Purpose To develop diagnostic reference levels (DRLs) and achievable doses (ADs) for the 10 most common adult computed tomographic (CT) examinations in the United States as a function of patient size by using the CT Dose Index Registry. Materials and Methods Data from the 10 most commonly performed adult CT head, neck, and body examinations from 583 facilities were analyzed. For head examinations, the lateral thickness was used as an indicator of patient size; for neck and body examinations, water-equivalent diameter was used. Data from 1 310 727 examinations (analyzed by using SAS 9.3) provided median values, as well as means and 25th and 75th (DRL) percentiles for volume CT dose index (CTDI), dose-length product (DLP), and size-specific dose estimate (SSDE). Applicable results were compared with DRLs from eight countries. Results More than 46% of the facilities were community hospitals; 13% were academic facilities. More than 48% were in metropolitan areas, 39% were suburban, and 13% were rural. More than 50% of the facilities performed fewer than 500 examinations per month. The abdomen and pelvis was the most frequently performed examination in the study (45%). For body examinations, DRLs (75th percentile) and ADs (median) for CTDI, SSDE, and DLP increased consistently with the patient's size (water-equivalent diameter). The relationships between patient size and DRLs and ADs were not as strong for head and neck examinations. These results agree well with the data from other countries. Conclusion DRLs and ADs as a function of patient size were developed for the 10 most common adult CT examinations performed in the United States. RSNA, 2017.

Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.Golgi biogenesis involves membrane tethering by two multifunctional proteins, GRASP65 and GRASP55, which are differentially localized to cis and medial Golgi cisternae, respectively. GRASP65 is associated with cis Golgi cisternae via both binding to the cis-localized golgin GM130 and insertion of its myristoylated N terminus (1, 2). GRASP55 is primarily on medial Golgi cisternae and binds medial-localized golgin-45 and other proteins and is myristoylated and palmitoylated (3, 4). Each protein contains a conserved N-terminal GRASP domain that mediates self-association, which results in the formation of homotypic tethering complexes that link analogous cisternae in adjacent ministacks (5-8).The GRASP region is predicted to contain two PDZ-like domains (4, 9). PDZ domains are ubiquitous globular proteinprotein interaction modules featuring a hydrophobic binding groove, which interacts with the C terminus of its target ligand, although recognition of internal sequences has also been observed (10, 11). Although recent work supports the presence of PDZ domains within the GRASP module (8), secondary structure predictions indicate significant mismatches to the typical organization of ␤-strands and ␣-helices found in eukaryotic PDZs.Toward elucidating the structural mechanism of GRASPmediated tethering, we solved the crystal structure of the GRASP domain of GRASP55. Although the GRASP domain was indeed composed of two PDZ domains, the domains were circularly permuted, resulting in overall folds that were structurally more similar to prokaryotic PDZs. This unusual arrangement of a metazoan PDZ revealed that the key ␤2 strands of the binding grooves lay outside of the previously predicted PDZ-like regions. Significantly, an internal ligand sequence mapped in GRASP65 that binds to its first PDZ domain formed a surface projection that appeared to fit inside a deep depression within the PDZ1-binding pocket. Taken together, these data suggest a unique internal PDZ ligand interaction on opposite sides of the molecule and imply a multimeric tethering mechanism that mediates Golgi biogenesis. MATERIALS AND METHODSConstructs-GFP-ActA (8) was cloned into GRASP55 pCS-2 (12) using XbaI. To generate His-tagged GRASP55, GR...

Gram-negative bacteria belonging to the Brucella species cause chronic infections that can result in undulant fever, arthritis and osteomyelitis in humans. Remarkably, Brucella sp. genomes encode a protein, named TcpB, that bears significant homology with mammalian TIR (Toll/IL-1 receptor) domains and whose expression causes degradation of the phosphorylated, signal competent form of the adapter MAL. This effect of TcpB is mediated through its Box 1 region and has no effect on other TLR adapter proteins such as MyD88 or TRIF. TcpB also does not affect a mutant, signal-incompetent form of MAL that cannot be phosphorylated. Interestingly the presence of TcpB leads to enhanced polyubiqitination of MAL which is likely responsible for its accelerated degradation. A Brucella abortus mutant lacking TcpB fails to reduce levels of MAL in infected macrophages. Therefore TcpB represents a unique pathogen-derived molecule that suppresses host innate-immune responses by specifically targeting an individual adapter molecule in the TLR signaling pathway for degradation.

Formation of the ribbon-like membrane network of the Golgi apparatus depends on GM130 and GRASP65, but the mechanism is unknown. We developed an in vivo organelle tethering assaying in which GRASP65 was targeted to the mitochondrial outer membrane either directly or via binding to GM130. Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction. Point mutation within the predicted binding groove of the GRASP65 PDZ domain blocked both tethering and, in a gene replacement assay, Golgi ribbon formation. Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.

The Golgi complex processes secretory proteins and lipids, carries out protein sorting and signaling, and supports growth and composition of the plasma membrane. Golgi complex size likely is regulated to meet the demands of each function, and this may involve differential changes of its distinct subdomains. Nevertheless, the primary size change is elongation of the Golgi ribbon-like network as occurs during Golgi complex doubling for mitosis and during differentiation involving upregulated secretion. One hypothesis states that Golgi complex size is set by the abundance of secretory cargo and Golgi complex components that, through binding vesicle coat complexes, drive vesicle coat formation to alter Golgi complex influx and efflux. Regulation of transport factors controlling Golgi membrane traffic is also observed and may control Golgi complex size, but more work is needed to directly link these events to Golgi complex size regulation, especially during differentiation of specialized cell types.

Biogenesis of the Golgi apparatus is likely mediated by the COPI vesicle coat complex, but the mechanism is poorly understood. Modeling of the COPI subunit ␤COP based on the clathrin adaptor AP2 suggested that the ␤COP C terminus forms an appendage domain with a conserved FW binding pocket motif. On gene replacement after knockdown, versions of ␤COP with a mutated FW motif or flanking basic residues yielded a defect in Golgi organization reminiscent of that occurring in the absence of the vesicle tether p115. Indeed, ␤COP bound p115, and this depended on the ␤COP FW motif. Furthermore, the interaction depended on E 19 E 21 in the p115 head domain and inverse charge substitution blocked Golgi biogenesis in intact cells. Finally, Golgi assembly in permeabilized cells was significantly reduced by inhibitors containing intact, but not mutated, ␤COP FW or p115 EE motifs. Thus, Golgi organization depends on mutually interacting domains in ␤COP and p115, suggesting that vesicle tethering at the Golgi involves p115 binding to the COPI coat.

GRASP65 links cis-Golgi cisternae via a homotypic, N-terminal PDZ interaction, and its mitotic phosphorylation disrupts this activity. Neither the identity of the PDZ ligand involved in the GRASP65 self-interaction nor the mechanism by which phosphorylation inhibits its interaction is known. Phospho-mimetic mutation of known cyclin-dependent kinase 1/cyclin B sites, all of which are in the C-terminal "regulatory domain" of the molecule, failed to block organelle tethering. However, we identified a site phosphorylated by Polo-like kinase 1 (PLK1) in the GRASP65 N-terminal domain for which mutation to aspartic acid blocked tethering and alanine substitution prevented mitotic Golgi unlinking. Further, using interaction assays, we discovered an internal PDZ ligand adjacent to the PLK phosphorylation site that was required for tethering. These results reveal the mechanism of phosphoinhibition as direct inhibition by PLK1 of the PDZ ligand underlying the GRASP65 self-interaction.

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