Natural resistance‐associated macrophage protein 1 (NRAMP1) is a putative membrane protein that dominates natural resistance to infection. An NRAMP1‐glutathione S‐transferase fusion protein was used to test the ability of the NRAMP1 NH 2 ‐terminal domain to bind to taxol‐stabilized microtubules. Co‐sedimentation analysis showed that the fusion protein binds to microtubules. Although the NH 2 ‐terminal domain of the NRAMP1 molecule has structural homology with the Pro‐rich region of microtubule‐associated protein 4 (MAP4), the presence of the MAP4 microtubule‐binding domain fragment had little effect on the binding of the fusion protein to microtubules.
Endosomal recycling of the mammalian iron transporter DMT1 is assumed to be important for efficient and rapid uptake of iron across the endosomal membrane in the transferrin cycle. Here, we show that the retromer, a complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network, is required for endosomal recycling of DMT1-II, an alternative splicing isoform of DMT1. Bacterially expressed Vps26-Vsp29-Vsp35 trimer, a retromer cargo recognition complex, specifically binds to the cytoplasmic tail domain of DMT1-II in vitro. In particular, this binding is dependent on a specific hydrophobic motif of DMT1-II, which is required for its endosomal recycling. DMT1-II colocalizes with the Vps35 subunit of the retromer in TfR-positive endosomes. Depletion of the retromer by siRNA against Vps35 leads to mis-sorting of DMT1-II to LAMP2-positive structures, and expression of siRNA-resistant Vps35 can rescue this effect. These findings demonstrate that the retromer recognizes the recycling signal of DMT1-II and ensures its proper endosomal recycling.
LexA protein is a represssor of several chromosomal genes involved in the SOS response in Escherichia coli.In previous experiments, we found that LexA protein may also be a repressor of the colicin E l gene.We now present evidence that the purified LexA protein strongly repressed the in vitro transcription of the colicin E l gene.As determined in DNase I protection experiments, LexA protein bound with a high affinity to the approximately 40-base pair long sequence between the Pribnow box and the start codon of the colicin E l gene.The sequence of the binding site was composed of two overlapped "SOS boxes" to which the LexA protein bound in a cooperative manner. ~~ ~~Colicin E l is an antibiotic protein encoded by a plasmid, ColE1.The gene for colicin E l is dormant under ordinary conditions but becomes active with regard to colicin E l production on treatments that damage DNA or inhibit DNA replication.The colicin E l induction is considered to be an "SOS reponse," which includes phenomena such as enhanced DNA repair capacity, induced mutagenesis, inhibition of cell division (filamentation), cessation of respiration, and prophage induction (1, 2).The SOS response is controlled co-' R. Brent, unpublished observations.
Abstract Background Heme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species through the Fenton reaction. Humans can absorb, transport, store, and recycle iron without an excretory system to remove excess iron. Two candidate heme transporters and two iron transporters have been reported thus far. Heme incorporated into cells is degraded by heme oxygenases (HOs), and the iron product is reutilized by the body. To specify the processes of heme uptake and degradation, and the reutilization of iron, we determined the subcellular localizations of these transporters and HOs. Results In this study, we analyzed the subcellular localizations of 2 isoenzymes of HOs, 4 isoforms of divalent metal transporter 1 (DMT1), and 2 candidate heme transporters--heme carrier protein 1 (HCP1) and heme responsive gene-1 (HRG-1)--in non-polarized and polarized cells. In non-polarized cells, HCP1, HRG-1, and DMT1A-I are located in the plasma membrane. In polarized cells, they show distinct localizations: HCP1 and DMT1A-I are located in the apical membrane, whereas HRG-1 is located in the basolateral membrane and lysosome. 16Leu at DMT1A-I N-terminal cytosolic domain was found to be crucial for plasma membrane localization. HOs are located in smooth endoplasmic reticulum and colocalize with NADPH-cytochrome P450 reductase. Conclusions HCP1 and DMT1A-I are localized to the apical membrane, and HRG-1 to the basolateral membrane and lysosome. These findings suggest that HCP1 and DMT1A-I have functions in the uptake of dietary heme and non-heme iron. HRG-1 can transport endocytosed heme from the lysosome into the cytosol. These localization studies support a model in which cytosolic heme can be degraded by HOs, and the resulting iron is exported into tissue fluids via the iron transporter ferroportin 1, which is expressed in the basolateral membrane in enterocytes or in the plasma membrane in macrophages. The liberated iron is transported by transferrin and reutilized for hemoglobin synthesis in the erythroid system.
