tseng et al, 2021). Thus, the phenotypes associated with loss of Ub-binding by Bro1 could be due to either the inability to serve as a receptor to bind and sort Ub-cargo or the inability to have that Ub-cargo -or some other ubiquitinated component (Watanabe and Hatakeyama, 2017;Crespo-Yanez et al, 2018) -control Vps4 activity.…”

“…, 2012 ). Recently, studies in yeast have discovered that uncoupling the ability of Bro1 to bridge its full cohort of protein interactions leads to formation of ILVs that lack Ub-cargo ( Tseng et al. , 2021 ).…”

“…Moreover, the V domain alone can stimulate the activity of Vps4 in vitro through contact with the Vps4 MIT domain. This stimulation of Vps4 can be further enhanced when the Bro1 V domain (V Bro1 ) binds Ub ( Tseng et al. , 2021 ).…”