The role of DMQ(9) in the long-lived mutant clk-1

Abstract

Introduction: Ubiquinone (UQ) is a redox active lipid that transfers electrons from complex I or II to complex III in the electron transport chain (ETC). The long-lived Caenorhabditis elegans mutant clk-1 is unable to synthesize its native ubiquinone, and accumulates high amounts of its precursor, 5-demethoxyubiquinone-9 (DMQ(9)). In clk-1, complexes I-III activity is inhibited while complexes II-III activity is normal. We asked whether the complexes I-III defect in clk-1 was caused by: (1) a defect in the ETC; (2) an inhibitory effect of DMQ(9); or (3) a decreased amount of ubiquinone.

Methods: We extracted the endogenous quinones from wildtype (N2) and clk-1 mitochondria, replenished them with exogenous ubiquinones, and measured ETC activities.

Results: Replenishment of extracted mutant and wildtype mitochondria resulted in equal enzymatic activities for complexes I-III and II-III ETC assays. Blue native gels showed that supercomplex formation was indistinguishable between clk-1 and N2. The addition of a pentane extract from clk-1 mitochondria containing DMQ(9) to wildtype mitochondria specifically inhibited complexes I-III activity. UQ in clk-1 mitochondria was oxidized compared to N2.

Discussion: Our results show that no measurable intrinsic ETC defect exists in clk-1 mitochondria. The data indicate that DMQ(9) specifically inhibits electron transfer from complex I to ubiquinone.

Figure 1. The amount of UQ in pentane extracted/replenished N2 and clk-1 mitochondria

(A) The amount of UQ measured in: L-N2 = lyophilized and rehydrated N2 mitochondria; E-N2 = pentane extracted N2 mitochondria; E-N2+UQ₉ = pentane extracted N2 mitochondria reconstituted with UQ₉. The majority of UQ measured in N2 mitochondria before extraction and after extraction and replenishment is UQ₉. (B) The same experimental procedures were performed using mitochondria isolated from clk-1 grown on UQ₉. The majority of UQ measured in clk-1 mitochondria before extraction is the biosynthetic precursor of UQ₉, DMQ₉. L-clk-1 = lyophilized and rehydrated mitochondria from clk-1 grown on UQ₉. E-clk-1 = pentane extracted mitochondria from clk-1 grown on UQ₉. E-clk-1+UQ₉ = pentane extracted mitochondria from clk-1 grown on UQ₉ replenished with UQ₉(C) The same experimental procedures were performed using mitochondria isolated from clk-1 grown on UQ₁₀. The majority of UQ measured in clk-1 mitochondria before extraction is the biosynthetic precursor of UQ₉, DMQ₉. L-clk-1 = lyophilized and rehydrated mitochondria from clk-1 grown on UQ₁₀. E-clk-1 = pentane extracted mitochondria from clk-1 grown on UQ₁₀. E-clk-1+UQ₁₀ = pentane extracted mitochondria from clk-1 grown on UQ₁₀ replenished with UQ₁₀. Data shown here are means +/− standard deviation. Each sample was measured in duplicate from 3 – 5 independent pentane extraction/replenishment experiments. Numbers over largest peaks refer to the values (nmoles/g protein) of DMQ_X or UQ_X of the particular experiment and are added for clarification.

Figure 2. clk-1 mitochondria showed the same electron transport activities as N2 mitochondria after removing DMQ_9.

After pentane extraction and replenishment with UQ₉, activities of (A) complex I-III and (B) complex II-III were measured in N2 and clk-1 mitochondria. The activities were normalized to either NFR (for I-III) or CII (for II-III) activity. After pentane extraction and replenishment with UQ₉, the normalized I-III (A) and II-III (B) activities showed no difference between clk-1 and N2 mitochondria. After replenishment with UQ₁₀, N2 mitochondria had significantly higher activity complex I-III than clk-1 mitochondria. (C) Pentane extracted/replenished clk-1 mitochondria, regardless of whether they were replenished with UQ₉ or UQ₁₀, showed a greater percentage of recovery in complex I-III activity than did N2 when compared with lyophilized and rehydrated mitochondria (* indicates p < 0.05). (D) Pentane extracted clk-1 and N2 mitochondria replenished with UQ₉ showed a similar percentage of recovery of compex II-III activity compared to lyophilized samples. clk-1 mitochondria replenished with UQ₁₀ showed a greater proportional increase in II-III activity than did N2. Data shown here are means +/− standard deviations. Each sample was measured in duplicate from 4 – 7 pentane extraction/replenishment experiments. L-mito= lyophilized mitochondria. E-mito = pentane-extracted mitochondria. +UQ₉ or +UQ₁₀ refers to the ubiquinone used for replenishment.

Figure 3. The organization of respiratory complexes in N2 and clk-1

Representative digitonin based blue native gel (from n=3 independent gels) of mitochondrial proteins from N2 and clk-1 as described in Suthammarak et.al. (Suthammarak et al. 2009). The numbers in the right panel indicate the approximate molecular masses (kDa) of the corresponding protein bands. On the left are the identities of the bands as determined by mass spectrometry in N2. (Suthammarak et al. 2009). No differences were noted between N2 and clk-1.

Figure 4. The quinone profile in mitochondria replenished with UQ₉ and DMQ₉

DMQ₉ was extracted from clk-1 mitochondria and added with UQ₉ to pentane-extracted N2 mitochondria. Mitochondria incorporated DMQ₉ along with UQ₉. L-N2 = lyophilized N2 mitochondria. E-N2 = pentane-extracted N2 mitochondria. E-N2+UQ₉ = pentane extracted N2 mitochondria reconstituted with UQ₉. E-N2+UQ₉+DMQ₉ = pentane extracted N2 mitochondria reconstituted with UQ₉ and DMQ₉ extracted from clk-1 mitochondria.

Figure 5. Mitochondrial activities measured with or without DMQ₉

Complex I-III (A) and II-III (B) activities of UQ₉-replenished mitochondria were measured with or without DMQ₉ added to the replenishment mixture. The complex I-III and II-III activities shown here were normalized to NFR and CII activities, respectively. Complex I-III activity was decreased in the presence of DMQ₉ (p = 0.05). Complex II-III activity was indistinguishable between mitochondria with or without DMQ₉. Data shown here are mean +/− standard deviation. Each sample was measured in duplicate from 3 – 5 pentane extraction/replenishment experiments. E-N2+UQ₉ = pentane extracted N2 mitochondria reconstituted with UQ₉. E-N2+UQ₉+DMQ₉ = pentane extracted N2 mitochondria reconstituted with UQ₉ and DMQ₉ extracted from clk-1 mitochondria.

Figure 6. The percentage of reduced UQ₉ is decreased in clk-1 compared with N2

The percentage of reduced UQ₉ was determined in clk-1 and N2 worms. The percentage of reduced UQ₉ in the clk-1 mutant is 57.7 ± 8.7% of the total UQ, which is lower than that in N2 (74.6 ± 9.1 %, p < 0.05). Data shown here are means +/− standard deviations from 4–6 different worm cultures of N2 and clk-1.