Endosome-Mitochondria Interface Controls Intracellular Iron Trafficking in Erythroid Cells

In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe2+ into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. An opposing view is that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria ("kiss-and-run" hypothesis; Ponka Blood 89:1, 1997). Despite the longevity of the prevailing opinion, experimental evidence (Zhang et al. Blood 105:368, 2005; Sheftel et al. Blood 110: 125, 2007) only supports the latter hypothesis. Using 3D live confocal imaging of reticulocytes following their incubation with MitoTracker Deep Red (MTDR) and Alexa Green Transferrin (AGTf), we have demonstrated transient endosome-mitochondria interactions. We have also documented these interactions by a novel method exploiting flow sub-cytometry to analyze reticulocyte lysates labeled with MTDR and AGTf. We have thusly identified a population of particles labeled with both fluorescent markers, representing endosomes interacting with mitochondria. FACS followed by 2D confocal microscopy confirmed the association of both organelles in the double-labeled population. In the current study, we examined whether reticulocyte mitochondria interact with transferrin (Tf) in a cell-free system. Lysates of reticulocytes previously labeled with MTDR were incubated with AGTf for various time intervals. Examination of lysates by 2D confocal microscopy revealed a time-dependent increase in the number of mitochondria in contact with fluorescent Tf. This can be prevented by the presence of excess, unlabeled Fe2-Tf, but not by albumin (Fig.1). Moreover, the addition of unlabeled Fe2-Tf to reticulocyte lysates removed AGTf from mitochondria, indicating that mitochondria from reticulocyte lysates are associated with TfR that can reversibly bind Tf. In addition, we demonstrate that endosomes containing mutated recombinant holotransferrin, which cannot release iron, remain associated with mitochondria, while endosomes containing mutated recombinant apotransferrin, which cannot bind iron, are not associated with mitochondria. Our findings indicate that endosomes containing holo-Tf promote their attachment to, and drive the detachment of apo-Tf-endosomes from, mitochondria, respectively. By co-immunoprecipitation assay (from murine eryhroleukemia [MEL] cells and reticulocytes lysates), we purified the voltage-dependent anion channel 2 (VDAC2), which is located at the outer membrane of the mitochondrion (Graham, et al. Curr Top Dev Biol. 59: 87, 2004) with DMT1. We confirmed the colocalization of VDAC2 and DMT1 in MEL cells and reticulocytes by both immunofluorescence and confocal microscopy. Moreover, we found a significant decrease in the number of mitochondria in contact with Tf-endosomes after depletion of VDAC2 in MEL cells or after treatment of reticulocyte lysates with the mitochondrial uncoupler CCCP, further supporting the concept of a physical interaction between endosomes and mitochondria. To examine a possible role of DMT1-VDAC2 interactions in iron trafficking, we depleted MEL cells of VDAC2 or inhibited VADC2 using erastin (a specific VDAC2 inhibitor that alters its gating) followed by the measurement of 59Fe incorporation from 59Fe-Tf into heme. Our finding of decreased 59Fe incorporation into heme of MEL cells with silenced or inhibited VDAC2 supports the idea that this outer-membrane mitochondrial protein is involved in the interaction of endosomes with mitochondria. We are currently continuing to delineate the molecular mechanisms involved in endosome-mitochondria interactions focusing on the "signal(s)" that direct iron-carrying endosomes towards mitochondria, the players involved in the docking of endosomes to mitochondria and the "signal(s)" that determine the detachment of iron-free endosomes from mitochondria. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Interaction of Transferrin-Endosomes with Mitochondria: Implications for Iron Transport to Ferrochelatase in Erythroid Cells

