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Structural basis for ESCRT-III protein autoinhibition.

Bajorek M, Schubert HL, McCullough J, Langelier C, Eckert DM, Stubblefield WM, Uter NT, Myszka DG, Hill CP, Sundquist WI - Nat. Struct. Mol. Biol. (2009)

Bottom Line: Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member.The IST1 and CHMP3 structures also reveal that equivalent downstream alpha5 helices can fold back against the core domains.Mutations within the CHMP3 core-alpha5 interface stimulate the protein's in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory alpha5 helix from the core activates ESCRT-III proteins for assembly at membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Utah, Salt Lake City, Utah, USA.

ABSTRACT
Endosomal sorting complexes required for transport-III (ESCRT-III) subunits cycle between two states: soluble monomers and higher-order assemblies that bind and remodel membranes during endosomal vesicle formation, midbody abscission and enveloped virus budding. Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member. IST1 and its ESCRT-III binding partner, CHMP1B, both form higher-order helical structures in vitro, and IST1-CHMP1 interactions are required for abscission. The IST1 and CHMP3 structures also reveal that equivalent downstream alpha5 helices can fold back against the core domains. Mutations within the CHMP3 core-alpha5 interface stimulate the protein's in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory alpha5 helix from the core activates ESCRT-III proteins for assembly at membranes.

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Requirement for IST1-CHMP1 interactions during abscission. (a) The upper panel shows quantified abscission defects as reflected in the percentages of HeLa M cells with visible midbodies following siRNA treatment to deplete endogenous IST1 (lanes 2–9) and rescue with an empty vector control (lane 2, negative control) or with vectors expressing wild type IST1 (lane 3, positive control) or the designated IST1 mutants (lanes 4–9). Untreated cells are shown in lane 1. Error bars show standard deviations from three independent repetitions of the experiment. The middle panel is a western blot (anti-IST1) showing levels of soluble endogenous IST1 (lanes 1 and 2) or exogenously expressed IST1 proteins (lanes 3–9). The bottom panel is a western blot (anti-α-Tubulin) showing expression levels of endogenous α-Tubulin (loading control). CHMP1B binding phenotypes of the different IST1 proteins are shown below: strong (S), intermediate (I), or weak (W). (b) Immunofluorescence images showing the midbody phenotypes of cells from a designated subset of the experiments from a. Microtubules (anti-α-Tubulin, grey) and nuclei (SYTOX green) were stained for reference, and yellow arrowheads highlight midbodies. Scale bars are 10µm.
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Figure 5: Requirement for IST1-CHMP1 interactions during abscission. (a) The upper panel shows quantified abscission defects as reflected in the percentages of HeLa M cells with visible midbodies following siRNA treatment to deplete endogenous IST1 (lanes 2–9) and rescue with an empty vector control (lane 2, negative control) or with vectors expressing wild type IST1 (lane 3, positive control) or the designated IST1 mutants (lanes 4–9). Untreated cells are shown in lane 1. Error bars show standard deviations from three independent repetitions of the experiment. The middle panel is a western blot (anti-IST1) showing levels of soluble endogenous IST1 (lanes 1 and 2) or exogenously expressed IST1 proteins (lanes 3–9). The bottom panel is a western blot (anti-α-Tubulin) showing expression levels of endogenous α-Tubulin (loading control). CHMP1B binding phenotypes of the different IST1 proteins are shown below: strong (S), intermediate (I), or weak (W). (b) Immunofluorescence images showing the midbody phenotypes of cells from a designated subset of the experiments from a. Microtubules (anti-α-Tubulin, grey) and nuclei (SYTOX green) were stained for reference, and yellow arrowheads highlight midbodies. Scale bars are 10µm.

Mentions: To determine whether CHMP1 binding was necessary for IST1 cytokinesis function(s), we tested whether IST1 mutations that impaired CHMP1B binding also inhibited abscission. Depletion of endogenous IST1 increased the percentage of HeLa cells with visible midbodies from 4±1% to 20±3% (Fig. 5a, compare lanes 1 and 2 and Fig. 5b, panels 1 and 2), and this midbody arrest defect was largely (though not entirely) rescued by re-expression of a wild type, siRNA-resistant IST1 construct (8±2%, lane/panel 3)11. Midbody arrest was also corrected by an IST1 protein with a mutation in the tip of the α1/α2 hairpin that retained strong (S) CHMP1B binding (IST1R51D, 8±1%, lane/panel 4). In contrast, three weak-binding IST1 mutants failed to rescue midbody arrest (18–21%, Fig. 5a, lanes 7–9 and Fig. 5b, panels 5 and 6). Point mutations within the α5-core interface that blocked CHMP1B binding also blocked rescue of the midbody arrest defect. However, these mutant proteins partitioned almost exclusively into the insoluble/membrane-bound cellular fractions, and their failure to support abscission may therefore reflect either aberrant CHMP1 binding or IST1 protein mislocalization (see Supplementary Fig. 4 and the Supplementary Discussion). Finally, two IST1 mutants with intermediate (I) CHMP1B binding affinities rescued midbody arrest to an intermediate extent (12–13%, Fig. 5a lanes 5 and 6). Hence, there was an excellent correlation between the ability of different IST1 constructs to bind CHMP1B in vitro and to function in abscission, indicating that CHMP1 binding is required for IST1 abscission function(s).


Structural basis for ESCRT-III protein autoinhibition.

