Functional compartmentalization of the protein synthetic machinery in higher eukaryotes was studied using permeabilized with saponin or electroporated cells. For the first time the system of coupled permebilization-translation was developed for CHO cells and human fibroblasts demonstrating the same efficiency of translation process in intact and permeabilized cells. A possibility of compartmentalization of aminoacyl-tRNA (aa-tRNA) in CHO cells was tested by studying aa-tRNA distribution between intracellular and extracellular spaces of permeabilized cells. In double-label experiments 25-fold retardation inside permeabilized cells was observed for endogenous aa-tRNA while the ratio of exogenous aa-tRNA present inside and outside was similar to that of intracellular and extracellular volumes. Moreover, cellular aa-tRNA was protected against action of added RNAse A while exogenous aa-tRNA was easily hydrolysed by RNAse inside the cells. After homogenization of the cells no protection of endogenous aa-tRNA against RNAse was found. Thus, both distribution and RNAse protection experiments show structural compartmentalization of cellular aminoacyl-tRNA. One of the important mechanisms to realize potential advantages of compartmentalization may be channeling, or direct transfer of intermediates from one component to another in a metabolic chain. Besides the need for some structural organization, another evidence of channeling mechanism is the impossibility of exogenous intermediates to enter a channeled pathway. Thus, the level of utilization of cellular and exogenously added aa-tRNA for protein synthesis in perforated cells was studied. Contrary to a cell-free system, in which no advantage was found, about 15-fold preference for cellular aa-tRNA was detected in permeabilized cells, i.e. endogenous aa-tRNA was much more efficient precursor, than exogenous aa-tRNA, for protein biosynthesis. Thus, both structural and functional criteria for channeling are fulfilled in eukaryotic protein synthesis. We suggested that channeling of tRNA occurs in some labile structures - translational compartments which are destroyed during homogenization. To obtain the information about such compartments, namely, about possible changes in their organization under different cell conditions, the fluorescently labeled ( and ( subunits of elongation factor 1 (EF-1) were used as compartment markers. Distribution of proteins in permeabilized human fibroblasts VH25 was compared under regular and low energy (no phosphocreatine) conditions. The absence of phosphocreatine in the incubation mixture was shown to cause almost complete stop of translation. The same granular EF-1( localization in cytoplasm was found under normal and low-energy conditions while the cytoplasmic distribution of EF-1( was more granular under normal conditions (resembling that of EF-1() and was much more nucleus-concentrated under low energy conditions. Thus, it is shown that involvement of the important component of translation apparatus - EF-1( - into suggested translatio nal compartments may depend on the efficacy of protein biosynthesis in cell. Difference in EF-1( and EF-1( localization under low energy conditions may reflect different levels of immobilization of these proteins inside the cells, as earlier suggested. As far as we know these data are the first to reveal the correlation between depletion of energetic compound and the altered localization of a translation machinery component in eukaryotic cells. The possibility of aminoacyl-tRNA synthetase-EF-1( interaction, suggested by channeling hypothesis, was studied in vitro using rabbit liver EF-1(, phenylalanyl-tRNA synthetase (PheRS) and valyl-tRNA synthetase (ValRS)-EF-1H complex. EF-1( caused 2-fold stimulation of both valyl-tRNA and phenylalanyl-tRNA formation. The stimulation was rather specific since BSA and bacterial factor EF-Tu had no stimulatory capacity. Nevertheless, the mechanisms of EF-1-induced stimulation were quite different for two synthetases. Stimulation of ValRS required GTP strongly suggesting cla ssical ternary complex valyl-tRNA-GTP-EF-1( involvement. In that case EF-1( stimulatory effect appears to occur at the stage of aminoacyl-tRNA dissociation from the synthetase as suggested by channeling hypothesis. Interestingly, ValRS separated from EF-1H cannot be activated by EF-1( demonstrating the absolute requirement of the structure of whole valyl-tRNA synthetase-EF-1H complex for the stimulation. On the contrary, activation of PheRS can be induced by GDP form of EF-1( as well. Moreover, the stimulatory effect was observed already at the level of phenylalanine activation, suggesting factor-synthetase interaction to occur at some step before aminoacyl-tRNA dissociation. A special functional role was suggested for EF-1(-GDP complex, namely, to form EF-1(*GDP*tRNA complex and to deliver deacylated tRNA to aminoacyl-tRNA synthetase. The ability of EF-1(-GDP to form a complex with tRNA was proved by several independent techniques. Analysis of EF-1(-GDP endogenous fluorescence showed possible conformation al change of the protein in the presence of tRNAPhe. On the other side, partial RNAse hydrolysis and chemical modification studies indicated conformational changes of tRNAPhe and tRNALeu in the presence of EF-1(-GDP. Interestingly, the sites of tRNA interaction with EF-1(-GDP coincided well with known aminoacyl-tRNA sites of interaction with bacterial factor EF-Tu-GTP suggesting similarity of both complexes. Finally, the formation of quaternary complex EF-1(-GDP-tRNAPhe-PheRS was demonstrated by "band shift" technique. Thus, the principal possibility of two ways to transport tRNA between aminoacyl-tRNA synthetase and EF-1( was demonstrated. Deacylated tRNA can be delivered from EF-1(-GDP to synthetase (as in the case of PheRS), and aminoacyl-tRNA can be delivered from synthetase to EF-1(-GTP (as in the case of ValRS). On the basis of these findings a model of translation apparatus functioning is developed which suggests EF-1( to be a shuttle between ribosome and hypotetic macromolecular complex of synthetases and EF-1H. The concept of functional compartmentalization of mammalian translation apparatus is put forward. Functional compartmentalization is the formation of labile protein-protein and protein-RNA complexes which provide organization of the translation compartments and their efficient functioning. The definition for translational compartment is as follows: it is an assembly of macromolecular complexes which are dynamically converted during mRNA translation in some limited space.
