Tether mutations that restore function and suppress pleiotropic phenotypes of theC. elegans isp-1(qm150)Rieske iron–sulfur protein

Abstract

Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron-sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO 2 production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron-sulfur protein Rip1, revealing remarkable conservation of the structure-function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a "spring-loaded" model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity.itochondria are sites for adenosine 5′-triphosphate (ATP) production by oxidative phosphorylation, cellular calcium buffering, iron-sulfur cluster biogenesis, reactive oxygen species (ROS) formation, and regulation of apoptosis. Although inherited defects in mitochondrial function are most often associated with severe childhood disorders, a large number of age-related diseases such as heart disease, cancer, diabetes, obesity, and neurodegeneration have also been linked to mitochondrial dysfunction (1, 2).In Caenorhabditis elegans, multiple studies have demonstrated that reduced electron transport chain activity (ETC) can lead to increased lifespan. These include mutations in the coenzyme Q biosynthetic gene clk-1, the pyrophosphokinase gene tpk-1, and the Rieske iron-sulfur protein isp-1 (3-7). Following RNAi knockdown of ETC components, several other proteins have been implicated in lifespan extension, including HIF-1, GCN-2, CEP-1, CEH-23, TAF-4, AHA-1, CEH-18, JUN-1, NHR-27, and NHR-49 (8-12). In addition, it was proposed that the mitochondrial unfolded protein response (mtUPR) directly mediated lifespan extension from ETC inhibition (13); however, more recent work has suggested that induction of the mtUPR is neither necessary nor sufficient to extend li...

New functional and biophysical insights into the mitochondrial Rieske iron-sulfur protein from genetic suppressor analysis inC. elegans

“…As already noted the majority of the suppressors of isp-1 (qm150) were intragenic and restricted to a small 6 amino acid "tether" region of ISP-1 7 . This region is composed of the amino acids DQRALA, where D__A_A are conserved in a-proteobacteria, yeast, worm, chicken, and human.…”

“…10 The forces involved could be quantified by analysis of data reflecting changes in binding energy upon mutation of the tether region. 7,13 A further discussion of this can be found in the supplementary information of our report. 7 We noted that the forces calculated for the spring-loaded model could be applied to understand control of the reaction and gating of harmful bypass reactions.…”

“…7 We noted that the forces calculated for the spring-loaded model could be applied to understand control of the reaction and gating of harmful bypass reactions. 7 Why did evolution build the spring-loading into the reaction?…”

“…6 Fortunately, multiple mutant lines were identified from 3 saturated screens that showed partial suppression of both the delayed development and the lifespan extension, as well as several other phenotypes associated with the isp-1(qm150) allele. 7 We mapped these suppressing mutations to the tether region of the ISP, all but one within the tether helical span or its H-bonding cap (the chemical "spring") (Fig. 1C), which changes configuration in synchrony with mechanical movement of the ISP head to facilitate interaction with its redox partners 7,8 ( Fig.…”

“…7 We mapped these suppressing mutations to the tether region of the ISP, all but one within the tether helical span or its H-bonding cap (the chemical "spring") (Fig. 1C), which changes configuration in synchrony with mechanical movement of the ISP head to facilitate interaction with its redox partners 7,8 ( Fig. 2).…”