Comparison of Protein Synthesis in Mitochondria, Synaptosomes, and Intact Brain Cells

Abstract

Qualitative aspects of protein synthesis in organelles and intact cultured cells of brain origin were compared to clarify the distinction between synaptosomal and mitochondrial protein synthesis. Brain mitochondria and synaptosomes were isolated either on a traditional Ficoll-sucrose gradient or by a new Percoll gradient procedure, and were incubated in an amino acid incorporation system containing [35S]methionine, then electrophoresed on gradient slab gels. Autoradiography of the gels revealed that in the presence of cycloheximide both mitochondria and synaptosomes synthesized at least 17 proteins in the 6,000-50,000 MW range, and that incubation with chloramphenicol reduced or eliminated these bands. With minor variation these patterns in the low-molecular-weight region also resembled patterns obtained from cycloheximide-inhibited rat liver mitochondria and intact brain cells (cultured glia, glioma, and neuroblastoma). In the higher molecular weight region of the gels (greater than 50,000) banding patterns were more complex and tended to differ between organelles and intact cells. These polypeptides probably reflect nonmitochondrial protein synthesis, and their variable response to inhibitors may account for confusion in the literature with regard to the effects of inhibitors of protein synthesis in brain mitochondria and synaptosomes.

Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes

“…The fact that labeling of a large number of bands was not appreciably affected by CAP suggests that most ofthe protein synthesis in the synaptosomal fraction is nonmitochondrial. In general, these results confirm the report of Irwin (1985). RNase did not appreciably affect the pattern of labeled bands, although the overall labeling appeared to be slightly reduced (data not shown).…”

“…A more revealing way to evaluate the contribution of eucaryotic ribosomal versus mitochondrial protein synthesis uses gel fluorography to identify which protein species are labeled in the presence of the various inhibitors (Irwin, 1985). An example from one preparation that had been incubated with or without the inhibitors is illustrated in Figure 4.…”

Neurofilament Proteins Are Synthesized in Nerve Endings from Squid Brain

“…It is generally held that presynaptic proteins are synthesized on perikaryal polysomes and delivered to their sites of function by axoplasmic transport (for review, see Grafstein and Fornian, 1980). Although this view encompasses several theoretical difficulties (Nixon, I980 Alvarez, 1992;Crispino et al, 1993), the concept that protein synthesis occurs locally in the presynaptic terminals (Morgan and Austin, 1968;Gambetti et al, 1972;Gilbert, 1972;Ramirez et al, 1972;Deanin and Gordon, 1973) has not achieved wide acceptance (Irwin, 1985;Rao and Steward, 199 I), primarily due to the paucity of direct evidence that the protein synthetic activity of synaptosomal preparations derives from nerve endings rather than contaminating structures, such as dendrites or glial fragments.…”

Protein Modification by RNA‐Dependent Posttranslational Aminoacylation in Synaptoplasm

“…A controversy concerning de novo protein synthesis in axon terminals has existed for many years. Protein synthesis has been postulated to occur in squid axoplasm (Giuditta et al, 1977), isolated axons from cultured retinal explants (Koenig and Adams, 1982), and synaptosomes from rat brain (Hernandez et al, 1978;Irwin, 1985). These speculations have been based on the capacity of an isolated portion of the axon or terminal to incorporate radiolabelled free amino acids into their macromolecular structure.…”

“…Supporting this belief are the results of a study by Ingoglia et al (1983), who, using pure axoplasm from squid, were able to demonstrate the posttranslational addition of arginine, tyrosine, aspartic acid, lysine, and leucine into the macromolecular protein structure of squid axoplasm. In addition, synaptosomal studies suggest that amino acid addition, separate from mitochondria1 or ribosomal protein synthesis, occurs in axonal terminals (Droz and Barondes, 1969;Gambetti et al, 1972;Irwin, 1985).…”