In earlier studies it was shown that the mammalian translation system is highly organized in vivo and that the intermediates in the process, aminoacyl-tRNAs, are channeled-i.e., they are directly transferred from the aminoacyl-tRNA synthetases to the elongation factor to the ribosomes without dissociating into the cellular fluid. Here, we examine whether spent tRNAs leaving the ribosome enter the fluid phase or are transferred directly to their cognate aminoacyl-tRNA synthetases to complete a channeled tRNA cycle. Using a permeabilized CHO cell system that closely mimics living cells, we find that there is no leakage of endogenous tRNA during many cycles of translation, and protein synthesis remains linear during this period, even though free aminoacyl-tRNA is known to rapidly equilibrate between the inside and outside of these cells. We also find that exogenous tRNA and periodate-oxidized tRNA have no effect on protein synthesis in this system, indicating that they do not enter the translation machinery, despite the fact that exogenous tRNA rapidly distributes throughout the cells. Furthermore, most of the cellular aminoacyl-tRNA synthetases function only with endogenous tRNAs, although a portion can use exogenous tRNA molecules. However, aminoacylation of these exogenous tRNAs is strongly inhibited by oxidized tRNA; this inhibitor has no effect on endogenous aminoacylation. On the basis of these and the earlier observations, we conclude that endogenous tRNA is never free of the protein synthetic machinery at any stage of the translation process and, consequently, that there is a channeled tRNA cycle during protein synthesis in mammalian cells.tRNA plays a central role in translation, acting as the carrier of both the monomeric units of proteins, the amino acids, and the growing polypeptide chains (1). As a consequence of this central role, tRNAs must interact, in turn, with most of the components of the protein-synthesizing machinery-including the aminoacyl-tRNA synthetases, the elongation factors, and the ribosomes. Understanding how tRNA is transferred from one component to the next during its cycling is essential for a complete description of the translation process at the molecular level.Recent work, using a permeabilized Chinese hamster ovary (CHO) cell system, which closely mimics living cells, has provided strong evidence that the translation apparatus is highly organized in vivo (2). Thus, while large molecules can enter these cells, very little RNA or protein leaks out; moreover, only ions and other low-molecular-weight compounds need to be added back to sustain rates of protein synthesis comparable to those in living cells (2). Additional information supporting an organized translation system comes from the isolation of multienzyme complexes and other assemblies containing multiple translation components (3-7), from the interaction of these macromolecules with cytoskeletal elements (8-10), and from colocalization of these components in cells (11-13).The conclusion that the translation system ...

The protein biosynthetic machinery, consisting of a large number ofprotein and nucleic acid components, is among the most complex systems in the mammalian cell. Evidence suggests that these components may be highly organized within the cell (1, 2); however, only remnants of this organization are thought to survive the procedures used to prepare cell-free translation systems. Thus, although it has been shown that ribosomes and other translation components can interact with the cytoskeletal framework ofthe cell (3-8), and with each other (9-12), presumably reflecting associations that normally occur in vivo, an intact proteinsynthesizing system has not been isolated. Moreover, it is likely that the relatively low rates ofprotein synthesis usually obtained with cell-free systems (refs. 13 and 14 and references therein) are a consequence of disrupting the structural organization of the translation components that normally exists within the cell.These conclusions were reinforced by recent evidence suggesting that protein biosynthesis in mammalian cells is a channeled pathway-i.e., aminoacyl-tRNAs, the intermediates in the process, are directly transferred from the aminoacyl-tRNA synthetases, to the elongation factor, and then to the ribosomes, without dissociation into the cellular fluid (15,16 The total volume of cell suspension was measured by pipetting, and an equal volume of saponin solution (150 ,g/ml) in SU buffer was added. The suspension was gently mixed, incubated for 6 min at 37°C, cooled on ice, and centrifuged for 1 min at 420 x g (Sorvall) at 4°C, and the permeabilized cell pellet was resuspended in SU buffer.

