# The Role of Cardiolipin in Assembly and Function of the Mitochondrial Respirasome

> **NIH NIH R01** · UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON · 2020 · $467,450

## Abstract

Individual respiratory complexes of mitochondria are in dynamic equilibrium with higher order supercomplex
organizations that compose the respirasome. Physiological and pathological perturbations in the mitochondrial
cardiolipin (CL) pool directly affect and regulate this equilibrium. Aging, neurodegenerative diseases, heart
failure, ischemia/reperfusion, cancer and Barth Syndrome are associated with abnormal CL pools.
Saccharomyces cerevisiae mutants lacking CL display similar phenotypes to mammalian cells with reduced CL
levels making yeast an excellent model system. We were the first to report that formation of the yeast
tetrameric respiratory supercomplex (SC III2IV2) and cytochrome c (cyt c) channeling between complexes III
(CIII, bc1 complex) and IV (CIV, cyt c oxidase) within the SC are directly dependent on CL. Using our recently
acquired K2 Summit direct electron detector for single particle electron cryo-microscopy, we have significantly
improved the quality of EM images. We achieved a higher resolution 3D density map of the tetrameric SC than
we previously reported. We are now in a position to attain 3D density maps of SCs at an unprecedented sub-
nanometer resolution. We propose an innovative combination of high resolution structural determinations,
functional assays, genetic manipulation of yeast cells and novel lipid-dependent reconstitution studies to
establish the molecular basis for CL-dependent formation and function of respiratory SCs. Aim 1: A) obtain a
sub-nanometer resolution 3D density map for the tetrameric SC to establish the precise dimensions of the lipid-
filled gaps and the interface between CIII and CIV; B) determine the location(s) of bound cyt c in the tetrameric
SC to decipher how SC formation makes cyt c channeling possible; C) resolve the structure of the III2IV1 trimer
to understand how structural differences with the tetramer results in CL-independent formation and lack of cyt c
channeling in the trimer; D) perform structural studies of the SC tetramer lacking subunit Qcr6 of CIII coupled
with lipid analysis (under Aim 2) to determine whether Qcr6p maintains the lipid-filled gap between CIII and
CIV, which makes SC formation sensitive to CL levels. Aim 2: A) integrate structural with quantitative analysis
of CL and other lipids present in the above SCs; B) determine the features of CL that support tetrameric SC
formation and function using an innovative in vitro SC-reconstitution system employing structural analogs of
CL; C) mimic pathological conditions resulting in CL pool alterations to understand how CL levels and CL
oxidation affect SC structure and function. Aim 3: A) determine CIII and CIV surface exposed CL-binding sites
potentially responsible for SC formation using a photoactivatible CL analog; B) use Molecular Dynamic
Simulations to predict surface exposed CL-binding sites; C) employ site-directed mutagenesis at chemically
modified and predicted CL-binding sites that lie at the CIII-CIV interface fo...

## Key facts

- **NIH application ID:** 9829578
- **Project number:** 5R01GM115969-04
- **Recipient organization:** UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
- **Principal Investigator:** WILLIAM DOWHAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $467,450
- **Award type:** 5
- **Project period:** 2016-12-02 → 2021-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9829578

## Citation

> US National Institutes of Health, RePORTER application 9829578, The Role of Cardiolipin in Assembly and Function of the Mitochondrial Respirasome (5R01GM115969-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9829578. Licensed CC0.

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