Differential requirements for Alix and ESCRT-III in cytokinesis and HIV-1 release

Abstract

The ESCRT machinery functions in topologically equivalent membrane fission events, namely multivesicular body formation, the terminal stages of cytokinesis and HIV-1 release. Here, we show that the ESCRT-III-binding protein Alix is recruited to the midbody of dividing cells through binding Cep55 via an evolutionarily conserved peptide. Disruption of Cep55/Alix/ESCRT-III interactions causes formation of aberrant midbodies and cytokinetic failure, demonstrating an essential role for these proteins in midbody morphology and cell division. We also show that the C terminus of Alix encodes a multimerization activity that is essential for its function in Alix-dependent HIV-1 release and for interaction with Tsg101. Last, we demonstrate that overexpression of Chmp4b and Chmp4c differentially inhibits HIV-1 release and cytokinesis, suggesting possible reasons for gene expansion within the mammalian Class E VPS pathway.

Fig. 1.

Cep55 recruits Alix to the midbody through binding a conserved peptide within Alix's PRR. (A) Alix mutants were fused to the VP16-activation domain and tested for interaction with the indicated proteins fused to Gal4 DNA-binding domain by yeast two-hybrid assay (n = 3 ± SD). (B) HeLa cells were transfected with plasmids encoding mCh-Cep55 and either YFP-Alix^R or YFP-Alix^R P801V, P802D, fixed and stained with α-tubulin. (Scale bars: 10 μm.) (C) Sequence alignment of the Cep55-binding region of the Alix-PRR with the Cep55-binding peptide highlighted in red. (D) Alix, Alix^R δPRR, and Alix^R δPRR plus peptide were fused to the VP16 DNA-activation domain and tested for interactions with the indicated Gal4 DNA-binding domain fusions by yeast two-hybrid assay (n = 3 ± SD). (E) HeLa cells were transfected with plasmids encoding mCh-Cep55 and either YFP-Alix^R or YFP-Alix^R δPRR and Alix^R δPRR plus peptide, fixed, and stained with α-tubulin. (Scale bars: 10 μm.)

Fig. 2.

Recruitment of Alix and ESCRT-III to the midbody is essential for normal midbody morphology. (A) HeLa cells stably transduced with retroviral vectors encoding mCh or mCh-Alix^R fusions were treated with siRNA targeting Luciferase or Alix, and resolved cell lysates were analyzed with α-Alix or α-Hsp90 antibodies. Alternatively, cells were fixed, stained with α-tubulin, and scored for aberrant midbodies (n = 3 ±SD) and multinucleation (n > 4 ±SD). (B) Representative micrographs of aberrant midbodies from A, enlargement depicting α-tubulin channel. (Scale bars: 10 μm.) (C) Clonal HeLa cells stably expressing YFP-Cep55 were treated with siRNA targeting Luciferase or Alix. Cells were fixed and stained with α-tubulin. Enlargements depict the α-tubulin channel. (Scale bars: 10 μm.) Alternatively, cells were lysed and analyzed with α-Alix, α-Cep55, and α-Hsp90 antisera.

Fig. 3.

Differential requirements for Chmp4 isoforms in retroviral budding and cytokinesis. (A and B) Cells (293T) were cotransfected with plasmids encoding HIV-1 pNL/HXB and the indicated amount of YFP-Chmp4 constructs. Resolved cell lysates and released virions were examined by Western blot analysis with α-Gag antisera; lysates were also examined by Western blot analysis with α-GFP antisera. Quantification of virion release via infrared imaging is given. β-Galactosidase assay was performed on HeLa-TZM-bl cells infected with 293T supernatant (n = 3 ±SD). (C) HeLa cells were transfected with the indicated amounts of YFP-Chmp4 constructs. Cells were fixed, stained with α-tubulin, and scored for multinucleation (n = 3 ±SD).

Fig. 4.

Activities in the C terminus of Alix. (A and B) The indicated Alix constructs fused to the VP16-activation domain were tested for interactions with Alix or Tsg101 fused to the Gal4 DNA-binding domain by yeast two-hybrid assay (n = 3 ±SD). (C) The indicated Alix mutants were fused to the VP16-activation domain and tested for interactions with Tsg101, Cin85, and Chmp4a fused to the Gal4 DNA-binding domain by yeast two-hybrid assay (n = 3 ±SD). (D) Coprecipitation assays were performed on lysates of 293T cells cotransfected with plasmids encoding YFP-Tsg101, Myc-Vps28, and the indicated GST-Alix^R fusion proteins. Resolved cells lysates (input, IP) and normalized bead-bound fractions (pull down, PD) were analyzed with α-GFP antisera. PD fractions were analyzed by Coomassie staining to visualize GST-Alix^R expression, and relative binding was quantified by infrared imaging. (E) HeLa cells stably transduced with retroviral vectors encoding mCh or the mCh-Alix^R fusions were treated with siRNA targeting Luciferase or Alix. Cells were fixed, stained with α-tubulin, and scored for midbody arrest and multinucleation (n = 3 ±SD). Resolved cell lysates were analyzed with α-Alix and α-Hsp90 antibodies.

Fig. 5.

Alix multimerization is required for Alix-dependent HIV-1 release. Cells (293T) were cotransfected with plasmids encoding the indicated HA-tagged Alix^R constructs and an HIV-1 pNL/HXB p6(P7L, P10L) plasmid. Resolved cell lysates and released virions were examined by Western blot analysis with α-Gag antisera. Quantification of virion release (VR) via infrared imaging is given. (n = 3). Lysates were also examined by Western blot analysis with α-HA antisera. β-Galactosidase assay was performed on HeLa-TZM-bl cells infected with 293T supernatants (n = 3 ±SD).