FERARI is required for Rab11-dependent endocytic recycling (original) (raw)

2020, Nature Cell Biology

Endosomal transport is essential for cellular organization and compartmentalization and cell-cell communication. Sorting endosomes provide a crossroad for various trafficking pathways and determine recycling, secretion or degradation of proteins. The organisation of these processes requires membrane tethering factors to coordinate Rab GTPase function with membrane fusion. Here, we report a conserved tethering platform that acts in the Rab11 recycling pathways at sorting endosomes, which we name FERARI (Factor for Endosome Recycling And Rab Interactions). The Rab binding module of FERARI consists of Rab11FIP5/RFIP-2 and rabenosyn-5/RABS-5, while the SNARE interacting module comprises VPS45 and VIPAS39/SPE-39. Unexpectedly, the membrane fission protein EHD1/RME-1 is also a FERARI component. Thus, FERARI appears to combine fusion activity through the SM protein VPS45 with pinching activity through EHD1/RME-1 on SNX-1-positive endosomal membranes. We propose that coordination of fusion and pinching through a kiss-and-run mechanism drives cargo at endosomes into recycling pathways. 45 in E. coli. His-VPS-45 co-purified with SPE-39-GST (Figure 1a, Extended Data 3d). VPS-33.2, which together with SPE-39 is part of the CHEVI complex 27, 28 served as a positive control, while the HOPS specific VPS-33.1 did not bind SPE-39. These data indicate direct binding of SPE-39 to VPS-45. This interaction was confirmed by yeast-two-hybrid (Y2H) and pull-down experiments (Figure 1b, Extended Data 2d, d', Supplementary Table 3). Moreover, FERARI is conserved in mammalian cells (Figure 1c). VPS45 has been shown to directly interact with rabenosyn-5 29, 30. Similar to VPS45, rabenosyn-5 interacted with VIPAS39 (Figure 1b-e, Fig. Extended Data 1b, 1b', 2b, 2b', 2d, 2d'). Based on HOPS and CORVET, we speculated that FERARI might contain more subunits (Extended Data 1a). To identify additional components, we incubated the bacterially expressed SPE-39/VPS-45 complex with worm lysate and performed mass spectrometry (Supplementary Table 1), followed by Y2H assays and pulldowns from selected, streamlined candidates. We discovered two additional components of FERARI: the Epsin-homology domain (EHD) containing protein RME-1 and UNC-44 (Figure 1f, Supplementary Table 3). UNC-44 contains ankyrin motifs and a death domain. SPE-39 interacted well with RME-1 and the ankyrin motifs of UNC-44 (Figure 1f, Supplementary Table 3, Extended Data 1c, 1c',1d, 1d', 2a, 2a'). RME-1 is homologous to mammalian EHD1-4, and UNC-44 to ANK1-3. Since EHD1 has been shown to act at endosomes and EHD2 at the plasma membrane 31, 32 , we focused on EHD1. EHD1 co-precipitated and co-localized with rabenosyn-5 (Figure 1h, Extended Data 4a, b). Much less is known about ANK1-3. However, ANK3 appears to be the ANK protein most highly expressed in HeLa cells (Extended Data 4d). ANK3 co-precipitated with endogenous VIPAS39 (Figure 1g). Finally, endogenous VIPAS39 was pulled-down with all other Cell 3 Cell 2 Cell Spermatheca c b wild-type spe-39(RNAi) vps-45(RNAi) wild-type spe-39(RNAi) vps-45(RNAi) Cell 3 Cell 2 Cell 1 Cell Cell 2 Cell 1 30 40 50 20 % yolk-GFP wt spe-39(RNAi)