Nramp metal transporters: Insights into their structure, function, and subcellular targeting

This thesis examines the molecular properties of Nramp proteins by centering on the two mammalian orthologs. Nramp1 (Slc11a1) is expressed in phagocytic cells and restricts replication of intracellular pathogens by removing divalent metals from the phagolysosome. Nramp2 (DMT1, Slc11a2) mediates uptake of dietary iron in the duodenum and aids in the acquisition of transferrin-associated iron in many cell types. The first half of this thesis explores structure-function relationships. In Chapter 2, the role of charged amino acids within the membrane-spanning segments of Nramp2 was examined by site-specific mutagenesis. These studies identified several invariant charged residues essential for metal transport and pH regulation of activity. In Chapters 3 and 4, the effects of two NRAMP2 mutations found in human patients suffering from severe congenital hypochromic microcytic anemia and iron overload were characterized in vitro. The first mutation was an E399D substitution in a region known as the "conserved transport motif" of the protein. The second mutation was an R416C substitution at an invariant residue in TM9. The effects of both mutations on expression, activity, and subcellular targeting were characterized. In both cases, a quantitative reduction in Nramp2 expression was found to be the cause of microcytic anemia and iron overload in the patients. The second half of this thesis focuses on the subcellular targeting of Nramp1 and 2. In Chapter 5, cytoplasmic signal(s) in Nramp2 responsible for its subcellular targeting/internalization from the plasma membrane were studied. This work led to the identification of a tyrosine-based motif in the carboxyl terminus of Nramp2 (YLLNT555-559) critical for the transporter's internalization from the cell surface and its recycling back to the plasma membrane. Chapter 6 explored differences in trafficking between two splicing isoforms of Nramp2 and found that one isoform (isoform 1) possessed differences in internalization/recycling which enabeled it to become enriched at the plasma membrane. In Chapter 7, the subcellular trafficking properties of Nramp1, including cytoplasmic sequences responsible for targeting to lysosomes, were investigated by using chimeric Nramp1/Nramp2 proteins. This work led to the identification of a tyrosine-based motif (YGSI15-18) in the amino terminus of Nramp1 that functions as a lysosomal targeting signal.