Influence of Min8 in respiration

To investigate the role of Min8 in respect of a potential connection to the Rcf-proteins, a double deletion strain was created in addition to min8∆: min8∆rcf2∆. Both proteins were found to localize adjacent to each other and the idea was that a specific phenotype could arise from a double knockout.

In a steady state analysis of the respective strains’ mitochondria, no discernible difference could be detected, comparing especially complex IV (Cox1, Cox12, Cox13, Cox5a/b) and complex III (Cor1, Rip1) subunits (Figure 3-11A). Also, Rcf-protein levels did not differ between wild-type, min8∆, rcf2∆ and min8∆rcf2∆. Similarly, the oxygen sensed state of the cell, determined by the ratio of the normoxic/hypoxic isoforms Cox5a and Cox5b, remained stable (Figure 3-11B).As a following characterization, the cellular respiration capacity was evaluated by performing a growth drop-test on non-fermentable media. Neither min8∆ nor min8∆rcf2∆ did develop a growth defect on non-fermentable carbon sources (Figure 3-11C). The double mutant rather displayed an enhanced growth ability but on both, glucose and glycerol media. Consequently, Min8 is not considered vital for respiration, neither in combination with Rcf2.

Figure 3-11: Min8 is not essential for respiration.

A) Isolated mitochondria of wild-type (Wt), min8∆, rcf2∆, min8∆rcf2∆ were lysed in Laemmli-buffer and subjected to SDS-PAGE (10-16 %) and western blotting. Tom70 and Aco1 served as loading controls. Cor1 and Rip1 represent complex III, Cox1, Cox12 and Cox13 complex IV. The asterisk marks unspecific cross-reactions of the Cor1 antibody. B) As in A) but mitochondria are applied to urea SDS-PAGE (17.5 %) and western blotting. C) Wild-type, min8∆, rcf2∆, min8∆rcf2∆ cells are spotted on plates in a serial dilution, containing glucose or glycerol media, and grown at 30 °C and 37 °C.

For specifying the role of Min8 and its position at the cytochrome c oxidase, different BN analyses were carried out. To ensure that no complex IV constituent was missed in the prior steady state experiment, the first assessment was performed with DDM solubilized mitochondria. Assembled supercomplexes are resolved into complex IV plus the complex III

A B

C

dimer and the overall amount of complexes can be estimated. While complex IV can be detected as two distinct forms, IV* and IV, Cox13 is only part of the slower migrating complex IV*

(Figure 3-12A). This occurrence was discovered in previous studies and suggested complex IV*

additionally containing Cox13, Rcf1 and Rcf2 (Vukotic et al., 2012). In both complex IV detections, the level of complex IV forms did not change significantly. Similar results were obtained when the respective mitochondria were analyzed after digitonin solubilization (Figure 3-12B). Although a slight tendency of III2IV2-reduction was detected in the particular min8∆

strains (Figure 3-12B, lanes 2, 4, 6, 8, 10 and 12), it could not be proven as significant.

Figure 3-12: MIN8 deletion does not affect respiration capacity.

A) Isolated wild-type, min8∆, rcf2∆, min8∆rcf2∆ mitochondria are solubilized with 0.6 % DDM buffer and used for Blue-Native (BN)-PAGE (6-10 %). Complex IV is detected via Cox1 and Cox13, and complex III via Cyt1. B) As in A) but mitochondria were solubilized with 1 % digitonin buffer and subjected to BN-PAGE (4-10 %). Cox4 and Cox1 represent complex IV and Atp5 complex V. C) Oxygen consumption measurement of isolated wild-type and min8∆ mitochondria with the Oxygraph-2k (n=3). LEAK: non-phosphorylating state; State 3: upon addition of saturating ADP; OXPHOS: maximum respiration under coupled conditions; OXPHOS via CIV: complex IV activity in parallel to complex III inhibition. Error bars indicate mean ± SEM.

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Expecting a more sensitive outcome in a respirometry approach, min8∆ mitochondria were subjected to the oxygen consumption measurements with the Oxygraph-2k, in comparison to wild-type mitochondria. The oxygen consumption was determined in the different states LEAK, state 3, OXPHOS and OXPHOS via complex IV (Figure 3-12C). The terminology is guided by the Oxygraph-2k manufacturer’s specification (Gnaiger 2020).

First, mitochondria are applied to the oxygen chamber containing the respiration medium with appropriate conditions to maintain the mitochondria intact. Mutant and wild-type mitochondria are always measured next to each other to keep the conditions stable for comparison. Pyruvate and Malate are added to allow LEAK respiration – oxygen consumption without added phosphorylating substrates. By this, high proton leakage would be visible since protons cannot escape into the mitochondrial matrix along the membrane potential via complex V. State 3 allows ATPase performance and is triggered by adding ADP to saturating conditions. OXPHOS represents the maximum state of respiration under coupled conditions. For this, succinate is supplemented. Complex IV activity, essentially OXPHOS via complex IV, can be measured in the end by inhibiting complex III with antimycin A and shuttling electrons specifically to complex IV with TMPD and ascorbate. Ascorbate regenerates TMPD, but both undergo autoxidation which is appreciated by sodium azide addition and the residual oxygen consumption is subtracted in the end. All four measured states did not display any difference between min8∆ and wild-type mitochondria.

In the end, these basic characterizing experiments could not assign a specific role for Min8 as complex IV interactor: it is not essential for respiration, nor does it influence the arrangement or the capacity of the respiratory chain. However, the significant interaction with Cox12 detected by chemical crosslinking indicated a close interaction at the periphery of complex IV.

Consequently, we were wondering if Min8 even plays a role in its biogenesis.

3.3.2 Min8 affects Cox12 assembly into cytochrome c oxidase