In eukaryotic cells Rab/Ypt GTPases represent a family of key membrane traffic controllers that associate with their targeted membranes via C-terminally conjugated geranylgeranyl groups. GDP dissociation inhibitor (GDI) is a general and essential regulator of Rab recycling that extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. Here, we present the structure of a complex between GDI and a doubly prenylated Rab protein. We show that one geranylgeranyl residue is deeply buried in a hydrophobic pocket formed by domain II of GDI, whereas the other lipid is more exposed to solvent and is skewed across several atoms of the first moiety. Based on structural information and biophysical measurements, we propose mechanistic and thermodynamic models for GDI and Rab escort protein-mediated interaction of RabGTPase with intracellular membranes.

9The cytosolic iron sulfur (Fe-S) assembly (CIA) pathway is required for the insertion 10 of Fe-S clusters into cytosolic and nuclear client proteins, including many DNA 11 metabolism proteins. The molecular mechanisms of client protein recognition and Fe-12 S cluster transfer remain unknown. Here we report crystal structures of the CIA 13 targeting complex and cryo-EM reconstructions of the complex bound either to the 14 DNA replication factor primase or the DNA helicase DNA2. The structures, combined 15 with biochemical, biophysical and yeast complementation assays, reveal an 16 evolutionarily conserved, bipartite client binding mode facilitated by the structural 17 flexibility of the MMS19 subunit. The primase Fe-S cluster is located ~70 Å away 18 from the catalytic cysteine in the CIA targeting complex, pointing to a 19conformationally dynamic mechanism of Fe-S cluster transfer. Altogether, our studies 20 suggest a model for Fe-S cluster insertion and thus provide a mechanistic framework Main 1 Iron-sulfur (Fe-S) clusters, found in all three domains of life 1 , stabilize protein folds, 2 facilitate electron transfer processes, and sense oxygen or iron levels in cells. Many 3 eukaryotic DNA replication and repair proteins, including B-family DNA polymerases 4 α, δ, ε and ζ, the DNA primase large subunit (PriL), and DNA helicases XPD, RTEL1, 5 FANCJ, DNA2, and DDX11, contain Fe-S clusters [2][3][4][5][6][7][8] . Although the precise roles of 6 the Fe-S clusters in many of these proteins remain enigmatic, loss of the Fe-S cluster 7 often leads to reduced protein stability and functionality 7,9,10 , and disease-causing 8 mutations have been mapped to the Fe-S domains of XPD, FANCJ, and MUTYH 11 . 9The biogenesis of eukaryotic Fe-S proteins is a highly regulated, multi-step 10 process, which is conserved from yeast to human 12,13 . The mitochondrial iron sulfur 11 cluster (ISC) machinery is required for the biogenesis of all mitochondrial Fe-S 12 proteins, while the cytosolic iron sulfur assembly (CIA) pathway is essential for the 13 maturation of cytosolic and nuclear Fe-S proteins. The CIA pathway depends on the 14 mitochondrial ISC machinery, which is thought to generate a sulfur-containing 15 precursor that is exported into the cytosol 14 . As a first step in the CIA pathway, the 16 [4Fe-4S] cluster is assembled on the NUBP1/NUBP2 scaffold complex (Nbp35-Cfd1 17 in yeast) [15][16][17][18] . The cluster is then thought to be transferred onto the intermediate 18 carrier protein CIAO3 (Nar1 in yeast) 19,20 . In the next step, the CIA targeting complex 19 (CTC), consisting of MMS19, CIAO1, and CIAO2B (MET18, CIA1, and CIA2B in 20 yeast; Fig. 1a), recognizes client apo-proteins through direct physical interactions 21 and mediates the insertion of the Fe-S cluster. MMS19 is an adaptor subunit required 22 for the recognition of diverse client proteins, including essential DNA replication and 23 repair factors, by direct physical interactions 21 . Cells lacking MMS19 exhibit 24 pleiotropic phenotypes including geno...