Delivery of iron (Fe) to most cells occurs following the binding of diferric transferrin (Tf) to its cognate receptors on the cell membrane following which the Tf-receptor complexes are internalized via endocytosis. Iron is then released from Tf within endosomes by a combination of Fe3+ reduction by Steap3 and a decrease in pH (~ pH 5.5). Subsequently, Fe2+ is transported through the endosomal membrane by DMT1. In erythroid cells, more than 90% of Fe has to enter mitochondria where ferrochelatase, the final enzyme in the heme biosynthetic pathway that inserts Fe2+ into protoporphyrin IX, resides. The intracellular path of iron from endosomes to ferrochelatase is still obscure or, at best, controversial. The prevailing opinion is that Fe, after its export from endosomes, spreads into the cytosol, from where the metal mysteriously finds its way into mitochondria. An opposing view is that the highly efficient transport of Fe toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria ("kiss-and-run" hypothesis;Ponka Blood 89:1, 1997). Despite the longevity of the prevailing opinion, experimental evidence (Richardson et al. Blood 87:3477, 1996; Zhang et al. Blood 105:368, 2005; Sheftel et al. Blood 110: 125, 2007) only supports the latter hypothesis, which sees favorable reception among Cell Biologists (McBride BMC Biology 13:8, 2015). Our laboratory has demonstrated, using both 2D and 3D live confocal imaging, that the intracellular Fe pathway in erythroid cells indeed involves a transient interaction of endosomes with mitochondria. To furtherdemonstrate the contact between these organelles, we have developed a novel method based on flow cytometry analysis ("flow sub-cytometry") of lysates obtained from reticulocytes with fluorescently labeled mitochondria (MitoTracker Deep Red; MTDR) and endosomes (Alexa Green Transferrin; AGTf). Using this strategy, we have identified three distinct populations: endosomes, mitochondria, and a population double-labeled with both fluorescent markers representing endosomes interacting with mitochondria. The size of the double-labeled population increases with the incubation time and plateaus in approximately 20 min. In this study, we examined whether reticulocyte mitochondria interact with Tf in a cell-free system. Lysates obtained by freeze-thawing of reticulocytes previously labeled with MTDR were incubated with AGTf for various time intervals. Examination of lysates by 2D confocal microscopy has revealed a time-dependent increase in the number of mitochondria being in contact with Tf-endosomes (fig 1: Images of mitochondria and endosomes; 20 min incubation with AGTf). This can be prevented by Fe2-Tf, but not by albumin, added to lysates. Moreover, the addition of unlabeled Fe2-Tf to reticulocyte lysates removed AGTf from mitochondria. We conclude that mitochondria from freeze-thawed reticulocyte lysates are associated with TfR that can reversibly bind Tf. We have also embarked on uncovering molecular partners involved in the endosome-mitochondria interactions. Using co-immunoprecipitation and pull-down strategies, we have attempted to detect proteins interacting with the intracellular loops of DMT1 that could be candidates for interactions with mitochondria. The co-immunoprecipitated proteins were separated based on their molecular weights, stained using Coomassie and/or Silver gel and identified by mass spectrometry followed by western blotting. We co-immunoprecipitated (from murine eryhroleukemia [MEL] cells and reticulocytes lysates) proteins that were pulled down with DMT1. One of the proteins that we have recognized is the voltage-dependent anion channel (VDAC), which is located at the outer membrane of the mitochondrion (Graham, et al. Curr Top Dev Biol. 59: 87, 2004). The identity of DMT1 was confirmed by western blotting using specific antibodies against VDAC. These results further support the concept of the physical interaction between endosomes and mitochondria. To examine a possible role of DMT1-VDAC interactions in iron trafficking, we silenced the expression of VDAC in MEL cells followed by the measurement of 59Fe incorporation from 59Fe-Tf into heme. Our finding of decreased 59Fe incorporation into heme of MEL cells with silenced VDAC supports the idea that this outer-membrane mitochondrial protein is involved in the interaction with endosomes. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Further Evidence for the “Kiss and Run” Hypothesis of Iron Delivery to Mitochondria in Erythroid Cells,

