This proposal addresses novel mitochondrial-cytoplasm signaling pathways in Saccharomyces cerevisiae as a model eukaryote. In the first mitochondrial-cytoplasm pathway, we discovered a new paradigm in copper trafficking distinct from metallochaperone copper shuttling. This pathway involves the candidate translocation of a copper-ligand (CuL) complex used in metallation of cytochrome c oxidase (CcO) and superoxide dismutase-1 (Sod1) within the mitochondria. In addition to yeast, the CuL complex is a significant source of mitochondrial copper in human fibroblasts, mouse liver and Arabidopsis. We postulate that translocation of the CuL complex into the mitochondria occurs through a yet unidentified mitochondrial transporter after Cu(I) is bound to an abundant free ligand in the cytoplasm.
We aim to elucidate the structure of the CuL complex, verify its translocation into the mitochondrial matrix and identify the transporters as three major objectives. Studies in these objectives will involve analytical and structural biology approaches to deduce the ligand structure and a combination of cellular and in vitro mitochondrial import studies to characterize the translocation pathway. We will attempt to identify the transporters through a genetic screen and the evaluation of a bank of respiratory deficient mutants. A second mitochondrial-cytoplasm pathway relates to iron sensing by the Aft1 transcriptional activator. Transcriptional activation by Aft1 is inhibited in iron-replete cells by a signal emanating from the mitochondrial exporter Atm1. Aft1 function is coupled to the mitochondrial iron status, as a number of yeast mutants exhibiting mitochondrial iron accumulation show constitutively active Aft1.
We aim to determine the mechanism by which iron-inhibition of Aft1 is blocked in these mutants. Iron sensing by Aft1 is dependent on two glutaredoxins and two newly identified proteins Fra1 and Fra2.
We aim to elucidate how these proteins mediate Fe-inhibition of Aft1. Human cells have homologs to Atm1 and Grx3/4 with the human ortholog of Atm1, ABCB7, having a clear role in iron homeostasis. Our recent studies suggest that an expandable matrix iron pool exists. This iron pool is expanded in yeast mutants impaired in iron-sulfur cluster biogenesis and can lead to mis-metallation of superoxide dismutase-2 (Sod2) and constitutive activation of the iron-responsive transcriptional activator Aft1. The unifying themes in this grant are the presence of bioavailable pools of copper and iron within the matrix and mitochondrial-cytoplasm cross talk as a key component in copper and iron homeostasis in eukaryotes including humans. Cross talk between mitochondria and the nucleus has wide implications in a number of metabolic diseases and disorders.

Public Health Relevance

Copper metallation of cytochrome oxidase and superoxide dismutase-1 by the copper- ligand complex within the mitochondria is relevant in human physiology. Deficiencies in cytochrome oxidase assembly lead to respiratory distress in humans presenting as neonatal cardiomyopathies. Mutants of the mitochondrial superoxide dismutase-1 are implicated in the etiology of familial amyotrophic lateral sclerosis. Dysfunction in the human ABCB7 mitochondrial iron exporter is known to cause X-linked sideroblastic anemia with ataxia.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083292-16
Application #
7663956
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Fabian, Miles
Project Start
2008-07-24
Project End
2012-04-30
Budget Start
2009-05-01
Budget End
2010-04-30
Support Year
16
Fiscal Year
2009
Total Cost
$263,375
Indirect Cost
Name
University of Utah
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
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Vest, Katherine E; Leary, Scot C; Winge, Dennis R et al. (2013) Copper import into the mitochondrial matrix in Saccharomyces cerevisiae is mediated by Pic2, a mitochondrial carrier family protein. J Biol Chem 288:23884-92
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Smith, Pamela M; Fox, Jennifer L; Winge, Dennis R (2012) Biogenesis of the cytochrome bc(1) complex and role of assembly factors. Biochim Biophys Acta 1817:276-86
Cui, Tie-Zhong; Smith, Pamela M; Fox, Jennifer L et al. (2012) Late-stage maturation of the Rieske Fe/S protein: Mzm1 stabilizes Rip1 but does not facilitate its translocation by the AAA ATPase Bcs1. Mol Cell Biol 32:4400-9
Winge, Dennis R (2012) Sealing the mitochondrial respirasome. Mol Cell Biol 32:2647-52
Frey, Avery G; Bird, Amanda J; Evans-Galea, Marguerite V et al. (2011) Zinc-regulated DNA binding of the yeast Zap1 zinc-responsive activator. PLoS One 6:e22535
Watts, Talina; Khalimonchuk, Oleh; Wolf, Rachel Z et al. (2011) Mne1 is a novel component of the mitochondrial splicing apparatus responsible for processing of a COX1 group I intron in yeast. J Biol Chem 286:10137-46
Li, Haoran; Mapolelo, Daphne T; Dingra, Nin N et al. (2011) Histidine 103 in Fra2 is an iron-sulfur cluster ligand in the [2Fe-2S] Fra2-Grx3 complex and is required for in vivo iron signaling in yeast. J Biol Chem 286:867-76

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