Cu is an essential nutrient for nearly all forms of life because it serves as an enzyme prosthetic group for catalyzing redox reactions or reactions involving O2 chemistry. These reactions are central to aerobic life. Our long term goal is to understand the metabolism of copper, especially when it is a limiting nutrient or in a situation of metabolic defect. Two decades ago, we discovered the paradigm of Cu sparing in the model organism, Chlamydomonas, where a Cu-independent protein can, in a situation of Cu-deficiency, replace an otherwise abundant Cu protein. The wide-spread occurrence of this mechanism in many microbial systems for various trace mineral nutrients (Fe, Zn, Mo, to list a few) is now well-established. Metal sparing mechanisms are likely to be important for the success of certain infectious bacteria and fungi in evading host defense systems, where metal sequestration is used as a defensive strategy. A new paradigm, discovered in the previous project period, is Cu "salvage", in which Cu is removed from a non-essential protein so that it can be recycled and used for the synthesis of an essential cuproprotein. In Chlamydomonas, replacement and salvage mechanisms are turned on in copper-deficient cells by a copper-sensing transcription factor, CRR1. Its target genes are associated with copper response elements, which serve as binding sites for the DNA binding domain of CRR1. We have identified all the CRR1 target genes in the Chlamydomonas genome by next gen transcriptome profiling (RNA-Seq).
In Specific Aim 1, we will undertake biochemical and reverse genetic analysis of select target genes, IRT2 (encoding a ZIP family FeII transporter), AOF1 (encoding a flavin amine oxidase), 142634 (encoding a down-regulated plastid-targeted metallochaperone) to further elaborate the copper sparing pathway, and of RSEP1 (encoding a thylakoid lumen protease) and CTR3 (encoding a soluble copper binding protein) to assess their function in the salvage pathway. In zinc-deficient Chlamydomonas cells, copper is hyper-accumulated in bio-inaccessible compartments, resulting in functional copper-deficiency. This phenotype has been observed recently in mammalian cells with disruptions in Cu homeostasis factors. Hyper- accumulation of Cu in Chlamydomonas requires CRR1.
In Aim 2, we will undertake biochemical characterization of these copper-loaded compartments and we will use high throughput screening methods for a classical genetic approach for the discovery of factors involved in loading and unloading Cu into these compartments. CRR1 is also required in Chlamydomonas for zinc homeostasis. Besides ZIP family transporters, two novel proteins, 123019 and 117548, with COG0523 domains (conserved in all kingdoms of life) are highly up-regulated in zinc-deficiency.
In Aim 3, we will distinguish the mechanism of regulation of the corresponding genes, identify zinc response elements, and use gain-of-function mutants (by over-expression) to deduce whether they are zinc chaperones.

Public Health Relevance

Mineral nutrients like Cu and Zn are required by all forms of life because the elements contribute to structures of molecules and they also provide catalytic centers;hence deficiencies, resulting from genetic defect or poor nutrition, can hinder normal metabolism. In this project, a model organism Chlamydomonas is used to discover mechanisms of Cu storage, sparing and salvage in Cu and Zn deficient cells. These mechanisms optimize the use of the mineral nutrients for the most important metabolic pathways.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM042143-20
Application #
8461624
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Maas, Stefan
Project Start
1989-06-01
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
20
Fiscal Year
2013
Total Cost
$376,130
Indirect Cost
$131,890
Name
University of California Los Angeles
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Hong-Hermesdorf, Anne; Miethke, Marcus; Gallaher, Sean D et al. (2014) Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas. Nat Chem Biol 10:1034-42
Bernal, Maria; Casero, David; Singh, Vasantika et al. (2012) Transcriptome sequencing identifies SPL7-regulated copper acquisition genes FRO4/FRO5 and the copper dependence of iron homeostasis in Arabidopsis. Plant Cell 24:738-61