Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity - PubMed (original) (raw)

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

Rik van der Kant et al. J Biol Chem. 2015.

Abstract

Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail in yeast where their sequential membrane targeting and assembly is well understood. Mammalian CORVET and HOPS subunits significantly differ from their yeast homologues, and novel proteins with high homology to CORVET/HOPS subunits have evolved. However, an analysis of the molecular interactions between these subunits in mammals is lacking. Here, we provide a detailed analysis of interactions within the mammalian CORVET and HOPS as well as an additional endosomal-targeting complex (VIPAS39-VPS33B) that does not exist in yeast. We show that core interactions within CORVET and HOPS are largely conserved but that the membrane-targeting module in HOPS has significantly changed to accommodate binding to mammalian-specific RAB7 interacting lysosomal protein (RILP). Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome-associated mutations in VPS33B selectively disrupt recruitment to late endosomes by RILP or binding to its partner VIPAS39. Within the shared core of CORVET/HOPS, we find that VPS11 acts as a molecular switch that binds either CORVET-specific TGFBRAP1 or HOPS-specific VPS39/RILP thereby allowing selective targeting of these tethering complexes to early or late endosomes to time fusion events in the endo/lysosomal pathway.

Keywords: Rab; endosome; intracellular trafficking; membrane fusion; transport.

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Figures

FIGURE 1.

Biochemical definition of mammalian CORVET, HOPS, and VPS33B-VIPAS39. A, MelJuSo lysates were immunoprecipitated (IP) by the indicated antibodies and analyzed by WB using indicated antibodies. Each panel represents an independent experiment. Upper left panel, MelJuSo cell lysates were generated (at which point a total lysate (TL) fraction was taken) and divided over five fractions, and an immunoprecipitation was performed on each lysate using one of the indicated antibodies (generating five experimental conditions, horizontal axis). The five experimental conditions were run on the same blot with the same exposures for each detection antibody. A separate gel was run for each detection antibody (on the vertical axis). From these blots cutouts were taken and grouped to compose the figure panel. Top right panel, same as the left panel, with three experimental conditions. The table (lower panel) summarizes the results from the IP experiments. A checkmark indicates interaction detected. ND = not detected; empty boxes, not tested. B, lysates of MelJuSo cells expressing eight tagged (experimental condition (exp)) or seven tagged subunits (control condition (ctrl)) not-expressing the GFP-tagged subunit) were immunoprecipitated (IP) with anti-GFP (to pull down VPS16, VPS11, or VPS33B) and analyzed by WB. Shown are total lysates for experimental and control lanes and the immunoprecipitated fraction for experimental and control lanes. (C) Summary of experimental data A-E, VIPAS39 and VPS33B do not interact with HOPS complex subunits.

FIGURE 2.

Interactions within the mammalian HOPS complex. A, structure of the yeast HOPS complex. Yeast HOPS interacts with Ypt-7 via Vps41 and the N terminus of Vps39. B, domain organization of the mammalian HOPS complex subunit orthologs. CLH, clathrin heavy chain repeat; CC, coiled coil; R, ring finger; V11C, PFAM VPS11 C terminus; V16N, PFAM VPS11 N terminus; V16C, PFAM VPS11 C terminus; V39_1, VPS39 domain 1; V39_2, VPS39 domain 2; CNH, citron homology. C, lysates of MelJuSo cells co-expressing tagged VPS constructs as indicated were immunoprecipitated (IP) with anti-GFP antibodies and analyzed by WB using anti-HA, anti-FLAG, anti-V5, and anti-GFP antibodies as indicated. Within each panel, experimental conditions were run on the same blot with the same exposures for each detection antibody, and cutouts were taken and grouped for presentation purposes. D, summarized results of Fig. 2_C. E_, MelJuSo cells expressing GFP-VPS16 constructs (green) and mRFP-RILP (red). Scale bars: 10 μm. Graphs show average correlation coefficient (CC) ± S.E. n > 25, between different VPS truncation constructs and RILP. F, detailed map of domain interactions and membrane targeting modules (domains required for RILP binding are in green) within the mammalian HOPS complex. G, interactions in the head domain of the mammalian HOPS complex superimposed on the known structure of the yeast HOPS complex. Asterisk indicates functional divergence (absence of lipid binding motif in mammalian VPS41) between yeast and mammalian HOPS.

FIGURE 3.

