Images of budding yeast through the fluorescent, confocal microscope.  These yeast cells were producing a green fluorescent protein (GFP) fusion of the plasma membrane iron permease, Ftr1p.  In addition, the vacuolar membrane is stained with a red fluorescent dye, FM4-64, while the nuclear chromatin is stained with a blue dye, DRAQ-5.  Regions at which the plasma and vacuolar membranes come into close contact appear yellow due the "addition" of the green from Ftr1p-GFP and the red from FM4-64.  It is possible that these points of contact are functional in regards to the trafficking of iron directly into the yeast vacuole.

Perspective on Research in Academia

    A primary responsibility of any PI of an academic research laboratory is the mentoring of the individuals in the lab who actually get the job done; this responsibility is not strongly different from that of a parent, doing what is necessary to make it possible for a daughter or son to establish her/himself as an independently functioning adult.  What I strive for my lab to provide is a mentoring environment that includes a variety of contemporary biochemical, cell biologic and chemical/biophysical approaches in the delineation of a variety of aspects of the cell biology of iron and copper.  My lab offers a collegial, helpful environment in which I require a high level of interaction and cooperation.  Each person in the lab has their "own" project, but each also collaborates on at least one other project with one or more other members of the lab, whether undergrad, grad, post-doc or "technician."  My "technicians" do not wash dishes - everyone does - but have their own projects, too, often involving a grad student or post-doc as "their" technician.  In learning collegiality and responsibility, the students and post-docs in the lab learn to become truly independent investigators who will, some day, be responsible for their own group of scientists whether in academia or industry.  Last, an important aspect of the way we think about problems in metals and cells is the fact that I'm a trained physical organic chemist.  My perspective on these problems is as a chemist; for me, it's all about structure and function, the How and Why about metallobiochemistry.  For us, the yeast or HEK cell producing a recombinant protein under control of a regulatable promoter is not different from adjusting the rate of a reaction in an Erlenmeyer flask by increasing or decreasing the temperature or pH.  We are Bio-Chemists.

Research Objectives

    The long term goal of the research conducted in my lab is to learn about the general principles that organisms use to acquire and metabolize the essential nutrient iron.  Since in eukaryotes, iron metabolism depends on the activity of copper-containing enzymes called ferroxidases, we examine the trafficking copper in cells as well.  The first challenge for a cell is to scavenge these two metals from the environment.  This is true for a yeast cell in culture, or for an epithelial cell in your intestine.  The second challenge is to efficiently and correctly partition these metals in the cell for subsequent utilization and storage.  Ultimately the cell or organism will have to regulate the accumulation of these metals and to ensure that they are not allowed to roam "free" since both are toxic.  

    Iron and copper are essential micronutrients.  They are required in fundamental cellular processes such as cellular respiration in all organisms, and for vital physiologic functions such as oxygen transport in blood and muscle, and the synthesis of the "elastic" material in blood vessels and  and "connective" material in joint ligaments.  However, these metals are also intrinsically toxic.  This toxicity results from their strong tendency to generate oxygen radicals which in turn destroy key cellular components.  The essentiality of these metals and their toxicity are illustrated by the diverse genetic disorders in copper and iron metabolism that result in a variety of human disease.

    There has been a dramatic increase in our understanding of the metabolism of copper and iron in eukaryotes in the past decade.  These rapid advances have resulted primarily from studies carried out in the yeast, Saccharomyces cerevisiae.  S. cerevisiae is a budding yeast, and is the common yeast that we use in brewing and baking.  Yeast and humans share approximately 95% of the same genes, and essentially all of the basic components required for the expression of these genes; the components of cell growth and differentiation; and the components needed for  the utilization of essential nutrients, including copper and iron.  Therefore, biochemical facts that we can establish in yeast are directly useful in our understanding of the same processes in humans.  And yeast have an advantage over nearly all other eukaryotes as an experimental, test system because of the ease with which the remarkable techniques of molecular biology can be used to carry out well-controlled experiments.  However, in the past year, we have established the use of mammalian cells in suspension culture as hosts for the expression of heterologous proteins and for the same type of cell biology experiments we've been carrying out in yeast.  Working in my lab, students and post-docs become well-acquainted with the genetic, cell biologic and biochemical techniques used in working with both yeast and mammalian cells in culture.

