Ferritin is an iron-binding protein composed of two subunits, H and L. Twenty-four of these subunits assemble to form apoferritins whose subunit composition varies in a characteristic way in different tissues. Using recombinant proteins, we have assessed the role of H and L subunits in mouse ferritin function and compared these to human ferritin subunits. We report that mouse ferritin subunits exhibit considerable functional similarity to their human counterparts, including a prominent role of the H subunit in the facilitation of rapid iron uptake, and a key role of amino acid residues Glu-62 and His-65 in this process. In addition, amino acid residues important to assembly of the protein are conserved between mouse and human, permitting the formation of fully functional hybrid proteins containing both mouse and human subunits. However, murine and human ferritin H subunits also evidenced substantial functional differences; murine ferritin H showed a consistent reduction in iron uptake activity relative to human ferritin H. Creation of chimeric human/mouse ferritin H subunits by "helix swapping" mapped the domain of the protein critical to this activity difference to the DE helix. These findings suggest a novel functional role for carboxyl-terminal domains of the ferritin H subunit.Ferritin is a protein which has as its principal function the intracellular storage of iron in a nontoxic and bioavailable form (see Refs. 1 and 2 for review). Ferritin is ubiquitously distributed in the animal and plant kingdoms and has recently been described in bacteria (3). Mammalian ferritin is composed of two subunits, termed H and L. Twenty-four of these subunits assemble to form the apoferritin protein.In mammals, ferritin is found in most tissues. However, the composition of ferritin varies in a consistent and tissue-specific way. For example, liver contains ferritin that is predominantly of the L subunit type, whereas heart contains ferritin rich in the H subunit. This biodistribution, as well as the evolutionary conservation of a dual subunit structure, supports the hypothesis that the H and L ferritin subunits may play different and complementary roles within the protein (4). Experiments using H-rich and L-rich ferritin prepared from natural sources (5, 6), as well as recent work with recombinant proteins (7-9), have supported this concept of a functional distinction between ferritin H and L subunits and revealed a prominent role of the H subunit in rapid iron oxidation (7), and the involvement of the L subunit in protein stability and iron mineralization (8, 9).Mouse cells and mouse models have been widely used to study iron homeostasis in health and disease (e.g. Refs. 10 -14). For the most part, however, biological inferences concerning ferritin function and iron metabolism in these models have been based on analogy to human ferritin. Indeed, substantial sequence similarity exists between mouse and human ferritin subunits; mouse L and human L exhibit 82% similarity (15), whereas human and mouse ferritin H are 93% identic...

We describe a strategy for the creation of recombinant ferritin heteropolymers which mimic the natural heterogeneity of this protein. This method entailed the co-expression of cDNA for both ferritin H and ferritin L subunits in a single bacterium using either a bicistronic vector, in which both cDNAs were expressed from the vector, or a dual vector expression strategy, in which each subunit was expressed from a separate compatible plasmid in a single bacterial host. Electron microscopy and sucrose density gradient centrifugation demonstrated that ferritin assembled spontaneously in such bacteria to form catalytically active proteins of the expected size and shape. Isoelectric focusing revealed that protein isolated from any of these bacteria exhibited a restricted heterogeneity in subunit composition. Such multi-subunit recombinant ferritins spontaneously assembled in bacteria may be useful in further studies of ferritin assembly and function. Our results further suggest that varying expression levels is a simple way to alter levels of individual components within a multi-subunit recombinant protein, and that this approach may be of general utility in assessing the contribution of individual components to the function of multi-subunit proteins or protein complexes.

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting and committed step in serotonin biosynthesis. Within this enzyme, two distinct domains have been hypothesized to exist, an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. In the present experiments, the functional boundary between the putative domains was defined using deletion mutagenesis. A full-length cDNA clone for rabbit TPH was engineered for expression in bacteria. Five amino-terminal deletions were constructed using PCR, i.e., Nz~50, Nz~60, N~90,Nz~106, and NL~~116 (referring to the number of amino acids deleted from the amino terminus). Enzymatic activity was determined for each mutant after expression in bacteria. Whereas deletion of 116 amino acids (Nz~116) abolished enzyme activity, all of the other amino-terminal deletions exhibited increased specific activity relative to the recombinant wild-type TPH. The ability of the cyclic AMP-dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined. Deletion of the first 60 amino-terminal residues abolished the ability of the enzymeto serve as a substrate for PKA, yet the native and N~50enzymes were phosphotylated. Moreover, a serine-58 point mutant (S58A) was not phosphorylated by PKA. In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine-58. In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase.