The COP9 signalosome (CSN) is an evolutionarily conserved multi-protein complex that interfaces with the ubiquitin-proteasome pathway and plays critical developmental roles in both animals and plants. Although some subunits are present only in an ∼320-kDa complex-dependent form, other subunits are also detected in configurations distinct from the 8-subunit holocomplex. To date, the only known biochemical activity intrinsic to the complex, deneddylation of the Cullin subunits from Cullin-RING ubiquitin ligases, is assigned to CSN5. As an essential step to understanding the structure and assembly of a CSN5-containing subcomplex of the CSN, we reconstituted a CSN4-5-6-7 subcomplex. The core of the subcomplex is based on a stable heterotrimeric association of CSN7, CSN4, and CSN6, requiring coexpression in a bacterial reconstitution system. To this heterotrimer, we could then add CSN5 in vitro to reconstitute a quaternary complex. Using biochemical and biophysical methods, we identified pairwise and combinatorial interactions necessary for the formation of the CSN4-5-6-7 subcomplex. The subcomplex is stabilized by three types of interactions: MPN-MPN between CSN5 and CSN6, PCI-PCI between CSN4 and CSN7, and interactions mediated through the CSN6 C terminus with CSN4 and CSN7. CSN8 was also found to interact with the CSN4-6-7 core. These data provide a strong framework for further investigation of the organization and assembly of this pivotal regulatory complex. Journal of Molecular Biology Impaired Folding of the Mitochondrial Small TIM Chaperones Induces Clearance by the i-AAA Protease Michael J. Baker1, 2, Ved P. Mooga1, Bernard Guiard3, Thomas Langer2, Michael T. Ryan1, , , Diana Stojanovski1, , 1 Department of Biochemistry, La Trobe Institute for Molecular Science and ARC Centre of Excellence for Coherent X-ray Science, La Trobe University, Melbourne 3086, Australia 2 Institute for Genetics, University of Cologne, Cologne 50674, Germany 3 Centre de Génétique Moléculaire, CNRS, 91190 Gif-sur-Yvette, France The intermembrane space of mitochondria contains a dedicated chaperone network—the small translocase of the inner membrane (TIM) family—for the sorting of hydrophobic precursors. All small TIMs are defined by the presence of a twin CX3C motif and the monomeric proteins are stabilized by two intramolecular disulfide bonds formed between the cysteines of these motifs. The conserved cysteine residues within small TIM members have also been shown to participate in early biogenesis events, with the most N-terminal cysteine residue important for import and retention within the intermembrane space via the receptor and disulfide oxidase, Mia40. In this study, we have analyzed the in vivo consequences of improper folding of small TIM chaperones by generating site-specific cysteine mutants and assessed the fate of the incompletely oxidized proteins within mitochondria. We show that no individual cysteine residue is required for the function of Tim9 or Tim10 in yeast and that defective assembly of the small TIMs induces their proteolytic clearance from mitochondria. We delineate a clearance mechanism for the mutant proteins and their unassembled wild-type partner protein by the mitochondrial ATP-dependent protease, Yme1 (yeast mitochondrial escape 1). Structural Insights on the Plant Salt-Overly-Sensitive 1 (SOS1) Na+/H+ Antiporter Rafael Núñez-Ramírez1, †, María José Sánchez-Barrena2, , †, , Irene Villalta3, †, Juan F. Vega1, Jose M. Pardo3, Francisco J. Quintero3, Javier Martinez-Salazar1, Armando Albert2, , 1 BIOPHYM, Departamento de Física Macromolecular, Instituto de Estructura de la Materia, Consejo Superior de Investigaciones Científicas, Serrano 113bis, Madrid E-28006, Spain 2 Departamento de Cristalografía y Biología Estructural, Instituto de Química Física “Rocasolano”, Consejo Superior de Investigaciones Científicas, Serrano 119, Madrid E-