Model for the roles of GLRX3 and BOLA2 in mammalian cytosolic iron trafficking. The cytosolic iron chaperone PCBP1 acquires Fe(II) from the labile iron pool and forms an Fe(II)-bridged complex with BOLA2 that requires GSH binding for stability. This complex is proposed to interact with apo-GLRX3 to form a GLRX3-BOLA2 heterocomplex. Each Grx domain in GLRX3 forms a [2Fe-2S]-binding complex with a BOLA2 monomer and GSH leading to formation of a [2Fe-2S]2-bridged GLRX3-BOLA22-GSH2 heterotrimer. The sulfur source required to form the [2Fe-2S] clusters is unclear, possibly provided by the cytosolic isoform of the cysteine desulfurase NFS1 or exported from the mitochondrial ISC pathway. Both the [2Fe-2S]2-GLRX3-BOLA22 heterotrimer and the [2Fe-2S]2-GLRX32 homodimer can deliver [2Fe-2S] clusters to the CIA Fe-S cluster assembly protein CIAPIN1 in vitro, although both GLRX3 and BOLA2 are required for Fe incorporation into CIAPIN1 in cell-based assays. [2Fe-2S]2-GLRX32 may also deliver clusters to the CIA scaffold protein NUBP1, which requires reductive coupling of [2Fe-2S]2+ clusters to form [4Fe-4S]2+ clusters. CIAPIN1, its functional electron transfer partner NDOR1, and NUBP1 are all components of the CIA machinery that assembles and delivers [4Fe-4S] clusters to target proteins in the cytosol and nucleus.