Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein–GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70–containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.

Eukaryotic cells use cytoskeletal motor proteins to transport many different intracellular cargos. Numerous kinesins and myosins have evolved to cope with the various transport needs that have arisen during eukaryotic evolution. Surprisingly, a single cytoplasmic dynein (a minus end-directed microtubule motor) carries out similarly diverse transport activities as the many different types of kinesin. How is dynein coupled to its wide range of cargos and how is it spatially and temporally regulated? The answer could lie in the several multifunctional adaptors, including dynactin, lissencephaly 1, nuclear distribution protein E (NUDE) and NUDE-like, Bicaudal d, Rod–ZW10–Zwilch and Spindly, that regulate dynein function and localization.

Dynactin, a large multisubunit complex, is required for intracellular transport by dynein; however, its cellular functions and mechanism of action are not clear. Prior studies suggested that dynactin increases dynein processivity by tethering the motor to the microtubule through its own microtubule binding domains. However, this hypothesis could not be tested without a recombinant source of dynactin. Here, we have produced recombinant dynactin and dynein in Saccharomyces cerevisiae, and examined the effect of dynactin on dynein in single-molecule motility assays. We show that dynactin increases the run length of single dynein motors, but does not alter the directionality of dynein movement. Enhancement of dynein processivity by dynactin does not require the microtubule (MT) binding domains of Nip100 (the yeast p150 Glued homolog). Dynactin lacking these MT binding domains also supports the proper localization and function of dynein during nuclear segregation in vivo. Instead, a segment of the coiled-coil of Nip100 is required for these activities. Our results directly demonstrate that dynactin increases the processivity of dynein through a mechanism independent of microtubule tethering.microtubule ͉ motor protein ͉ nuclear segregation ͉ p150 Glued ͉ single molecule D ynactin, a large (Ϸ1.2 MD) complex, was first identified as an activator of dynein-mediated, minus-end-directed vesicle transport (1, 2), and has subsequently been shown to be essential for nearly every cellular function of cytoplasmic dynein (3, 4). Several dynactin alleles have been linked to human neurological disease, which most likely results from a defect in intracellular trafficking (5, 6). Dynactin is composed of a filament of the actin-related protein Arp1, capped at each end by additional subunits. The barbed end subcomplex contains a dimer of the largest subunit, p150 Glued (Nip100 in yeast), which binds to dynein directly (4). The Nterminus of p150Glued contains a CAP-Gly domain and a basic region, both of which have been shown to bind microtubules (MTs) (7-9), followed by a coiled-coil that projects as a 24-nm stalk from the Arp1 filament (10, 11).Two general mechanisms have been proposed through which dynactin could aid the cellular function of dynein. First, many studies have provided evidence that dynactin is important for localizing cytoplasmic dynein to its proper intracellular cargo (4). Dynactin also might modulate dynein motor activity, an idea that has been explored through several in vitro motility assays. Dynactin has been proposed to increase the processivity of dynein, based on findings that dynactin increases the run length of dynein-coated beads in vitro (12). Increased processivity might be important in the cell for uninterrupted transport over long distance or for transport under high load. However, because dynactin and dynein were nonspecifically adsorbed onto beads in this study, it could not be determined whether dynein-dynactin complexes were observed, or if dynactin bound separately from dynein on the bead surface ...

SUMMARY The mitochondrion maintains and regulates its proteome with chaperones primarily inherited from its bacterial endosymbiont ancestor. Among these chaperones is the AAA+ unfoldase ClpX, an important regulator of prokaryotic physiology with poorly defined function in the eukaryotic mitochondrion. We observed phenotypic similarity in S. cerevisiae genetic interaction data between mitochondrial ClpX (mtClpX) and genes contributing to heme biosynthesis, an essential mitochondrial function. Metabolomic analysis revealed that 5-aminolevulinic acid (ALA), the first heme precursor, is five-fold reduced in yeast lacking mtClpX activity, and total heme is reduced by half. mtClpX directly stimulates ALA synthase in vitro by catalyzing incorporation of its cofactor, pyridoxal phosphate. This activity is conserved in mammalian homologs; additionally, mtClpX depletion impairs vertebrate erythropoiesis, which requires massive upregulation of heme biosynthesis to supply hemoglobin. mtClpX therefore is a widely conserved stimulator of an essential biosynthetic pathway, and employs a previously unrecognized mechanism for AAA+ unfoldases.

Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism.

The Hepatitis Delta Virus (HDV) ribozyme was the first RNA enzyme proposed to use a proton-transfer mechanism for catalysis. Previous biochemical evidence suggested that the genomic HDV ribozyme promotes cis-cleavage using cytosine 75 whose pK(a) is perturbed within the active site. Here we present further biochemical evidence for the involvement of C75 in proton transfer, as well as evidence to support a plausible mechanism for C75 pK(a) perturbation. Nucleotide analogue interference mapping (NAIM) experiments with C analogues having altered N3 pK(a)s demonstrate the importance of C75 ionization in the HDV cis-cleavage reaction. pH-dependent interference rescue with C analogues having enhanced N3 acidity indicates that C75 is the only cytidine residue that must be protonated for ribozyme activity. Furthermore, interference analysis with pseudoisocytidine, a charge-neutral mimic of a C with a protonated N3, shows a pattern consistent with proton transfer, possibly from the C75 N3 to the 5'-oxyanion leaving group during the cis-cleavage reaction. Strong pH-independent inhibition of ribozyme function also occurs at C75 with a C analogue that lacks the N4 amino group, implicating the exocyclic amine in critical interactions in the active site. Interactions with the amino group may play an important role in perturbing the C75 N3 pK(a). Protonation of C41 has been proposed to be important for ribozyme activity; however, no interference at C41 was observed in this analogue series, which argues against a functional role for C41 protonation. These data support a model wherein C75 of the genomic HDV ribozyme acts as a general acid during its cis-cleavage reaction, and provide a glimpse into how RNAs, in a manner similar to protein enzymes, might employ local environmental electronic modulation to catalyze reactions.

5-Aminolevulinic acid synthase (ALAS) catalyzes the first step in heme biosynthesis. We present the crystal structure of a eukaryotic ALAS from Saccharomyces cerevisiae. In this homodimeric structure, one ALAS subunit contains covalently bound cofactor, pyridoxal 5'-phosphate (PLP), whereas the second is PLP free. Comparison between the subunits reveals PLP-coupled reordering of the active site and of additional regions to achieve the active conformation of the enzyme. The eukaryotic C-terminal extension, a region altered in multiple human disease alleles, wraps around the dimer and contacts active-site-proximal residues. Mutational analysis demonstrates that this C-terminal region that engages the active site is important for ALAS activity. Our discovery of structural elements that change conformation upon PLP binding and of direct contact between the C-terminal extension and the active site thus provides a structural basis for investigation of disruptions in the first step of heme biosynthesis and resulting human disorders.

The mitochondrial matrix GTPase NOA1 is a nuclear encoded protein, essential for mitochondrial protein synthesis, oxidative phosphorylation and ATP production. Here, we demonstrate that newly translated NOA1 protein is imported into the nucleus, where it localizes to the nucleolus and interacts with UBF1 before nuclear export and import into mitochondria. Mutation of the nuclear localization signal (NLS) prevented both nuclear and mitochondrial import while deletion of the N-terminal mitochondrial targeting sequence (MTS) or the C-terminal RNA binding domain of NOA1 impaired mitochondrial import. Absence of the MTS resulted in accumulation of NOA1 in the nucleus and increased caspase-dependent apoptosis. We also found that export of NOA1 from the nucleus requires a leptomycin-B sensitive, Crm1-dependent nuclear export signal (NES). Finally, we show that NOA1 is a new substrate of the mitochondrial matrix protease complex ClpXP. Our results uncovered an unexpected, mandatory detour of NOA1 through the nucleolus before uptake into mitochondria. We propose that nucleo-mitochondrial translocation of proteins is more widespread than previously anticipated providing additional means to control protein bioavailability as well as cellular communication between both compartments.