Table 1.
Yeast two-hybrid analysis of human epithelial breast cancer proteins that interact with mitoNEET.
Fig 1.
BiFC analysis of mNT-NAF-1 interaction.
A. Representative images of: (1) Positive control for NAF-1 homodimer interaction using co-expression of NAF-1-YFPc and NAF-1-YFPn with ER tracker localization. (2) Positive control for mNT homodimer interaction using co-expression of mNT-YFPc and mNT-YFPn with mitochondrial (Mito) tracker localization. (3) mNT-NAF-1 interaction following co-expression of NAF-1-YFPc and mNT-YFPn with ER tracker localization. (4) mNT-NAF-1 interaction following co-expression of NAF-1-YFPc and mNT-YFPn with Mito tracker localization. (5) Negative control for mNT-NAF-1 interaction using co-expression of mNT-YFPn and soluble sNAF-1-YFPc with mitochondrial (Mito) tracker localization. B. The different split-YFP/mNT/NAF-1/sNAF-1 vectors used for the in vivo analysis of mNT-NAF-1 interaction shown in A. C. A comparison between the BiFC signal obtained with the mNT-NAF-1 interaction (co-transfection with NAF-1-YFPc and mNT-YFPn; mNT-NAF-1) and the BiFC signal obtained with the mNT-sNAF-1 interaction (co-transfection with sNAF-1-YFPc and mNT-YFPn; mNT-sNAF-1). Vector construction, transfection and imaging are described in Materials and Methods. ***, p≤0.001.
Fig 2.
2Fe-2S cluster transfer from holo-mNT to apo-NAF-1.
Apo-NAF-1 was incubated at 37°C for 20 min with β-mercaptoethanol and holo-mNT, and chromatographed on a native gel as described in [31] with the modification described in Materials and Methods. Red-colored bands in the upper native gels are indicative of the [2Fe–2S] cluster presence in the two proteins. Blue-colored bands in the lower duplicate gel are the result of Coomassie Blue staining to confirm the presence and levels of the two proteins.
Fig 3.
DCA analysis and complex model of NAF-1-mNT interaction.
A. A cartoon representation of the mNT-NAF-1 complex, with mNT on the left (blue) and NAF-1 on the right (pink). This figure was generated by aligning the PDB crystal structures of mNT (PDB 2QH7) and NAF-1 (PDB 3FNV) with the result of the simulation. The distance between the iron-sulfur clusters is shown to be about 12.6 Å (highlighted in red). B. Surface representation of the mNT-NAF-1 complex demonstrating the close fit between the two proteins. A closer view of this lock-and-key part of the interface is shown in S3 Fig. The DCA couplings are depicted as green lines. A full list of the couplings, along with their approximate distances is included in S1 Table.
Fig 4.
Mitochondrial membrane potential (MMP), labile iron and ROS measurements in cancer cell lines with suppressed mNT and/or NAF-1 expression.
Compared to single suppression of mNT [mNT(-)] or NAF-1 [NAF-1(-)], double shRNA suppression of mNT and NAF-1 [mNT(-)/NAF-1(-)] stable lines do not result in a significantly larger impairment in MMP (A), mitochondrial labile iron (B) and mitochondrial ROS (C) accumulation. The iron chelator DFP (100 μM) is shown to recover the effect of mNT and/or NAF-1 suppression on MMP, mitochondrial labile iron and ROS in all lines in a similar manner (A-C). All measurements were compared to control MDA-MB-231 cell lines transfected with the same shRNA vector containing a scrambled RNA. The expression levels of the mNT and NAF-1 proteins in all lines are shown (D), and Coomassie blue loading controls in (E). ***P <0.001.
Fig 5.
Overlap between transcripts altered in cancer cells with suppressed mNT [mNT(-)] or NAF-1 [NAF-1(-)] expression.
A. Venn diagram showing the overlap between transcripts altered (p≤0.05) in cancer cells with suppressed mNT [mNT(-)] or NAF-1 [NAF-1(-)] expression detected with RNA-seq analysis. B. KEGG annotation of transcripts common to cells with suppressed mNT [mNT(-)] or NAF-1 [NAF-1(-)] expression.
Fig 6.
A hypothetical model for the interaction of mNT with NAF-1 in cells.
mNT is shown to accept Fe-S clusters from the mitochondria and transfer them to NAF-1. The flow of clusters from mNT to NAF-1 is shown to be used by NAF-1 to regulate different processes such as apoptosis/autophagy activation, as well as cellular proliferation. The cluster relay between mNT and NAF-1 is proposed to link mitochondrial Iron/ROS/Fe-S homeostasis and function with the regulation of cell death/proliferation by the NAF-1/BCL-2/MAPK/PI3K-Akt pathways.