CC2D1A is a regulator of ESCRT-III CHMP4B

Abstract

Endosomal sorting complexes required for transport (ESCRTs) regulate diverse processes ranging from receptor sorting at endosomes to distinct steps in cell division and budding of some enveloped viruses. Common to all processes is the membrane recruitment of ESCRT-III that leads to membrane fission. Here, we show that CC2D1A is a novel regulator of ESCRT-III CHMP4B function. We demonstrate that CHMP4B interacts directly with CC2D1A and CC2D1B with nanomolar affinity by forming a 1:1 complex. Deletion mapping revealed a minimal CC2D1A-CHMP4B binding construct, which includes a short linear sequence within the third DM14 domain of CC2D1A. The CC2D1A binding site on CHMP4B was mapped to the N-terminal helical hairpin. Based on a crystal structure of the CHMP4B helical hairpin, two surface patches were identified that interfere with CC2D1A interaction as determined by surface plasmon resonance. Introducing these mutations into a C-terminal truncation of CHMP4B that exerts a potent dominant negative effect on human immunodeficiency virus type 1 budding revealed that one of the mutants lost this effect completely. This suggests that the identified CC2D1A binding surface might be required for CHMP4B polymerization, which is consistent with the finding that CC2D1A binding to CHMP4B prevents CHMP4B polymerization in vitro. Thus, CC2D1A might act as a negative regulator of CHMP4B function.

Figure 1

CC2D1B interacts with CHMP4B in vitro. (A) Schematic drawing of the domain organization of CC2D1 isoforms. Both isoforms contain 4 DM14 domains followed by a C2 domain. (B) Schematic drawing of the CC2D1A and B constructs tested for CHMP4B interaction by SEC and ITC. Complex formation is indicated with a +. (C) SEC (S-200 column) in combination with RI (refractive index) and MALLS (Multi Angle Laser Light Scattering) analyses of CC2D1B(1-601) in complex with CHMP4B(7-105) reveals a 1:1 complex. The inset shows the complex eluted from the SEC column (upper band CC2D1B(1-601) and lower band, CHMP4B(7-105). (D) MALLS (S-75 column) analysis of the CC2D1A(346-455)-CHMP4B(23-97) complex shows a molecular mass of 18.2 kDa. The calculated molecular mass of the complex is 20.9 kDa. (E) Pull down of CC2D1A(346-455) wild type and mutant (mut; CC2D1A__mut) by MBP-CHMP4B(7-105), shows that the CC2D1A mutations abrogate CHMP4B binding. Lanes 1 and 2 input of CC2D1A(346-455) wild-type and mutant, respectively; lane 3 pull down of CC2D1A346-455) wild-type and lane 4 of the mutant; lanes 5 to 7 shows the controls, MBP-CHMP4B(7-105) alone, CC2D1A(346-455) and mutant alone. Molecular mass markers are indicated.

Figure 2

Crystal structure of the helical hairpin of CHMP4B. (A) Stereo image of the electron density map calculated based on the SAD phases without density modification. (B) Ribbon diagram of the CHMP4B helical hairpin containing residues 23-97. Note that the crystallized construct contained four extra residues at the N-terminus, which are in a helical conformation. (C) Stereo images of CHMP4B (blue) and CHMP3 (salmon) (Protein Data bank (PDB) ID 3FRT) based on superpositioning of the Cα atoms. (D) Stereo images of CHMP4B (blue) and IST1 (cyan) (PDB ID 3FRR) based on superpositioning of the Cα atoms. (E) Superpositioning of the Cα atoms of CHMP4B (blue) and Vps20 (gray) (CHMP6) (PDB ID 3FTU; the CHMP4B residues affecting CC2D1A interaction are labeled in red and the Vps20 residues involved in ESCRT-II Vps25 interaction are shown in yellow.