    Thus, we also use mammalian cells in culture in our studies on the trafficking and distribution of iron within the eukaryotic cell.  The major focus of these experiments is the production of the ferroxidase enzymes expressed by both humans and lower eukaryotes, and in the examination of their mechanism of action in the mobilization of iron for use in the subsequent biosynthesis of heme. 

At the Copper '04 meeting, Ischia, October, 2004

Coupling Fe(II) Oxidation to Fe(III) Uptake by Fet3p and Ftr1p - One of the most exciting new insights into iron metabolism in eukaryotes is our new understanding of the essential role that copper plays.  At the most practical level, all of us - yeast included - need copper in order to correctly utilize the iron in our diets.  This need is due to the fact that iron uptake and metabolism depends on the enzymic action of a copper enzyme.  Yeast and humans have at least two of these enzymes.  In yeast, these are known as Fet3p and Fet5p while in humans they are known as ceruloplasmin and hephaestin.  Humans that lack the former have severe problems with tissue damage due to iron that is not correctly localized in the cells.  Patients who lack the latter enzyme are iron starved because hephaestin is needed to get the iron in our intestine into the blood stream.  Yeast have  similar "diseases" if they lack one or the other of their Fet proteins.  Both of these proteins work together with an iron permease to transport iron across a membrane.  Fet3p works together with Ftr1p to transport iron into the yeast cell.  A model for how Fet3p and Ftr1p work together in iron transport is illustrated below; as this cartoon shows, iron and copper uptake rely on the activity of a plasma membrane metalloreductase, Fre1p.  A recent review on this system can be obtained by clicking here.  One objective of our current research is to test this model of iron trafficking and then to target it in the development of new anti-fungal agents useful in the treatment of infections due to human pathogens like Candida albicans and Cryptococus neoformans.

    Biophysical studies of Fet3p, in particular, are an important part of this work.  This has included the efforts of our collaborators, John Hart and his associate, Alex Taylor, who have just finished the structure of this important iron-trafficking enzyme. The structure of Fet3p is shown as a ribbon diagram below.  One example of our structure-function work on Fet3p can be found by following this link.

Intracellular Iron Trafficking: Managing Iron in Eukaryotic Cells - In complementary studies, we are examining the pathways of iron trafficking inside the yeast cell following its uptake via the Fet3p, Ftr1p complex.  Little is know about the state of this nutrient prior to its incorporation into heme or iron-sulfur clusters, processes that occur in the mitochondria.  In yeast, this iron is stored in the vacuole from which it is mobilized in times of need.  The focus of this work is the identification of the ligands in the yeast cytosol that coordinate the iron - which is likely to be in the ferrous state - and the resulting trafficking pathways that target this complex to the sites of storage (the vacuole) and utilization, for example, the activation in the cytosol of iron-containing enzymes like ribonucleotide reductase and methyl sterol oxidase.  These studies make use of a combination of cell biologic and spectroscopic approaches including confocal fluorescence microscopy and electron paramagnetic resonance spectroscopy.  A recent paper on the cycling of iron into and out of the vacuole can be found here.

