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Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of sitedirected TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.

DNA polymerase epsilon (pol epsilon) is a multiple subunit complex consisting of at least four proteins, including catalytic Po12p, Dpb2p, Dpb3p, and Dpb4p. Pol epsilon has been shown to play essential roles in chromosomal DNA replication. Here, we report reconstitution of the yeast pol epsilon complex, which was expressed and purified from baculovirus-infected insect cells. During the purification, we were able to resolve the pol epsilon complex and truncated Po12p (140 kDa), as was observed initially with the pol epsilon purified from yeast. Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity, and stimulation by proliferating cell nuclear antigen indicates that the reconstituted pol epsilon is functionally identical to native pol epsilon purified from yeast and is therefore useful for biochemical characterization of the interactions of pol epsilon with other replication, recombination, and repair proteins. Identification and characterization of a proliferating cell nuclear antigen consensus interaction domain on Po12p indicates that the motif is dispensable for DNA replication but is important for methyl methanesulfonate damage-induced DNA repair. Analysis of the putative zinc finger domain of Po12p for zinc binding capacity demonstrates that it binds zinc. Mutations of the conserved cysteines in the putative zinc finger domain reduced zinc binding, indicating that cysteine ligands are directly involved in binding zinc.

Monoclonal antibodies were previously used to identify two glycoproteins, called fasciclin I and II (70 and 95 kDa, respectively), which are expressed on different subsets of axon fascicles in the grasshopper (Schistocerca americana) embryo. Here the monoclonal antibodies were used to purify these two membrane-associated glycoproteins for further characterization. Fasciclin II appears to be an integral membrane protein, where fasciclin I is an extrinsic membrane protein. The amino acid sequences of the amino terminus and fragments of both proteins were determined. Using synthetic oligonucleotide probes and antibody screening, we isolated genomic and cDNA clones. Partial DNA sequences of these clones indicate that they encode fasciclins I and II.