Bajorek M, Schubert HL, McCullough J, Langelier C, Eckert DM, Stubblefield WM, Uter NT, Myszka DG, Hill CP, Sundquist WI - Nat. Struct. Mol. Biol. (2009)

Requirement for IST1-CHMP1 interactions during abscission. (a) The upper panel shows quantified abscission defects as reflected in the percentages of HeLa M cells with visible midbodies following siRNA treatment to deplete endogenous IST1 (lanes 2–9) and rescue with an empty vector control (lane 2, negative control) or with vectors expressing wild type IST1 (lane 3, positive control) or the designated IST1 mutants (lanes 4–9). Untreated cells are shown in lane 1. Error bars show standard deviations from three independent repetitions of the experiment. The middle panel is a western blot (anti-IST1) showing levels of soluble endogenous IST1 (lanes 1 and 2) or exogenously expressed IST1 proteins (lanes 3–9). The bottom panel is a western blot (anti-α-Tubulin) showing expression levels of endogenous α-Tubulin (loading control). CHMP1B binding phenotypes of the different IST1 proteins are shown below: strong (S), intermediate (I), or weak (W). (b) Immunofluorescence images showing the midbody phenotypes of cells from a designated subset of the experiments from a. Microtubules (anti-α-Tubulin, grey) and nuclei (SYTOX green) were stained for reference, and yellow arrowheads highlight midbodies. Scale bars are 10µm.
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Related In: Results  -  Collection

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Figure 5: Requirement for IST1-CHMP1 interactions during abscission. (a) The upper panel shows quantified abscission defects as reflected in the percentages of HeLa M cells with visible midbodies following siRNA treatment to deplete endogenous IST1 (lanes 2–9) and rescue with an empty vector control (lane 2, negative control) or with vectors expressing wild type IST1 (lane 3, positive control) or the designated IST1 mutants (lanes 4–9). Untreated cells are shown in lane 1. Error bars show standard deviations from three independent repetitions of the experiment. The middle panel is a western blot (anti-IST1) showing levels of soluble endogenous IST1 (lanes 1 and 2) or exogenously expressed IST1 proteins (lanes 3–9). The bottom panel is a western blot (anti-α-Tubulin) showing expression levels of endogenous α-Tubulin (loading control). CHMP1B binding phenotypes of the different IST1 proteins are shown below: strong (S), intermediate (I), or weak (W). (b) Immunofluorescence images showing the midbody phenotypes of cells from a designated subset of the experiments from a. Microtubules (anti-α-Tubulin, grey) and nuclei (SYTOX green) were stained for reference, and yellow arrowheads highlight midbodies. Scale bars are 10µm.
Mentions: To determine whether CHMP1 binding was necessary for IST1 cytokinesis function(s), we tested whether IST1 mutations that impaired CHMP1B binding also inhibited abscission. Depletion of endogenous IST1 increased the percentage of HeLa cells with visible midbodies from 4±1% to 20±3% (Fig. 5a, compare lanes 1 and 2 and Fig. 5b, panels 1 and 2), and this midbody arrest defect was largely (though not entirely) rescued by re-expression of a wild type, siRNA-resistant IST1 construct (8±2%, lane/panel 3)11. Midbody arrest was also corrected by an IST1 protein with a mutation in the tip of the α1/α2 hairpin that retained strong (S) CHMP1B binding (IST1R51D, 8±1%, lane/panel 4). In contrast, three weak-binding IST1 mutants failed to rescue midbody arrest (18–21%, Fig. 5a, lanes 7–9 and Fig. 5b, panels 5 and 6). Point mutations within the α5-core interface that blocked CHMP1B binding also blocked rescue of the midbody arrest defect. However, these mutant proteins partitioned almost exclusively into the insoluble/membrane-bound cellular fractions, and their failure to support abscission may therefore reflect either aberrant CHMP1 binding or IST1 protein mislocalization (see Supplementary Fig. 4 and the Supplementary Discussion). Finally, two IST1 mutants with intermediate (I) CHMP1B binding affinities rescued midbody arrest to an intermediate extent (12–13%, Fig. 5a lanes 5 and 6). Hence, there was an excellent correlation between the ability of different IST1 constructs to bind CHMP1B in vitro and to function in abscission, indicating that CHMP1 binding is required for IST1 abscission function(s).

Bottom Line: Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member.The IST1 and CHMP3 structures also reveal that equivalent downstream alpha5 helices can fold back against the core domains.Mutations within the CHMP3 core-alpha5 interface stimulate the protein's in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory alpha5 helix from the core activates ESCRT-III proteins for assembly at membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Utah, Salt Lake City, Utah, USA.

ABSTRACT
Endosomal sorting complexes required for transport-III (ESCRT-III) subunits cycle between two states: soluble monomers and higher-order assemblies that bind and remodel membranes during endosomal vesicle formation, midbody abscission and enveloped virus budding. Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member. IST1 and its ESCRT-III binding partner, CHMP1B, both form higher-order helical structures in vitro, and IST1-CHMP1 interactions are required for abscission. The IST1 and CHMP3 structures also reveal that equivalent downstream alpha5 helices can fold back against the core domains. Mutations within the CHMP3 core-alpha5 interface stimulate the protein's in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory alpha5 helix from the core activates ESCRT-III proteins for assembly at membranes.

Show MeSH
Related in: MedlinePlus