Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO(2) in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone RbcX(2). RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by RbcX(2). As revealed by the structure of a RbcL(8)-(RbcX(2))(8) assembly intermediate, RbcX(2) acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates RbcL(8) core assembly. Finally, addition of RbcS results in RbcX(2) release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly.

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The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly.

N-formylmethionyl-leucyl-phenylalanine, a potent chemotactic peptide for human polymorphonuclear leukocytes, is less chemotactic for human blood monocytes. Esterification of the N-formylated tripeptide enhances its chemotactic activity for monocytes by more than 4 logs, whereas a decrease by 3 logs is observed for polymorphonuclear leukocytes. These results indicate the participation of the C-terminal carboxyl group in chemotaxis of different cell types. We have also observed the selective inhibition of chemotactic responsiveness of human blood monocytes by a clinically useful antirheumatic drug, sodium aurothiomalate. Since this is the first in vitro cell migration model of inflammation in which gold compounds have demonstrated activity in micromolar concentrations, it suggests a site of action for this antirheumatic drug.In 1975 Schiffman et al. reported that a number of small synthetic N-formylmethionyl peptides were strongly chemotactic for both neutrophils and macrophages (1). They subsequently found that the N-formyl tripeptides were more effectivc: than dipeptides in their chemotactic activity, lysosomal enzyme-releasing ability in rabbit neutrophils (2), histamine-releasing ability in human basophils (3), and receptor binding on rabbit neutrophils (4). However, it is not clear whether these tripeptides are equally active in chemotaxis against both human polymorphonuclear leukocytes (PMN) and blood monocytes (MNC). We are. now reporting that the formylated tripeptides are poor chemoattractants for MNC and that esterification of the formylated tripeptides increases their chemotactic activity against MNC by more than four orders of magnitude. In addition, several antiinflammatory and antirheumatic drugs were examined for their inhibitory activity in chemotaxis of PMN and MNC. MATERIALS AND METHODSN-formylmethionyl-leucyl-phenylalanine (FM LP) was obtained from Bachem (Marina Del Rey. California). The methyl ester of FMLP (FMLP-M) was synthesized by routine methods of peptide synthesis; M P 132-135'; [ylDZh-19.4 (C = 0.5, DMF); C,H,S elemental analysis within 0.3% of theory: chromatographically pure in two standard solvent systems for peptides (5). Sodium aurothiomalate was obtained from Aldrich Chemicals and sodium aurothioglucose from Sigma Chemicals.Blood was drawn in sterile syringes with 0.38% of sodium citrate/ml of blood from healthy human volunteers who had not ingested any antiinflammatory drugs including aspirin for at least 7 days. After low-speed centrifugation (200 X g for 15 minutes), the platelet-enriched plasma was removed and the cells were resuspended in Dulbecco's phosphate-buffered saline (Grand Island Biological Co, Grand Island, New