In mitochondria Fe-S clusters, prosthetic groups critical for the activity of many proteins, are first assembled on Isu, a 14 kDa scaffold protein, and then transferred to recipient apo-proteins. The assembly process involves interaction of Isu with both Nfs1, the cysteine desulfurase serving as a sulfur donor, and the yeast frataxin homolog (Yfh1) serving as a regulator of desulfurase activity and/or iron donor. Here, based on the results of biochemical experiments with purified wild-type and variant proteins, we report that interaction of Yfh1 with both Nfs1 and Isu are required for formation of a stable tripartite assembly complex. Disruption of either the Yfh1-Isu or the Nfs1-Isu interaction destabilizes the complex. Cluster transfer to recipient apo-protein is known to require the interaction of Isu with the J-protein/Hsp70 molecular chaperone pair, Jac1 and Ssq1. Here we show that Yfh1 interaction with Isu involves the PVK sequence motif, which is also the site key for the interaction of Isu with Hsp70 Ssq1. Coupled with our previous observation that Nfs1 and Jac1 binding to Isu is mutually exclusive due to partially overlapping binding sites, we propose that such mutual exclusivity of cluster assembly factor (Nfs1/Yfh1) and cluster transfer factor (Jac1/Ssq1) binding to Isu has functional consequences for the transition from the assembly process to the transfer process, and thus regulation of the biogenesis of Fe-S cluster proteins.