VPS11 interacts with CORVET specific subunits. A, MelJuSo cells co-expressing mRFP-RILP (red) and GFP-TGFBRAP1 or HA-VPS8 (green) were fixed, stained with antibodies against EEA1 (and anti-HA for VPS8), and imaged by CLSM. Scale bars: 10 μm. Graphs show average correlation coefficient (CC) ± S.E n > 25, for RILP or EEA1 and GFP, TGFBRAP1, or VPS8. Asterisks indicate significance to GFP control (****, p ≤ 0.0001; * p ≤ 0.05; ns = not significant). B, MelJuSo cells co-expressing GFP-TGFBRAP1 or HA-VPS8 were fixed, stained with antibodies against EEA1 (and anti-HA for VPS8), and imaged by CLSM. Scale bars: 10 μm. C, lysates of MelJuSo cells expressing GFP or GFP-VPS11 were immunoprecipitated (IP) with anti-GFP and analyzed by WB using anti-GFP and anti-TGFBRAP1 antibodies. Experimental conditions were run on the same blot with the same exposures for each detection antibody, and cutouts were taken and grouped for presentation purposes. TL, total lysate. D, MelJuSo cells expressing mRFP-VPS11 (blue) or co-expressing mRFP-VPS11 (blue) and GFP-TGFBRAP1 (green) were fixed, stained with antibodies against EEA1 (red), and imaged by CLSM. Scale bars: 10 μm. Graphs show average correlation coefficient (CC) ± S.E n > 25, between VPS11 and EEA1 ± ectopically expressed TGFBRAP1 (****, p ≤ 0.0001).

FIGURE 4.

CORVET- and HOPS-specific assembly via VPS11. A and B, lysates of HEK293 cells co-expressing GFP-VPS11 constructs (as indicated) and HA-TGFBRAP1 (A) or MYC-mRFP-VPS39 (B) constructs were immunoprecipitated (IP) with anti-GFP before SDS-PAGE and WB and probed with anti-GFP and anti-HA or anti-MYC antibodies. TL, total lysate. C, detailed map of interactions between TGFBRAP1, VPS39, and VPS11. TGFBRAP1 and VSP11 both bind to the same region in VPS11, indicating competitive binding. Domains that contribute to RILP binding are indicated in green. D, MelJuSo cells expressing GFP-VPS11 (blue) and HA-RILP (red) ± GFP-TGFBRAP1 (green) were fixed, stained with antibodies against HA and FLAG and imaged by CLSM. Scale bars: 10 μm. Graphs show average correlation coefficient (CC) ± S.E (n > 25) between VPS11 and RILP in the ± ectopically expressed TGFBRAP1 (****, p ≤ 0.0001).

FIGURE 5.

VPS33B and VIPAS39 are not HOPS complex subunits and mutations in VPS33B differentially affect VPS33B interactions with VIPAS39 and RILP. A, lysates of MelJuSo cells silenced for indicated HOPS subunits were analyzed by WB using anti-VPS16, anti-VPS33B, anti-VIPAS39, anti-VPS11, anti-VPS33, and anti-actin (as the loading control) antibodies as indicated. Within each panel, experimental conditions were run on the same blot with the same exposures for each detection antibody, and cutouts were taken and grouped for presentation purposes. B, Immunoprecipitates (IP) with anti-GFP from lysates of MelJuSo cells co-expressing GFP-VPS33B mutants and HA-VIPAS39 (as indicated) were analyzed by WB using anti-GFP and anti-HA antibodies. Experimental conditions were run on the same blot with the same exposures for each detection antibody, and cutouts were taken and grouped for presentation purposes. TL, total lysate. C, MelJuSo cells expressing GFP-VPS33B constructs (green) and mRFP-RILP (red) were fixed and imaged by CLSM. Scale bars: 10 μm. Correlation coefficient was calculated from plot profiles measuring RILP intensity and GFP intensity over a vector through the cells. Graphs show the average correlation coefficient ± S.E (n > 25) between VPS33B constructs and RILP (*, p ≤ 0.05; ****, p ≤ 0.0001, ns = not significant, when compared with VPS33B wt). D, summary of VPS33B domains involved in interactions with RILP (green) and VIPAS39. Asterisks indicate Leu-30 (L30) residue (mutated in ARC syndrome) required for VPS33B recruitment to RILP.

FIGURE 6.

Model of membrane binding specificity of the CORVET and HOPS complex and their conversion during maturation. A, model of the mammalian HOPS complex superimposed on the yeast structure, depicting RILP-binding domains. Asterisks indicate poorly conserved regions in the N terminus of VPS39 and loss of the lipid binding motif of VPS41, which have altered membrane targeting in the mammalian complex. CLH, clathrin heavy chain repeat; CC, coiled coil; R, ring finger; CNH, citron homology. B, the CORVET complex binds to RAB5 on EE and within this complex, TGFBRAP1 binding to VPS11 prevent association of VPS11 to RILP (1). 2, to allow RAB7-RILP binding, TGFBRAP1 is replaced by VPS39, possibly controlled by CCZ1/MON1 and TGFβ signaling. Replacement of VPS8 with VPS41 adds an additional RILP binding motif, completing the conversion of CORVET to HOPS complex from RAB5- to RAB7- during endosomal maturation (3). Both the head and tail of mammalian HOPS can bind the homodimer RAB7-RILP. There may be two conditions; an inactive conformation where HOPS binds back to RILP on the same vesicle failing to contact a fusion partner and an active conformation in which RILP-HOPS contacts other vesicles (LE, phagosomes or autophagosomes containing HOPS interactors such as ARL8b, PLEKHM1, or RILP) for tethering and subsequent fusion.

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Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity - PubMed (original) (raw)