Proof of INAC*CBP interaction

All previously performed experiments did not reveal a defect in complex III biogenesis upon deletion of INA complex components (Lytovchenko et al., 2014).

72

The steady-state levels of complex III, as well as the activity of the mature enzyme in ina mutants, never differed from that of wild type mitochondria. Moreover, detailed analyses of cbp mutants did not indicate any impairment of F₁F_o-ATP synthase (Gruschke et al., 2011; Gruschke et al., 2012). However, our data clearly show that the INA complex associates with Cbp3, Cbp6, Cbp4 and newly translated cytochrome b. The absence of a clear phenotype for either complex III or V in ina and cbp mutants respectively makes the function of this association unclear.

Figure 2.32 INAC interaction with Cbps is not a post-lysis artifact. [³⁵S]Ina22 was im-ported into wild type and Cbp3^ProtA mitochondria (lanes 6 and 7). Mitochondria were lysed separately and the two lysates were mixed (lane 8). Then, the mixed mi-tochondrial lysate was subjected to affinity chromatography with either anti-Cbp3 antibodies (lane 10), IgG coupled beads (lane 9) or control serum (lane 11) raised against Pam18. Samples were analyzed by SDS-PAGE, followed by autoradiogra-phy (upper panel) and Western blotting with the indicated antibodies (three lower panels). Please note that anti-Ina22 antibodies recognize radioactively labeled Ina22.

Therefore, we asked whether the observed interaction of the INA complex with Cbp proteins indeed happensin vivo or whether we observe an unspecific protein ag-gregation in post lysis mitochondria. To check if the latter was the case, the following experiment was performed. First, in vitro synthesized radioactively labeled Ina22 was imported into WT and Cbp3^ProtA mitochondria and the mitochondria were sub-jected to IgG affinity chromatography. Clearly, imported Ina22 was co-isolated with Cbp3 (Fig. 2.32, lanes 1-5), showing that [³⁵S]Ina22 can be co-isolated with Cbp3 in principle. Next, [³⁵S]Ina22 was imported into wild type, but not into Cbp3^ProtA mitochondria. After this, mitochondria were solubilized separately and the lysate of wild type mitochondria (with imported Ina22) was mixed with Cbp3^ProtA lysate (with no radiolabeled Ina22). The obtained mixed lysate (Fig. 2.32, lane 8) was sub-jected to affinity chromatography, either with beads coupled to IgG (should isolate Cbp3^ProtA), antibodies against endogenous Cbp3 (should isolate both tagged and

RESULTS

non-tagged Cbp3), or control serum (α-Pam18, would still react with Protein A).

Elution fractions were analyzed by SDS-PAGE, followed by autoradiography and Western blotting. As the result shows, imported Ina22 was co-imunoprecipitated only with endogenous Cbp3 protein, but not with Cbp3^ProtA (Fig. 2.32. lanes 9 and 10). The latter proves that the Cbp3-Ina22 interaction does not happen due to protein aggregation in post lysis mitochondria and therefore confirms that the newly identified INAC*CBP complex is not an artifact.

74

The mitochondrial F₁F_o-ATP synthase is the terminal enzyme of OXPHOS system that uses the proton gradient across the inner mitochondrial membrane to drive ATP synthesis. Structurally, this enzyme can be divided into two parts – the hydrophilic F₁, which produces ATP, and the hydrophobic F_o, which forms a membrane proton channel and transmits the energy from proton movement to the catalytic centers. The proton channel is formed by mitochondrial-encoded Atp6 and Atp9 proteins and, on its own, is not involved in ATP synthesis. Nuclear-encoded subunits are required to couple proton movement across the membrane to ATP production. Without this coupling, the proton gradient across the inner mitochondrial membrane would dissipate without productive ATP synthesis and, therefore, only heat would be generated. Some hibernating animals, as well as children, actually use an uncoupled mitochondrial respiration in brown fat tissue for this purpose (Nicholls et al., 1978; Kozak et al., 1988; Kalinovich et al., 2017).

However, in the majority of eukaryotic cells, uncoupled respiration has deleterious consequences.

It is not surprising that expression and assembly of nuclear- and mitochondrial-encoded F₁F_o-ATP synthase subunits should be coordinated in order to avoid the overproduction of individual F₁ and F_o modules. When present in excess in mito-chondria, the F₁ and F_o modules will hydrolyze ATP and dissipate the membrane potential, respectively. Moreover, the biogenesis of the Atp9/Atp6 channel requires special regulation, as its formation prior to association with nuclear-encoded sub-units is highly unfavorable. According to the recently proposed model of F₁F_o-ATP synthase assembly, Atp9 associates with Atp6 in a final assembly step, when nuclear-encoded subunits are already positioned (Rak et al., 2011). However, the mechanism by which the last step is controlled remains unidentified.

