The divalent metal transporter (DMT1, also known as NRAMP2 or DCT1) is the likely target for regulation of intestinal iron absorption by iron stores. We investigated changes in intestinal DMT1 expression after a bolus of dietary iron in iron-deficient Belgrade rats homozygous for the DMT1 G185R mutation (b/b) and phenotypically normal heterozygous littermates (+/b). Immunofluorescent staining with anti-DMT1 antisera showed that DMT1 was located in the brush-border membrane. Duodenal DMT1 mRNA and protein levels were six- and twofold higher, respectively, in b/b rats than in +/b rats. At 1.5 h after dietary iron intake in +/b and b/b rats, DMT1 was internalized into cytoplasmic vesicles. At 1.5 and 3 h after iron intake in +/b and b/b rats, there was a rapid decrease of DMT1 mRNA and a transient increase of DMT1 protein. The decrease of DMT1 mRNA was specific, because ferritin mRNA was unchanged. After iron intake, an increase in ferritin protein and decrease in iron-regulatory protein binding activity occurred, reflecting elevated intracellular iron pools. Thus intestinal DMT1 rapidly responds to dietary iron in both +/b and b/b rats. The internalization of DMT1 may be an acute regulatory mechanism to limit iron uptake. In addition, the results suggest that in the Belgrade rat DMT1 with the G185R mutation is not an absolute block to iron.

The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes. Cell lines were generated that overexpressed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT-and Rab7 Q67L-expressing cells contained a reduced number of vesicles the size of postlysosomes (>2.5 ,um) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, >90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal a-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted a-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H+-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.

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