Electric pulse-induced precipitation of biological macromolecules in electroporation.

We found that electric discharge through solution of biological macromolecules (DNA, RNA and proteins) causes precipitation of significant portions of these macromolecules. This precipitation is a consequence of the interaction of biological macromolecules with the metal ions solubilized from the anode plate by the electric pulse, and occurs in both absence and presence of the cells in poration medium. Precipitated fractions of macromolecules sediments at the centrifugation speed used to pellet eukaryotic cells and does not dissolve when washed with buffer. Our data indicate a complication of the direct evaluation of electroporation efficiency based on the assumption that electroporated biological macromolecules which remain associated with the cells after several washes, are successfully electroinjected into the cytoplasm of cells.

Efficient mammalian protein synthesis requires an intact F-actin system.

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.

A channeled tRNA cycle during mammalian protein synthesis.

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.

Supramolecular organization of the mammalian translation system.

Although evidence suggests that the protein synthetic machinery is organized within cells, this point has been difficult to prove because any organization that might exist is lost upon preparation of the cell-free systems usually used to study translation in vitro. To examine this process under conditions more representative of the intact cell, we have developed an active protein-synthesizing system using Chinese hamster ovary (CHO) cells permeabilized with the plant glycoside saponin. This procedure renders cells permeable to trypan blue and exogenous tRNA, but there is little release of endogenous macromolecules. Protein synthesis in this system proceeds at the same rate as that in intact cells and is about 40-fold faster than that in a cell-free system prepared from the same cells. Active protein synthesis in this system requires the addition of only Mg2+, K+, and creatine phosphate, with a small further stimulation by ATP and an amino acid mixture; no exogenous macromolecules are necessary. The proteins synthesized in this system are indistinguishable from those made by the intact cell, and the channeling of aminoacyl-tRNA observed in vivo is maintained. Our data suggest that the permeabilized cell system retains the protein-synthesizing capabilities of the intact cell and presumably its internal structure as well. Studies with this system demonstrate that the protein-synthesizing apparatus is highly organized and that its macromolecular components are not freely diffusible in mammalian cells.