Osteopontin (OPN) is a cytokine with multiple functions, including immune defense mechanisms against invading microorganisms. OPN-deficient mice are impaired in clearing intracellular pathogens, suggesting an important role of OPN during phagocytosis, but it remains to be defined how OPN may enhance this innate immune process. Here, we demonstrate that OPN binds to monocytes, but not resting T cells, NK cells, or B cells, and mediates chemoattraction of IL-1-activated human monocytes. Moreover, OPN binds in a specific manner to all known serotypes of the two bacterial species Streptococcus agalactiae and Staphylococcus aureus and opsonizes these bacteria for phagocytosis. We identify the integrin αXβ2 (CD11c/CD18), which is highly expressed on the cell surface of monocytes, as a novel OPN receptor. To eliminate the contribution from other molecular interactions between the bacteria and the phagocyte, we show that OPN-coated synthetic beads are phagocytosed in an αXβ2 integrin-dependent manner. The ligand recognition does not involve the RGD motif previously reported to support binding of OPN to integrins. Taken together, these data identify the αXβ2 integrin as a novel OPN receptor that is required for OPN-mediated phagocytosis, thereby elucidating an important mechanism of an innate immune function of OPN.

The mammalian protein synthesizing system is highly organized in vivo, and its substrate, tRNA, is channeled throughout the translation process. However, the cellular components responsible for this organization are not known. To examine this question a series of studies was carried out using intact and permeabilized Chinese hamster ovary cells. We show that cold shock dramatically reduces the protein synthetic capacity of these cells by as much as 95%. The loss of activity can be reversed by a short recovery period under conditions that allow energy metabolism to occur; transcription and translation during the recovery period are not needed. While individual components of the translation apparatus are not inactivated by the cold shock, the supramolecular organization of the system appears to be altered and F-actin levels are found to decrease. Resumption of protein synthesis during the recovery period coincides closely with the restoration of F-actin to normal levels. Moreover, disruption of actin filaments, but not microtubules, also leads to a major reduction in translation. These data support the conclusion that the cellular microfilament network plays an important role in the structure and function of the translation system and that perturbations of this network can have profound effects on protein synthesis.Considerable evidence has now accumulated showing that the mammalian protein synthetic machinery is highly organized in vivo, both structurally and functionally (1-3). For example, recent work with a permeabilized Chinese hamster ovary cell (CHO) 1 system, that closely mimics the living cell, demonstrated that a channeled tRNA cycle functions during the translation process (3), i.e. aminoacyl-tRNA and tRNA, the intermediates in the process, are directly transferred from the aminoacyl-tRNA synthetases, to the elongation factor, to the ribosome, and back to the synthetases, without dissociation into the cellular fluid. Such studies suggest that the various parts of the translation apparatus must associate as tRNA is transferred from one component to the next. In fact, since only small amounts of RNA and protein leak out of permeabilized cells under conditions in which large, exogenously-added macromolecules can enter, it is likely that the components of the translation machinery are permanently organized into a supramolecular structure (2). Additional evidence for an organized translation machinery comes from the isolation of a multienzyme aminoacyl-tRNA synthetase complex and other assemblies which contain multiple translation components (4 -8). Interactions among various individual components of the translation system also have been demonstrated in vitro, supporting the idea that similar associations occur in vivo (9 -12). Thus, elucidating the components responsible for this supramolecular organization and how they influence protein synthesis is essential for a complete understanding of the translation process in vivo.Several pieces of information suggest that the cytoskeletal elements of the cell serv...

Multiple sclerosis (MS) is an inflammatory disease where phagocytic cells infiltrate the nerve tissue and act as terminal agents in destruction of the myelin sheath. However, the mechanism that triggers the ability of these cells to recognize myelin remains obscure. We show that myelin basic protein (MBP), a major autoantigen in MS, is a potent and specific ligand for the integrin αMβ2 (Mac-1, CD11b/CD18) expressed mainly on phagocytic cells. MBP undergoes a dramatic conformational change when liberated from the lipid-rich environment of the myelin sheath. The MS drug glatiramer acetate mimics the conformationally labile regions of MBP, interacts in the unfolded state strongly with αMβ2, and inhibits the MBP binding to αMβ2. Our study reveals a link between MBP, glatiramer acetate, and the αMβ2 integrin, and suggests a new model for MS pathogenesis based on the recognition of unfolded MBP by the αMβ2 integrin.