Currently these projects are funded by two grants from the National Institutes of Health :

DK053820-08  "Fet3p (Ferroxidase) and Ftr1p (Permease) in Iron Uptake in Yeast"

DK077826-01  "Managing Ionic Iron: Molecular Architecture and Mechanism of Cell Iron Metabolism"

Ferroxidases in Human Health and Disease - In humans, a deficit of the ferroxidase, ceruloplasmin,  leads to a variety of pathologies including - ultimately - loss of neural function in central nervous system (CNS).  Levels of hCp are determined genetically in the case of the disorders aceruloplasminemia and Wilson's disease, and can also be linked to reduced copper uptake and/or correct copper trafficking.  The molecular basis of the Wilson's disorder is linked also to the mis-trafficking of copper to the sites of hCp activation leading to a decline in the activity of hCp in the patient.  Ferroxidases like hCp and yFet3p have strong activity towards cuprous copper, also; the importance of this metal oxidase activity is that both Fe(II) and Cu(II) are strong pro-oxidants, catalyzing the formation of reactive oxygen species (ROS), well-known to cause damage to many cell components.  In the case of the CNS, this metal-dependent chemistry is linked to neurodegeneration.  We are working on two aspects of this link between metallooxidase and neurodegeneration.  First, we are producing and examining in detail the structure-function characteristics of the ceruloplasmin proteins found in patients with aceruloplasminemia.  In this work we have developed a mammalian cell system to produce these mutant proteins.  Second, we are developing strategies to turn yFet3p and hCp into protein therapeutics to be used to supplement the metallooxidase activity in a patient so as to suppress if not prevent the cell death due to the ROS generated in the mis-management of the ferrous and cuprous ions in the CNS. 

Currently this project is funded by a grant from the National Institutes of Health :

RR024178-01  "Production of Recombinant Eukaryotic Ferroxidases as Protein Therapeutics"

Techniques used in my laboratory

Immunoprecipitation; Immunocytochemistry; Indirect Immunofluorescence  

    We have used the latter technique to establish the orientation and topology of the Ftr1p permease in the yeast plasma membrane.  By comparing the exposure of an epitope in permeabilized versus unpermeabilized yeast spheroplasts (yeast cells without their cell walls), and by inserting the epitope at various sites in the permease's primary sequence, we can show which parts of this sequence are outside of the cell, and which are inside.  For example, an epitope at the carboxyl-terminus is exposed to antibody only in permeabilized cells.  This is seen in the confocal fluorescence images below.

Based on a series of such results, we have proposed the following model for Ftr1p. 

The epitope insertions are indicated, as are a number of motifs that mutagenesis studies have shown are required for iron permeation through Ftr1p.  A PDF of this work can be downloaded by clicking here.

Live cell imaging using fluorescent fusion proteins and confocal fluorescence microscopy; FRET (fluorescence resonance energy transfer) 

   We have made extensive use of GFP, YFP and CFP fusions to Fet3p and Ftr1p to 'track' these two proteins in their trafficking in the yeast cell.  These two proteins form a complex in the plasma membrane, and our images show that for either protein to traffic to the membrane, the other must be present, also.  If only one is produced, it remains localized to the perinuclear region of the cell, most likely in the endoplasmic reticulum.  This is dramatically shown in the image below in which Fet3p:GFP is produced in an ftr1∆ strain, thus one that does not produce any Ftr1p.  The cell in the image is dividing and the nucleus and its surrounding membrane compartment are being segregated between mother and daughter cell. 

In contrast, when Fet3p:GFP is produced in a cell that is also producing Ftr1p, the fusion protein - along with its permease partner - traffics exclusively to the plasma membrane as shown below.

The interaction between Fet3p and Ftr1p in the cell has been examined by fluorescencen resonance energy transfer (FRET).  One way of quantifying FRET is in the confocal microscope in which the FRET signal is given by pixel density as shown in the image below.  What you see here is equivalent to ~10% FRET efficiency indicating the that FRET pairs are ~50Å apart, a reasonable distance for the protein pair illustrate in the cartoon above. 

DNA footprinting and gel retardation assays including EMSA, supershift assays

Yeast two hybrid analyses

Protein expression and purification

Enzyme kinetic analysis

Calorimetry

Spectroscopy including EPR, EXAFS, ESEEM, MCD, and RR done in collaboration