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Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells

View Article: PubMed Central - PubMed

ABSTRACT

The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1 supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1 supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.

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FRET measurements showing close proximity of CIV subunit CoxVIIIa and CIII-subunit k in a supercomplex.(a) Positions of CoxVIIIa-mRuby (acceptor, CIV-A) and CIIIk-Clover (donor, CIII-D) within a CICIII2CIV supercomplex. Only one subunit k of one CIII is labeled. (b) Fluorescence lifetime of Clover as donor in the presence and absence of acceptor mRuby. Pair 1: CoxVIIIa-mRuby as acceptor and CIIIk-clover as donor. Pair 2: modified ATeam37 composed of clover as donor and mRuby as acceptor was used as FRET control. Significance: ***P < 0.001 compared to CIIIk-Clover (ANOVA one-way).
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f4: FRET measurements showing close proximity of CIV subunit CoxVIIIa and CIII-subunit k in a supercomplex.(a) Positions of CoxVIIIa-mRuby (acceptor, CIV-A) and CIIIk-Clover (donor, CIII-D) within a CICIII2CIV supercomplex. Only one subunit k of one CIII is labeled. (b) Fluorescence lifetime of Clover as donor in the presence and absence of acceptor mRuby. Pair 1: CoxVIIIa-mRuby as acceptor and CIIIk-clover as donor. Pair 2: modified ATeam37 composed of clover as donor and mRuby as acceptor was used as FRET control. Significance: ***P < 0.001 compared to CIIIk-Clover (ANOVA one-way).

Mentions: We next tested possible colocalization of CIII and CIV in a supercomplex by Förster Resonance Energy Transfer (FRET) using CIII subunit k fused to Clover as donor and CoxVIIIa-mRuby2 as acceptor. The C-terminus of CIII k is localized at the p-side according to the supercomplex models and the distance between the fused Clover and CoxVIIIa-mRuby2 should be short enough to allow FRET (Fig. 4a). Cells were transiently transfected with donor only (CIIIk-Clover, CIII-D) or both constructs (CIII-D × CIV-A) and fluorescence lifetime images were recorded. As a control, ATeam37 was used that was constructed with Clover as donor and mRuby2 as acceptor within the same ATeam molecule. The lifetime of Clover was obtained from bi-exponential fits from the TCSPC decays. In the presence of the acceptor mRuby2, the lifetime of the donor significantly decreased. This indicates proximity of CIII and CIV in a supercomplex. The decrease in fluorescence lifetime was stronger in an ATeam control construct, since here the expression and proximity of both fluorescent proteins was guaranteed (Fig. 4b, Supplementary Fig. S10). The FRET results thus are in line with the preceding FLIM data with one sensor only at CoxVIIIa and suggest the existence of a CICIII2CIV supercomplex in situ.


Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells
FRET measurements showing close proximity of CIV subunit CoxVIIIa and CIII-subunit k in a supercomplex.(a) Positions of CoxVIIIa-mRuby (acceptor, CIV-A) and CIIIk-Clover (donor, CIII-D) within a CICIII2CIV supercomplex. Only one subunit k of one CIII is labeled. (b) Fluorescence lifetime of Clover as donor in the presence and absence of acceptor mRuby. Pair 1: CoxVIIIa-mRuby as acceptor and CIIIk-clover as donor. Pair 2: modified ATeam37 composed of clover as donor and mRuby as acceptor was used as FRET control. Significance: ***P < 0.001 compared to CIIIk-Clover (ANOVA one-way).
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Related In: Results  -  Collection

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f4: FRET measurements showing close proximity of CIV subunit CoxVIIIa and CIII-subunit k in a supercomplex.(a) Positions of CoxVIIIa-mRuby (acceptor, CIV-A) and CIIIk-Clover (donor, CIII-D) within a CICIII2CIV supercomplex. Only one subunit k of one CIII is labeled. (b) Fluorescence lifetime of Clover as donor in the presence and absence of acceptor mRuby. Pair 1: CoxVIIIa-mRuby as acceptor and CIIIk-clover as donor. Pair 2: modified ATeam37 composed of clover as donor and mRuby as acceptor was used as FRET control. Significance: ***P < 0.001 compared to CIIIk-Clover (ANOVA one-way).
Mentions: We next tested possible colocalization of CIII and CIV in a supercomplex by Förster Resonance Energy Transfer (FRET) using CIII subunit k fused to Clover as donor and CoxVIIIa-mRuby2 as acceptor. The C-terminus of CIII k is localized at the p-side according to the supercomplex models and the distance between the fused Clover and CoxVIIIa-mRuby2 should be short enough to allow FRET (Fig. 4a). Cells were transiently transfected with donor only (CIIIk-Clover, CIII-D) or both constructs (CIII-D × CIV-A) and fluorescence lifetime images were recorded. As a control, ATeam37 was used that was constructed with Clover as donor and mRuby2 as acceptor within the same ATeam molecule. The lifetime of Clover was obtained from bi-exponential fits from the TCSPC decays. In the presence of the acceptor mRuby2, the lifetime of the donor significantly decreased. This indicates proximity of CIII and CIV in a supercomplex. The decrease in fluorescence lifetime was stronger in an ATeam control construct, since here the expression and proximity of both fluorescent proteins was guaranteed (Fig. 4b, Supplementary Fig. S10). The FRET results thus are in line with the preceding FLIM data with one sensor only at CoxVIIIa and suggest the existence of a CICIII2CIV supercomplex in situ.

View Article: PubMed Central - PubMed

ABSTRACT

The assembly of respiratory complexes into macromolecular supercomplexes is currently a hot topic, especially in the context of newly available structural details. However, most work to date has been done with purified detergent-solubilized material and in situ confirmation is absent. We here set out to enable the recording of respiratory supercomplex formation in living cells. Fluorescent sensor proteins were placed at specific positions at cytochrome c oxidase suspected to either be at the surface of a CI1CIII2CIV1 supercomplex or buried within this supercomplex. In contrast to other loci, sensors at subunits CoxVIIIa and CoxVIIc reported a dense protein environment, as detected by significantly shortened fluorescence lifetimes. According to 3D modelling CoxVIIIa and CoxVIIc are buried in the CI1CIII2CIV1 supercomplex. Suppression of supercomplex scaffold proteins HIGD2A and CoxVIIa2l was accompanied by an increase in the lifetime of the CoxVIIIa-sensor in line with release of CIV from supercomplexes. Strikingly, our data provide strong evidence for defined stable supercomplex configuration in situ.

No MeSH data available.


Related in: MedlinePlus