Jac1p is a conserved, specialized J-protein that functions with Hsp70 in Fe-S cluster biogenesis in mitochondria of the yeast Saccharomyces cerevisiae. Although Jac1p as well as its specialized Hsp70 partner, Ssq1p, binds directly to the Fe-S cluster scaffold protein Isu, the Jac1p-Isu1p interaction is not well understood. Here we report that a C-terminal fragment of Jac1p lacking its J-domain is sufficient for interaction with Isu1p, and amino acid alterations in this domain affect interaction with Isu1p but not Ssq1p. In vivo, such JAC1 mutations had no obvious phenotypic effect. However, when present in combination with a mutation in SSQ1 that causes an alteration in the substrate binding cleft, growth was significantly compromised. Wild type Jac1p and Isu1p cooperatively stimulate the ATPase activity of Ssq1p. Jac1p mutant protein is only slightly compromised in this regard. Our in vivo and in vitro results indicate that independent interaction of Jac1p and the Isu client protein with Hsp70 is sufficient for robust growth under standard laboratory conditions. However, our results also support the idea that Isu protein can be "targeted" to Ssq1p after forming a complex with Jac1p. We propose that Isu protein targeting may be particularly important when environmental conditions place high demands on Fe-S cluster biogenesis or in organisms lacking specialized Hsp70s for Fe-S cluster biogenesis.