The recent identification of the INA complex partially shed some light on how F₁ might be connected to F_o. In this study, we extend upon previous knowledge and show that not only is the INA complex required for peripheral stalk and F1

biogenesis, but it is also involved in the formation of the Atp6/Atp9 proton channel, the final and most important step of F₁F_o-ATP synthase assembly.

DISCUSSION

3.1 Altered expression of mitochondrial-encoded F

F

-ATP synthase components in ina mutants

In this study, we show that deletion of the INA complex alters expression of the mitochondrial-encoded F₁F_o-ATP synthase subunits Atp6, Atp8 and Atp9. Impor-tantly, neither expression nor stability of the other mitochondrial-encoded subunits of the respiratory chain was affected, implying that the observed effect is F₁F_o-ATP synthase-specific. In vivo pulse-labeling with [³⁵S] methionine showed that the levels of Atp6 and Atp8 increase almost two-fold uponINA22 orINA17 deletion. As sta-bility of the newly-translated proteins increased only slightly compared to wild type, we concluded that their production was strongly enhanced and therefore resulted in greater protein abundance. Considering that Atp6 and Atp8 are encoded on the same bi-cistronic mRNA and are present in a one to one stochiometry within the mature complex V, their similar expression levels in various mutants is not surprising and was reported before (Rak and Tzagoloff, 2009; Rak et al., 2016).

Analyses of Atp9 levels in ina mutants gave contradicting results. In vivo pulse-labeling experiments did not reveal significant differences between the mutants and the wild type. However, when Atp9 was analyzed after in organello pulse-labeling with [³⁵S] methionine, we clearly saw a strong reduction in Atp9 levels, of both its monomeric and its oligomeric form. The reason for these results remains unclear. We can speculate that the Atp9 protein is highly unstable and, therefore, its oligomerization and efficient assembly into complex V is required to reduce its turnover. It is not unlikely that the half-life of Atp25 or of the Aep1/Aep2 proteins, required for Atp9 expression and stability (Zeng et al., 2008; Finnegan et al., 1995;

Payne et al., 1993; Ziaja et al., 1993), might be very short and so their constant import into mitochondria is required for efficient Atp9 biogenesis. While the required factors are constantly supplied by the cytoplasmic translation systemin vivo, their translation and import into purified mitochondriain vitro is of course not possible.

Thus, while the Atp9 ring is efficiently assembled into F₁F_o-ATP synthase and therefore stabilized in wild type mitochondria, its impaired assembly inina mutants, in combination with reduced amounts of stabilizing factors in purified mitochondria, might contribute to the observed two-fold decrease in Atp9 levels. However, we do not exclude that some yet unidentified Atp9 biogenesis factors contribute to the observed phenotype in ina mutants.

Neither our results, nor a previously performed extensive analysis of mitochon-drial ribosome-associated proteins, showed INAC association with the mitochonmitochon-drial translation system (Kehrein et al., 2015). Therefore, we exclude INAC association with mitochondrial ribosomes and its direct regulation of Atp6/Atp8 translation.

However, based on previously published data, it is known that Atp6/Atp8 expres-sion depends to a large extent on nuclear-encoded F₁F_o-ATP synthase subunits

76

(Goyon et al., 2008; Rak and Tzagoloff, 2009; Rak et al., 2016). The mechanism of Atp6/Atp8 translational regulation by a preassembled F₁ has been described (Rak and Tzagoloff, 2009; Rak et al., 2016). A preassembled F₁ is required to inhibit Smt1, a suppressor of Atp6/Atp8 translation, and therefore to stimulate Atp22 in-teraction with the ATP6/ATP8 mRNA (Rak et al., 2016). Taking significantly increased levels of free F₁ in ina mutants into consideration, it can be speculated that these higher F₁ levels contribute to a stronger translational stimulation. How-ever, this hypothesis requires further verification, as a direct dependence between the levels of free F₁ and Atp6/Atp8 translation rate was not previously shown.

Moreover, we can not exclude that ATP6/ATP8 mRNA levels are elevated in ina mutants which results in higher translation rates. Though no evidence for a potential role of INAC in ATP6/ATP8 transcription or mRNA maturation has been obtained so far.

To conclude, deletion of the INA complex components indirectly affects trans-lation of Atp6, Atp8 and Atp9. Further analysis is required to shed a light on the mechanistic details of the observed phenotype.

3.2 Atp6 processing defect as a marker for impaired F