SUMMARY The modular SCF ubiquitin ligases feature a large family of substrate receptors that enable recognition of diverse targets. However, how the repertoire of SCF complexes is sustained remains unclear. Real-time measurements of formation and disassembly indicate that SCFFbxw7 is extraordinarily stable but, in the Nedd8-deconjugated state, is rapidly disassembled by the cullin-binding protein Cand1. Binding and ubiquitylation assays show that Cand1 is a protein exchange factor that accelerates the rate at which Cul1–Rbx1 equilibrates with multiple F-box Protein–Skp1 modules. Depletion of Cand1 from cells impedes recruitment of new F-box proteins to pre-existing Cul1 and profoundly alters the cellular landscape of SCF complexes. We suggest that catalyzed protein exchange may be a general feature of dynamic macromolecular machines and propose a hypothesis for how substrates, Nedd8, and Cand1 collaborate to regulate the cellular repertoire of SCF complexes.

Toxoplasma gondii, which causes toxoplasmic encephalitis and birth defects, contains an essential chloroplast-related organelle to which proteins are trafficked via the secretory system. This organelle, the apicoplast, is bounded by multiple membranes. In this report we identify a novel apicoplast-associated thioredoxin family protein, ATrx1, which is predominantly soluble or peripherally associated with membranes, and which localizes primarily to the outer compartments of the organelle. As such, it represents the first protein to be identified as residing in the apicoplast intermembrane spaces. ATrx1 lacks the apicoplast targeting sequences typical of luminal proteins. However, sequences near the N terminus are required for proper targeting of ATrx1, which is proteolytically processed from a larger precursor to multiple smaller forms. This protein reveals a population of vesicles, hitherto unrecognized as being highly abundant in the cell, which may serve to transport proteins to the apicoplast.The apicoplast is a relict secondary chloroplast that resides within the cytosol of most apicomplexan parasites, including the intracellular pathogens Toxoplasma gondii and the malaria parasite Plasmodium. It is an essential organelle in these organisms, and since it does not have a counterpart in animal hosts, pathways known to function in the organelle have been considered as potential targets for prophylactic or therapeutic intervention for both toxoplasmosis and malaria (52). Indeed, specific antibiotics that target the transcription and translation systems of the apicoplast, including clindamycin and tetracycline, are known to be toxic to the parasites (9, 14, 23). Other small molecules inhibit key apicoplast metabolic pathways, such as isoprenoid biosynthesis (27) or fatty acid biosynthesis (22). Therefore, identification of additional essential molecules in the apicoplast could yield new drug targets.The apicoplast was acquired by secondary endosymbiosis and is surrounded by four membranes (30); the inner two presumably correspond to the two original chloroplast membranes, while the outer two are thought to be derived from the algal plasma membrane and the endocytic vacuole. Thus, there are eight distinct locations within the apicoplast: one for each of the four membranes, three for the intermembrane spaces, and one for the lumen of the organelle. Little is known about proteins that reside in nonluminal compartments. Since the apicoplast genome has few genes other than those involved in genetic functions (53), the vast majority of apicoplast proteins are encoded by nuclear genes. Many proteins have been predicted to localize to the apicoplast lumen on the basis of a predicted apicoplast targeting sequence (17, 42), as described below.To date, all proteins known to reside in the lumen of the apicoplast are synthesized with an N-terminal bipartite targeting sequence that routes them to the organelle. This sequence is composed of a signal peptide that diverts the nascent protein into the endoplasmic reticulum (ER). A...

SUMMARY Mitochondrial fission requires recruitment of dynamin-related protein 1 (Drp1) to the mitochondrial surface and activation of its GTP-dependent scission function. The Drp1 receptors MiD49 and MiD51 recruit Drp1 to facilitate mitochondrial fission, but their mechanism of action is poorly understood. Using X-ray crystallography, we demonstrate that MiD51 contains a nucleotidyl transferase domain that binds ADP with high affinity. MiD51 recruits Drp1 via a surface loop that functions independently of ADP binding. However, in the absence of nucleotide binding, the recruited Drp1 cannot be activated for fission. Purified MiD51 strongly inhibits Drp1 assembly and GTP hydrolysis in the absence of ADP. Addition of ADP relieves this inhibition and promotes Drp1 assembly into spirals with enhanced GTP hydrolysis. Our results reveal ADP as an essential cofactor for MiD51 during mitochondrial fission.

