ttm-1 Encodes CDF Transporters That Excrete Zinc from Intestinal Cells of C. elegans and Act in a Parallel Negative Feedback Circuit That Promotes Homeostasis Hyun Cheol Roh 1¤a , Sara Collier 1 , Krupa Deshmukh 1¤b , James Guthrie 2 , J. David Robertson 2,3 , Kerry Kornfeld 1 * 1 Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, United States of America, 2 Research Reactor Center, University of Missouri, Columbia, Missouri, United States of America, 3 Department of Chemistry, University of Missouri, Columbia, Missouri, United States of America Abstract Zinc is an essential metal involved in a wide range of biological processes, and aberrant zinc metabolism is implicated in human diseases. The gastrointestinal tract of animals is a critical site of zinc metabolism that is responsible for dietary zinc uptake and distribution to the body. However, the role of the gastrointestinal tract in zinc excretion remains unclear. Zinc transporters are key regulators of zinc metabolism that mediate the movement of zinc ions across membranes. Here, we identified a comprehensive list of 14 predicted Cation Diffusion Facilitator (CDF) family zinc transporters in Caenorhabditis elegans and demonstrated that zinc is excreted from intestinal cells by one of these CDF proteins, TTM-1B. The ttm-1 locus encodes two transcripts, ttm-1a and ttm-1b, that use different transcription start sites. ttm-1b expression was induced by high levels of zinc specifically in intestinal cells, whereas ttm-1a was not induced by zinc. TTM-1B was localized to the apical plasma membrane of intestinal cells, and analyses of loss-of-function mutant animals indicated that TTM-1B promotes zinc excretion into the intestinal lumen. Zinc excretion mediated by TTM-1B contributes to zinc detoxification. These observations indicate that ttm-1 is a component of a negative feedback circuit, since high levels of cytoplasmic zinc increase ttm-1b transcript levels and TTM-1B protein functions to reduce the level of cytoplasmic zinc. We showed that TTM-1 isoforms function in tandem with CDF-2, which is also induced by high levels of cytoplasmic zinc and reduces cytoplasmic zinc levels by sequestering zinc in lysosome-related organelles. These findings define a parallel negative feedback circuit that promotes zinc homeostasis and advance the understanding of the physiological roles of the gastrointestinal tract in zinc metabolism in animals. Citation: Roh HC, Collier S, Deshmukh K, Guthrie J, Robertson JD, et al. (2013) ttm-1 Encodes CDF Transporters That Excrete Zinc from Intestinal Cells of C. elegans and Act in a Parallel Negative Feedback Circuit That Promotes Homeostasis. PLoS Genet 9(5): e1003522. doi:10.1371/journal.pgen.1003522 Editor: David J. Eide, University of Wisconsin, United States of America Received December 7, 2012; Accepted April 6, 2013; Published May 23, 2013 Copyright: ß 2013 Roh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants from the National Institute of Health to KK (GM068598 and CA84271) and from National Science Foundation to the University of Missouri (BCS-0922374). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kornfeld@wustl.edu ¤a Current address: Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America ¤b Current address: Dupont, Hyderabad, India Introduction The trace element zinc is essential for all biological systems. Zinc is a well established structural and enzymatic cofactor required for the function of numerous proteins [1], and emerging evidence indicates that zinc functions as a signaling molecule in several biological processes, including development, immune response and synaptic transmission [2–4]. Organisms have evolved mechanisms that control zinc homeostasis and metabolism in individual cells and the entire body, and these mechanisms are critical for human health. Diets that contain deficient or excess zinc and result in impaired zinc homeostasis cause a wide range of defects in human health [5,6]. Furthermore, genetic mutations that cause aberrant zinc metabolism are implicated in a variety of human diseases, such as cancer, diabetes and neurodegenerative diseases [7–13]. Thus, understanding mechanisms of zinc homeo- stasis has important implications for human health. Zinc metabolism is regulated at the cellular and organismal levels. In eukaryotic cells, the movement of zinc ions across membranes is mediated by transmembrane zinc transporter proteins from two major families, Cation Diffusion Facilitator (CDF/ZnT/SLC30) and Zrt-, Irt-like protein (ZIP/SLC39) [14]. CDF proteins have six conserved transmembrane motifs (TMs) and transport cytoplasmic zinc out of the cell or into the lumen of intracellular organelles, thereby decreasing the level of cytoplasmic zinc. By contrast, ZIP proteins have eight conserved TMs and transport zinc in the opposite directions, thereby increasing the level of cytoplasmic zinc. It appears that almost all zinc ions in the cytoplasm are bound to small molecules or soluble proteins such as metallothioneins [15]. Metallothioneins are small, cysteine-rich proteins that bind up to seven zinc atoms and may function in zinc detoxification and storage. A critical question is how the activity of zinc transporters and metallothioneins are coordinately regulated to maintain zinc homeostasis. Some evidence indicates that, in response to high zinc conditions, the expression of specific CDF proteins and methallothioneins are induced to promote zinc excretion and sequestration in intracellular organelles and proteins, thereby protecting from zinc toxicity [16,17]. PLOS Genetics | www.plosgenetics.org 1 May 2013 | Volume 9 | Issue 5 | e1003522