ESCRTing the RABs through conversion

Abstract

The endosomal system is essential for the intra- and intercellular communication in cells and multicellular organisms. It is involved in the secretion of signaling factors and serves as a venue for signaling receptors from the plasma membrane, which are endocytosed after ligand binding. Many internalized receptor-ligand complexes and numerous other endocytosed proteins arrive at the Rab5-positive early endosome, where they will be sorted. Cargoes marked with ubiquitin are bound by endosomal sorting complex required for transport (ESCRT)-0 and ESCRT-I complexes to initiate their degradation. The remaining cargoes are recycled back to the plasma membrane or the trans-Golgi network. To degrade ubiquitinated cargoes, the early endosome has to mature into a late endosomal structure, the multivesicular body (MVB). This procedure requires the Rab5-to-Rab7 conversion, mediated by the RABEX5-MON1/CCZ1 RabGEF cascade. Moreover, cargoes destined for degradation have to be packaged into intraluminal vesicles (ILVs) through ESCRT-III and Vps4. The matured late endosome or MVB finally fuses with a lysosome to degrade the cargo. Although ESCRT-mediated ILV formation and Rab conversion are well-characterized processes during endosome maturation, it remained until recently unclear whether these processes are connected. Lately, several studies were published illuminating the relationship of ESCRT functions and Rab conversion. Here, we review the current knowledge on the role of the ESCRT machinery in cargo degradation and RABEX5 regulation and MON1/CCZ1-mediated Rab conversion during endosome maturation. Moreover, we propose a model on the regulatory role of ESCRT functions during endosome maturation.

Keywords: ESCRT; RABEX5; Rab GTPases; Rab conversion; endosome maturation.

Figure 1. Unified model of ESCRT functions regulating the progression of endosome maturation.

Hypothesis for co-ordination between Rab conversion and ILV formation by ESCRT during endosome maturation. Overall mechanisms such as recycling (right), ILV formation, and Rab5 to Rab7 exchange (left) are shown by large arrows or gradients. Recycling will proceed throughout the first stages of endosome maturation lasting into late endosomes (indicated by tubular structures). ILVs will accumulate until their degradation in endolysosomes. The surface of endosomes will be covered either with Rab5 (shown by a green outline) on early endosomes or Rab7 (magenta outlines) on late endosomes and lysosomal structures. The right part of the endosomes shows the ESCRT progression, while the left part shows the parallel events pertaining to Rab conversion and RABEX5. Early endosomal membranes contain large amounts of cargo (either for recycling or degradation) that will be progressively removed during maturation. The ESCRT machinery will proceed from cargo corralling (ESCRT-0 and ESCRT-I in purple and orange) to filament and ILV formation (ESCRT-III and Vps4) (transition by ESCRT-II is not shown for simplicity). In the left part of the model, RABEX5 is active as a Rab5 GEF with the help of RABAPTIN5. After cargo removal and PI(3)P concentration reach a critical level, RABEX5 is removed from the endosome by 1. losing its binding to ubiquitinated cargo, 2. being deubiquitinated by USP8, 3. having RABAPTIN5 removed through binding of HD-PTP and 4. MON1/CCZ1 binding to its membrane binding domain. The MON1/CCZ1 complex also activates Rab7 on the membrane to replace Rab5. On the late endosome, late ESCRTs will finish ILV formation, the last recycling cargo will be removed, and MON1/CCZ1 will dissociate from the membrane and the HOPS tethering complex will be recruited. When all these tasks are finished and sufficient PI(3,5)P₂ is accumulated on the membrane, fusion with the lysosome will occur, leading to degradation of the contents followed by endocytic lysosome reformation (ELR) (modified from [2,17]). ESCRT, endosomal sorting complex required for transport; ILV, intraluminal vesicle.

Figure 2. Co-ordination of RABEX5 localization and ESCRT machinery during cargo corralling and ILV formation.

