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Mitochondria have an essential role in the production and export of iron-sulfur clusters. The NEET protein family members mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1) are the only known iron-sulfur proteins at the interface of the outer-mitochondrial membrane (OMM) and the cytosol, with mNT localized to the OMM and NAF-1 on the mitochondria-associated ER membrane. Each has a similar homodimeric structure coordinating two 2Fe-2S clusters. We have shown previously that the NEETs have the ability to transfer 2Fe-2S clusters to a standard acceptor protein [1,2]. Additionally, mNT has been reported to acquire 2Fe-2S clusters from the mitochondria [3]; this is also likely the case for NAF-1. The localization, transfer ability and 2Fe-2S source suggest that these proteins act as conduits, transporting 2Fe-2S clusters from the mitochondria to cytosolic proteins. In this study we identify Anamorsin as the first known physiological acceptor of 2Fe-2S clusters from each of the NEET proteins [4]. Anamorsin is required for cytosolic iron-sulfur cluster biogenesis. We analyzed the kinetics of this reaction using UV-Vis spectroscopy and determined transfer rates that are similar to those of known human cluster transfer proteins. We also show by biolayer interferometry that these proteins form a direct protein-protein interaction, which is required for the transfer. Furthermore we found via microscale thermophoresis that mNT interacts with VDAC1, a pore protein in the OMM. We propose that the NEETs acquire 2F-2S clusters through VDAC1 and transfer them to the essential Anamorsin.[1] Zuris JA, et al. (2011) Proc Natl Acad Sci USA 108(32):13047-52.[2] Tamir S, et al. (2013) PLoS ONE 8(5):e61202.[3] Ferecatu I, et al. (2014) J Biol Chem 289(41):28070-86.[4] Lipper CH, et al. (2015) PLoS ONE (In press).