Abstract 3178 Delivery of iron (Fe) to most cells occurs following the binding of diferric transferrin (Tf) to its cognate receptors on the cell membrane. The Tf-receptor complexes are then internalized via endocytosis, and iron is released from Tf by a process involving endosomal acidification and reduction by Steap3. Iron is then transported across the endosomal membrane by the divalent metal transporter, DMT1. Unfortunately, the post-endosomal path of iron within cells remains elusive or is, at best, controversial. It has been commonly accepted that a low molecular weight intermediate chaperones iron in transit from endosomes to mitochondria and other sites of utilization; however, this much sought iron binding intermediate has never been identified. In erythroid cells, more than 90% of iron has to enter mitochondria where ferrochelatase, the final enzyme in the heme biosynthetic pathway that inserts Fe2+ into protoporphyrin IX, resides. Indeed, strong evidence exists for specific targeting of Fe toward mitochondria in developing red blood cells in which iron acquired from Tf continues to flow into mitochondria even when the synthesis of protoporphyrin IX is suppressed. Based on this, we have formulated the hypothesis that, in erythroid cells, a transient mitochondrion-endosome interaction is involved in Fe translocation to its final destination and have collected experimental support for this proposition (Zhang et al. Blood 105:368, 2005; Sheftel et al. Blood 110: 125, 2007). We have previously shown, using 3D live confocal imaging, that the iron delivery pathway in reticulocytes involves a transient interaction of endosomes with mitochondria. Moreover, we have demonstrated the interaction of these organelles by a novel method exploiting flow cytometry to analyze reticulocyte lysates labeled with Alexa Green Transferrin (AGTf) and MitoTracker Deep Red (MTDR). By using this new technique of flow subcytometry, we identified a double-labeled population representing endosomes interacting with mitochondria. The dynamic nature of this interaction was shown by chase experiments in which a time-dependent decrease of the double-labeled population was observed when reticulocytes were washed and re-incubated with unlabeled Fe2-Tf. Furthermore, we have shown that the iron status of endosomes governs the efficacy of endosome-mediated iron delivery to mitochondria. Experiments with heme, which feedback inhibited the release of iron from Tf in endosomes, slows the generation of the double-labeled population. Additionally, treatment of cells with heme in chase experiments retarded the dissociation of endosomes from mitochondria. In this study, we provide further evidence for an interaction between mitochondria with endosomes. Fluorescently-labeled mitochondria isolated from mouse reticulocytes with MTDR and AGTf were analyzed using 2D confocal microscopy. Results from these experiments confirmed that mitochondria indeed come in physical contact with endosomes. In addition, we have used different constructs of fluorescently labeled, recombinant human Tf, which either remain permanently bound to iron (recombinant diferric-transferrin; rTf) or cannot bind to iron (recombinant apotransferrin; rapoTf), in flow subcytometry studies. As expected, these studies showed that reticulocytes incubated with MTDR and rapoTf failed to produce a double-labeled population in uptake experiments. Interestingly, when reticulocyte lysates were incubated with MTDR and rTf, compared to controls using wild type human Tf, the size of the double-labeled population was decreased. This suggests that failure of iron release from rTf may interfere with the process of endocytosis or endosomal trafficking. Disclosures: No relevant conflicts of interest to declare.

From Rabbit Reticulocytes to Clam Oocytes: In Search of the System That Targets Mitotic Cyclins for Degradation

By the late 1980s, the basic biochemistry of ubiquitin-mediated protein degradation had already been elucidated by studies that used reticulocyte lysates. However, the scope and biological functions of this system remained largely obscure. Therefore, I became interested at that time in the mechanisms by which mitotic cyclins are degraded in exit from mitosis. Using a cell-free system from clam oocytes that faithfully reproduced cell cycle stage–specific degradation of cyclins, we identified in 1995 a large ubiquitin ligase complex that targets mitotic cyclins for degradation. Subsequent studies in many laboratories showed that this ubiquitin ligase, now called the anaphase-promoting complex/cyclosome, has centrally important roles in many aspects of cell cycle control.

The UL31 and UL34 Gene Products of Herpes Simplex Virus 1 Are Required for Optimal Localization of Viral Glycoproteins D and M to the Inner Nuclear Membranes of Infected Cells