(A) Domain structure of RABEX5. The domains correspond to the same-colored domains in (B). Ubiquitin-binding domains (consisting of a Zink Finger and an additional ubiquitin-binding motif) are shown in red, membrane binding domains (with helical bundle [HB]) are in brown (they can be bound by MON1), GEF domain and coiled-coil domain are colored in green. (B) Hypothetical co-ordination model of Rab conversion and ESCRT-mediated ILV formation during endosome maturation. General concentration of different factors is indicated on the sides to underscore the progression through the maturation process. On the left, Rab5 (green) will be replaced by Rab7 (magenta), with a period of time where both can be found on endosomes (white). On the right: PI(3)P will accumulate and then decrease again on late endosomes (purple bar), free ubiquitinated cargo will decrease and be packed into ILVs (red bar), RABEX5 will be removed from endosomes (green bar) and MON1/CCZ1 will only be present for a short time as indicated (dark brown bar). The progression through endosome maturation is shown in four steps (I–IV). I. Early endosome with high levels of recycling cargo and ubiquitinated cargo for degradation. On the left side, recruited RABEX5 is shown binding to ubiquitinated cargo and directly to the membrane. RABAPTIN5 is binding to the coiled-coil region of RABEX5 and activating its GEF activity to recruit Rab5. RABEX5 is also ubiquitinated (red dot), which increases its affinity to endosomal membranes. On the right side, ESCRT-0 is binding to ubiquitinated cargo. Its recruitment is aided by the recognition of PI(3)P. HD-PTP binds to ESCRT-0 through its Bro1 domain. II. Cargo corralling is increased by the presence of ESCRT-I that contains additional binding sites for ubiquitinated cargo. While TSG101 is directly recruited by HRS (in ESCRT-0), it is also bound by HD-PTP through the coiled-coil domain. The positive feedback loop keeping Rab5 firmly activated is depicted on the left side. III. Maturing endosome shows USP8 deubiquitinating RABEX5, which will lead to a loss of association with the endosomal membrane. Moreover, USP8 attracts MON1/CCZ1. USP8 is also involved in later steps of ILV formation as indicated by a dashed line arrow pointing to the right. At this point, most of the recycling cargo will be gone and the degradation cargo will begin to be packed into late ESCRT filament structures (shown on the right). ESCRT-II will be recruited and in turn start filament formation of CHMP6 and CHMP4 (ESCRT-III). This filament will now be bound by the Bro1 domain of HD-PTP. ESCRT-0 might by this point have left the assembly. The later replacement of the CHMP4 filament with a CHMP3/CHMP2 filament will release the Bro1 domain, which will then be able to bind RABAPTIN5 as indicated by the dashed line arrow pointing to the left. IV. Late endosome membranes will contain very little recycling cargo and almost no ubiquitinated degradation cargo. Cargo for degradation will be mostly moved into forming or finished ILVs and is deubiquitinated by USP8. The binding of RABEX5 to this cargo will not be possible any more. Deubiquitination by USP8 will have destabilized RABEX5 further. Additionally, HD-PTP binding to RABAPTIN5 will reduce the GEF activity of RABEX5, interrupting the positive feedback loop with Rab5 to keep RABEX5 on the membrane. Last but not least, the high PI(3)P concentration on the membrane will enhance the recruitment of MON1/CCZ1, which also binds to the HB of RABEX5 and displaces the adjacent membrane binding domain to fully remove RABEX5 from endosomal membranes. Rab7 will be activated and recruited onto the membrane by the GEF activity of CCZ1, and a late endosome environment will be established (Figure combined from [17,23,24,26]). ESCRT, endosomal sorting complex required for transport; HD-PTP, His domain protein tyrosine phosphatase; ILV, intraluminal vesicle.

Figure 3. The ESCRT machinery at a glance.

A simplified overview of ILV formation by the ESCRT machinery in endosomes highlighting the possible roles of ESCRT in Rab conversion. The three main points of intersection between the two pathways are indicated (corresponding to Figure 1). The ESCRT complexes are arranged by their sequence of action from left to right. The whole machinery will most probably never be arranged in this way because early ESCRTs will leave, while late ESCRTs will only arrive later in the process (as shown in more detail in Figure 2B). All major ESCRT subunits are shown and color coded according to the complex they belong to (ESCRT-0 to Vps4) (see also Table 1). Ubiquitinated cargo is depicted (with a red UBQ). Binding domains (BD) for ubiquitin (UBQ-BD), PI(3)P (PI(3)P-BD and GLUE-D) and PI (3,5)P₂ (PI(3,5)P₂-BD) are depicted as domains belonging to the respective proteins. The red arrow shows cargo deubiquitination by USP8 (taken from [17]). Recruitment of ESCRT-III and Vps4 subunits corresponds approximately to the sequence shown from left to right. ESCRT, endosomal sorting complex required for transport; ILV, intraluminal vesicle.