Zinc is an essential nutrient because of the important roles this metal plays as a catalytic and structural cofactor. Several zinc-dependent proteins reside within organelles such as the endoplasmic reticulum (ER), Golgi, and mitochondria. Therefore, transporter proteins are needed to distribute zinc into intracellular compartments. Because excess zinc can be toxic, cells also require homeostatic mechanisms to control the intracellular levels of free or labile zinc in the cytosol and within organelles. Cellular zinc homeostasis is achieved by several mechanisms including the control of zinc uptake, efflux, vesicular storage, and binding by metallothionein. In this proposal, another important facet of zinc homeostasis is considered, i.e. the control of zinc levels within intracellular organelles. These processes will be studied using the yeast Saccharomyces cerevisiae as a model eukaryotic cell. Preliminary results have raised five central hypotheses that provide the foundation for the specific aims of this proposal: 1) The yeast Msc2 and Zrg17 proteins form a heteromeric complex that transports zinc into the ER.
In Aim 1, the composition of this complex will be assessed and its zinc transport activity will be characterized. 2) It is proposed that zinc finger domains will make useful probes of zinc status in the ER and other compartments of living cells.
In Aim 2, new in vivo zinc sensors will be developed based on fluorescence resonance energy transfer (FRET). These and other assays to be developed will be used to define the role of Msc2/Zrg17 and other transporters in maintaining ER zinc homeostasis. 3) Zinc transport into the ER via the Msc2/Zrg17 complex is regulated in response to zinc status by both transcriptional and post-translational control mechanisms.
In Aim 3, the molecular mechanisms of these regulatory systems will be dissected and their roles in maintaining ER zinc status determined. 4) It is proposed that zinc transport into the ER is mediated by transporters in addition to the Msc2/Zrg17 complex.
In Aim 4, other zinc transporters for this compartment will be identified. 5) Several studies of mammalian cells showed that zinc deficiency leads to increased oxidative stress, the source of which has long been a mystery.
In Aim 5, the hypothesis that the oxidative stress of zinc deficiency arises from the loss of ER zinc homeostasis will be tested.
These aims represent a cohesive and comprehensive analysis of zinc homeostasis in the secretory pathway of eukaryotic cells.7.

Public Health Relevance

The processes of intracellular zinc transport and homeostasis within organelles are essential for basic cellular function, physiology, and human health. Despite this importance, however, we know little about the transporters involved and how they are regulated in response to zinc status and other factors. As a result of our proposed studies, we will obtain a fundamental understanding of zinc metabolism in the secretory pathway of all eukaryotes and generate new probes of intracellular zinc that will be useful in our studies as well as in the analysis of zinc metabolism in mammals and other organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM056285-15S1
Application #
8708248
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1997-09-30
Project End
2014-07-31
Budget Start
2011-09-01
Budget End
2014-07-31
Support Year
15
Fiscal Year
2013
Total Cost
$107,196
Indirect Cost
$35,196
Name
University of Wisconsin Madison
Department
Nutrition
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Carvalho, Sandra; Barreira da Silva, Rosa; Shawki, Ali et al. (2015) LiZIP3 is a cellular zinc transporter that mediates the tightly regulated import of zinc in Leishmania infantum parasites. Mol Microbiol 96:581-95
Mith, Oriane; Benhamdi, Asma; Castillo, Teddy et al. (2015) The antifungal plant defensin AhPDF1.1b is a beneficial factor involved in adaptive response to zinc overload when it is expressed in yeast cells. Microbiologyopen 4:409-22
Eide, David J (2014) Bacillithiol, a new role in buffering intracellular zinc. Mol Microbiol 94:743-6
MacDiarmid, Colin W; Taggart, Janet; Kerdsomboon, Kittikhun et al. (2013) Peroxiredoxin chaperone activity is critical for protein homeostasis in zinc-deficient yeast. J Biol Chem 288:31313-27
Ehrensberger, Kate M; Mason, Carter; Corkins, Mark E et al. (2013) Zinc-dependent regulation of the Adh1 antisense transcript in fission yeast. J Biol Chem 288:759-69
Jeong, Jeeyon; Eide, David J (2013) The SLC39 family of zinc transporters. Mol Aspects Med 34:612-9
Frey, Avery G; Eide, David J (2012) Zinc-responsive coactivator recruitment by the yeast Zap1 transcription factor. Microbiologyopen 1:105-14
Jeong, Jeeyon; Walker, Joel M; Wang, Fudi et al. (2012) Promotion of vesicular zinc efflux by ZIP13 and its implications for spondylocheiro dysplastic Ehlers-Danlos syndrome. Proc Natl Acad Sci U S A 109:E3530-8
North, Matthew; Steffen, Janet; Loguinov, Alex V et al. (2012) Genome-wide functional profiling identifies genes and processes important for zinc-limited growth of Saccharomyces cerevisiae. PLoS Genet 8:e1002699

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