ABSTRACT UL31 and UL34 of herpes simplex virus type 1 form a complex necessary for nucleocapsid budding at the inner nuclear membrane (INM). Previous examination by immunogold electron microscopy and electron tomography showed that pUL31, pUL34, and glycoproteins D and M are recruited to perinuclear virions and densely staining regions of the INM where nucleocapsids bud into the perinuclear space. We now show by quantitative immunogold electron microscopy coupled with analysis of variance that gD-specific immunoreactivity is significantly reduced at both the INM and outer nuclear membrane (ONM) of cells infected with a UL34 null virus. While the amount of gM associated with the nuclear membrane (NM) was only slightly (P = 0.027) reduced in cells infected with the UL34 null virus, enrichment of gM in the INM at the expense of that in the ONM was greatly dependent on UL34 (P < 0.0001). pUL34 also interacted directly or indirectly with immature forms of gD (species expected to reside in the endoplasmic reticulum or nuclear membrane) in lysates of infected cells and with the cytosolic tail of gD fused to glutathione S-transferase in rabbit reticulocyte lysates, suggesting a role for the pUL34/gD interaction in recruiting gD to the NM. The effects of UL34 on gD and gM localization were not a consequence of decreased total expression of gD and gM, as determined by flow cytometry. Separately, pUL31 was dispensable for targeting gD and gM to the two leaflets of the NM but was required for (i) the proper INM-versus-ONM ratio of gD and gM in infected cells and (ii) the presence of electron-dense regions in the INM, representing nucleocapsid budding sites. We conclude that in addition to their roles in nucleocapsid envelopment and lamina alteration, UL31 and UL34 play separate but related roles in recruiting appropriate components to nucleocapsid budding sites at the INM.

RNase/Anti-RNase Activities of the Bacterial parD Toxin-Antitoxin System

ABSTRACT The bacterial parD toxin-antitoxin system of plasmid R1 encodes two proteins, the Kid toxin and its cognate antitoxin, Kis. Kid cleaves RNA and inhibits protein synthesis and cell growth in Escherichia coli. Here, we show that Kid promotes RNA degradation and inhibition of protein synthesis in rabbit reticulocyte lysates. These new activities of the Kid toxin were counteracted by the Kis antitoxin and were not displayed by the KidR85W variant, which is nontoxic in E. coli. Moreover, while Kid cleaved single- and double-stranded RNA with a preference for UAA or UAC triplets, KidR85W maintained this sequence preference but hardly cleaved double-stranded RNA. Kid was formerly shown to inhibit DNA replication of the ColE1 plasmid. Here we provide in vitro evidence that Kid cleaves the ColE1 RNA II primer, which is required for the initiation of ColE1 replication. In contrast, KidR85W did not affect the stability of RNA II, nor did it inhibit the in vitro replication of ColE1. Thus, the endoribonuclease and the cytotoxic and DNA replication-inhibitory activities of Kid seem tightly correlated. We propose that the spectrum of action of this toxin extends beyond the sole inhibition of protein synthesis to control a broad range of RNA-regulated cellular processes.

Deletion of Ser-171 causes inactivation, proteasome-mediated degradation and complete deficiency of human transaldolase

Homozygous deletion of three nucleotides coding for Ser-171 (S171) of TAL-H (human transaldolase) has been identified in a female patient with liver cirrhosis. Accumulation of sedoheptulose 7-phosphate raised the possibility of TAL (transaldolase) deficiency in this patient. In the present study, we show that the mutant TAL-H gene was effectively transcribed into mRNA, whereas no expression of the TALΔS171 protein or enzyme activity was detected in TALΔS171 fibroblasts or lymphoblasts. Unlike wild-type TAL-H–GST fusion protein (where GST stands for glutathione S-transferase), TALΔS171–GST was solubilized only in the presence of detergents, suggesting that deletion of Ser-171 caused conformational changes. Recombinant TALΔS171 had no enzymic activity. TALΔS171 was effectively translated in vitro using rabbit reticulocyte lysates, indicating that the absence of TAL-H protein in TALΔS171 fibroblasts and lymphoblasts may be attributed primarily to rapid degradation. Treatment with cell-permeable proteasome inhibitors led to the accumulation of TALΔS171 in whole cell lysates and cytosolic extracts of patient lymphoblasts, suggesting that deletion of Ser-171 led to rapid degradation by the proteasome. Although the TALΔS171 protein became readily detectable in proteasome inhibitor-treated cells, it displayed no appreciable enzymic activity. The results suggest that deletion of Ser-171 leads to inactivation and proteasome-mediated degradation of TAL-H. Since TAL-H is a regulator of apoptosis signal processing, complete deficiency of TAL-H may be relevant for the pathogenesis of liver cirrhosis.