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Mechanisms of iron release from lysosomes
Author(s) -
Nguyen Annie,
Zhao Ningning,
Morrison Chevaun,
Gonzalez Angelica,
Sauble Eric,
La Alice,
Linder Maria C.,
Knutson Mitchell
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.921.11
Subject(s) - dmt1 , endosome , cytoplasm , chemistry , transporter , microbiology and biotechnology , colocalization , ferritin , transfection , autophagy , bafilomycin , hek 293 cells , biochemistry , intracellular , gene knockdown , ferric , biology , apoptosis , organic chemistry , gene
Stored cellular Fe is made available and recycled at least partly through lysosomal degradation of cytoplasmic ferritin (Ft) after autophagy, but how Fe returns to the cytoplasm is unknown. Divalent metal transporter 1 (DMT1) is associated not just with transferrin‐related endosomes but also with lysosomes, and the latter is true for Zip 8, which might also be an Fe transporter. To begin the determine whether one or both DMT1 and Zip8 might be involved, we first established that Zip 8 transfection into HEK cells (with low endogenous levels) enhanced Fe uptake, determined with 59 Fe‐citrate. We then knocked down expression of Zip 8 with siRNA in rat hepatoma cells, and studied the accumulation of 59 Fe in lysosomes separated from cytoplasmic Ft on iodixanol gradients (Kidane et al, Am J Physiol 291: C445, 2006). To track and move Ft into lysosomes, we pretreated cells with 59 Fe‐ferric ammonium citrate for 24h then induced Fe depletion with deferoxamine (as previously). Compared with scrambled siRNA, 80‐90% knockdown of Zip 8 mRNA was associated with about 35% greater retention of 59 Fe in lysosomes. Using confocal microscopy on HepG2 cells, we also determined that Fe depletion rapidly increased colocalization of DMT1 with the lysosomal marker (LAMP2). We conclude that both of these transporters could be involved in the return of lysosomal Fe to the cytoplasm. Supported in part by PHS Grants RO1 HD 46949 (MCL) and RO1DK080706 (MK).