The localization of tail-anchored (TA) proteins, whose transmembrane domain resides at the extreme C terminus, presents major challenges to cellular protein targeting machineries. In eukaryotic cells, the highly conserved ATPase, guided entry of tail-anchored protein 3 (Get3), coordinates the delivery of TA proteins to the endoplasmic reticulum. How Get3 uses its ATPase cycle to drive this fundamental process remains unclear. Here, we establish a quantitative framework for the Get3 ATPase cycle and show that ATP specifically induces multiple conformational changes in Get3 that culminate in its ATPase activation through tetramerization. Further, upstream and downstream components actively regulate the Get3 ATPase cycle to ensure the precise timing of ATP hydrolysis in the pathway: the Get4/5 TA loading complex locks Get3 in the ATP-bound state and primes it for TA protein capture, whereas the TA substrate induces tetramerization of Get3 and activates its ATPase reaction 100-fold. Our results establish a precise model for how Get3 harnesses the energy from ATP to drive the membrane localization of TA proteins and illustrate how dimerization-activated nucleotide hydrolases regulate diverse cellular processes.

Toxoplasma gondii is an obligate intracellular parasite that resides in the cytoplasm of its host in a unique membrane-bound vacuole known as the parasitophorous vacuole (PV). The membrane surrounding the parasite is remodeled by the dense granules, secretory organelles that release an array of proteins into the vacuole and to the PV membrane (PVM). Only a small portion of the protein constituents of the dense granules have been identified, and little is known regarding their roles in infection or how they are trafficked within the infected host cell. In this report, we identify a novel secreted dense granule protein, GRA14, and show that it is targeted to membranous structures within the vacuole known as the intravacuolar network and to the vacuolar membrane surrounding the parasite. We disrupted GRA14 and exploited the knockout strain to show that GRA14 can be transferred between vacuoles in a coinfection experiment with wild-type parasites. We also show that GRA14 has an unexpected topology in the PVM with its C terminus facing the host cytoplasm and its N terminus facing the vacuolar lumen. These findings have important implications both for the trafficking of GRA proteins to their ultimate destinations and for expectations of functional domains of GRA proteins at the host-parasite interface.Capable of infecting essentially any warm-blooded vertebrate, Toxoplasma gondii is one of the most successful pathogens on the planet (20, 39). Toxoplasma infects nearly onethird of the human population and causes potentially fatal disease in immunocompromised individuals and congenitally infected neonates (20). This protozoan parasite also causes ocular disease in immunocompetent individuals who are either congenitally or postnatally infected (46). As an obligate intracellular parasite, Toxoplasma enters the host cell into a nonfusogenic vacuole (the parasitophorous vacuole [PV]), in which the parasite replicates in the cytoplasm of its host. The PV membrane (PVM) is porous to small molecules (less than 1,300 Da) but otherwise serves as a boundary between the host and parasite during its intracellular survival (36).Toxoplasma invasion is mediated by a trio of specialized secretory organelles, named the micronemes, rhoptries, and dense granules, which contribute to the parasite's ability to initiate and sustain infection within its host. The first proteins secreted are from the micronemes, which release molecular adhesins that interact with the parasite's actin-myosin motor to provide the driving force for invasion (24). The rhoptries are then released and help to establish the nascent PV and modulate host cell processes (4). Lastly, proteins from the dense granules that are implicated in the remodeling and maintenance of the PV for intracellular survival are secreted (29).The precise role of dense granule proteins (GRAs) in the T. gondii life cycle is still largely unknown. To date, two groups of GRA proteins have been identified. The first group contains proteins that lack homology to organisms other than closely relat...