Figure 3

CC2D1A binds to the N-terminal end of the CHMP4B helical hairpin. (A) The molecular surface of CHMP4 is shown in two orientations and charged residues are indicated. The two patches affecting CC2D1A interaction are circled in green; CHMP4B_mut1 carries the R28A, R30A, D31R and E33R mutations and CHMP4B_mut2 has E90R, E94R and E97R mutated. (B) Co-purification of CC2D1A(346-455) and wild-type or mutant MBP-CHMP4B(7-105); lane 1, wild-type MBP-CHMP4B(7-105); lane 2 MBP-CHMP4B(7-105)_mut1; lane 3, MBP-CHMP4B(7-105)_mut2; lane 4, MBP-CHMP4B(7-105)_mut1.2.

Figure 4

CC2D1A prevents CHMP4B polymerization. Sucrose gradient analyses of MBP-CHMP4B_{ΔC_Alix}, CHMP4B_ΔC-Alix and CC2D1A. (A) CC2D1A(346-455); (B) MBP-CHMP4B_{ΔC_Alix}; (C) MBP-CHMP4B_{ΔC_Alix} after TEV protease cleavage; (D) MBP-CHMP4B_{ΔC_Alix} was incubated with CC2D1A(346-455) and then subjected to TEV protease cleavage; (E) MBP-CHMP4B_{ΔC_Alix} was incubated with CC2D1A__mut and then subjected to TEV protease cleavage. (F) CC2D1A(309-494) and (G) CHMP4B_{ΔC_Alix} polymers were incubated with CC2D1A(309-494).

Figure 5

Mutations in CHMP4B annul the dominant negative effect of C-terminally truncated CHMP4B. (A) (Left panel) Expression of wild type CHMP4B_1-153FLAG exerts a strong dominant negative effect on HIV-1 budding (lane 2) as compared to the vector control (lane 1). Expression of CHMP4B_1-153mut1 shows that it is still dominant negative (lane 3), while CHMP4B_1-153mut2 lost the dominant negative effect (lane 4). (Right panel) lane 1 vector only control; lane 2, expression of wild type CHMP4B_1-153FLAG exerts a strong dominant negative effect and lane 3, the double mutant CHMP4B_1-153mut1.2 is no longer dominant negative. (B) Western blot revealing the intracellular Gag processing corresponding to the panels shown in (A). (Lower panel), Western blot showing the expression levels of the CHMP4B constructs.

Figure 6

The mut1.2 surface patch is important for plasma membrane localization and polymerization of CHMP4B in vitro. Confocal microscopy of CHMP4B(1-153)-flag localization in HEK293 cells. (A) CHMP4B(1-153)-flag wild type, (B) CHMP4B(1-153)-flag carrying the mut1, (C) the mut2 and (D) the mut1.2 surface patch mutations. Individual confocal Z-sections are shown. Nuclei have been stained with 4′6-diamidino-2-phenylindole. The scale bar represents 10 μm. (E) Sucrose gradient analyses of recombinant wild-type MBP-CHMP4B_ΔC-ALIX (top panel) and MBP-CHMP4B_ΔC-ALIX carrying the mut1.2 surface patch mutations (bottom panel). Molecular mass markers are indicated.

Figure 6

The mut1.2 surface patch is important for plasma membrane localization and polymerization of CHMP4B in vitro. Confocal microscopy of CHMP4B(1-153)-flag localization in HEK293 cells. (A) CHMP4B(1-153)-flag wild type, (B) CHMP4B(1-153)-flag carrying the mut1, (C) the mut2 and (D) the mut1.2 surface patch mutations. Individual confocal Z-sections are shown. Nuclei have been stained with 4′6-diamidino-2-phenylindole. The scale bar represents 10 μm. (E) Sucrose gradient analyses of recombinant wild-type MBP-CHMP4B_ΔC-ALIX (top panel) and MBP-CHMP4B_ΔC-ALIX carrying the mut1.2 surface patch mutations (bottom panel). Molecular mass markers are indicated.