Incorporation of 3H-amino acids into proteins in a partially purified fraction of axoplasm: evidence for transfer RNA-mediated, post- translational protein modification in squid giant axons

Abstract

Transfer RNA (tRNA) has been demonstrated to be present in axons of both invertebrates and the higher vertebrates, but nothing is known of its role in the metabolism of the axon. The present experiments were performed to determine whether tRNA functions in axons as a participant in post-translational protein modification of endogenous proteins. RNA was extracted from the axoplasm of squid giant axons and incubated with a variety of 3H-amino acids, aminoacyl-tRNA synthetases (obtained from squid optic lobe), and an appropriate reaction mixture. All of the amino acids tested were bound to an RNA fraction, but this reaction did not occur when samples were incubated in the presence of ribonuclease or in the absence of axoplasmic RNA. When radioactive RNA was chromatographed by polyacrylamide gel electrophoresis, the radioactivity comigrated with known tRNA markers, suggesting the presence of 3H-aminoacylated tRNA. Aminoacylation of RNA could also be demonstrated by incubating fresh axoplasm with labeled amino acids and a reaction mixture, minus exogenous aminoacyl-tRNA synthetases. These findings indicate the presence in axoplasm of a variety of species of aminoacyl-tRNAs as well as their corresponding synthetase enzymes. In the latter experiment no radioactivity was found associated with the protein fraction. This was also the finding when 3H-aminoacylated tRNA was either injected directly into the axon or incubated with extruded axoplasm. Thus, under the conditions described above, there is no evidence of transfer of amino acids from tRNA to proteins. In other experiments, axoplasm was pooled to a volume of 50 to 100 microliters, homogenized gently, and centrifuged at 150,000 X g for 1 hr. Some of the high speed supernatant was incubated with labeled amino acids and an appropriate reaction mixture, and the remainder was passed through an S-200 Sephacryl column before incubation with the same reaction mixture. There was no incorporation of amino acids into protein in the high speed supernatant fraction. However, in the S-200 purified fraction 3H-labeled Arg, Lys, Tyr, Leu, and Asp were all incorporated into proteins in amounts of 44, 30, 7, 5 and 3.5 times heat-inactivated controls. The reaction is not inhibited by Ca2+ or Ca2+-activated proteases, but appears to be dependent on the presence of tRNA. The addition of amino acids to protein is not protein synthesis since the reactions occurred in a partially purified fraction of the 150,000 X g supernatant, a fraction devoid of ribosomes and free amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins

“…The second argument is that many components of translation machinery are found in axons, including in squid giant axoplasm, and therefore this serves as prima facie evidence that translation must be occurring in this cellular compartment (Alvarez et al 2000;Giuditta et al 2008). These data include reports of abundant tRNAs (Black and Lasek 1977;Ingoglia et al 1983), mRNAs (Capano et al 1987;Gioio et al 1994;Kaplan et al 1992;Rapallino et al 1988), various elongation and initiation factors (Giuditta et al 1980;Giuditta et al 1986;Giuditta et al 1991;Kar et al 2013), rRNA (Giuditta et al 1980;Perrone-Capano et al 1999), and even polyribosomes that can be translated in vitro to produce various proteins (Giuditta et al 1991;Sotelo et al 1999). Given this apparent translation capacity of squid axoplasm it should then be possible for isolated axoplasm to synthesize proteins directly from radioactive amino acids.…”

Local Gene Expression in Axons and Nerve Endings: The Glia-Neuron Unit

“…The apparent lack of other cytoplasmic RNAs, notably rRNAs, confirmed that axons could not synthesize proteins and suggested that axoplasmic tRNA comprised only a few species serving a different function such as addition of activated amino acids to the native proteins (161). These data were contradicted by two independent observations.…”

“…First, selective assays of single tRNAs and aminoacyltRNA synthetases demonstrated that the giant axon contained as many as six different tRNAs and the corresponding synthetases. In other words, all the tRNA species assayed for were in the axoplasm (161). As these components could not be involved in other activities but translation processes, the findings contributed to tilt the balance in favor of axonal protein synthesis.…”

Slow axoplasmic transport under scrutiny

“…identifi ed as ribosomal, messenger and transfer (Edström et al, 1962;Black and Lasek, 1977;Koenig, 1979;Ingoglia et al, 1983;Jirikowski et al, 1990;Mohr and Richter, 1992;Wensley et al, 1995). Notwithstanding, the role of axoplasmic RNAs has been controversial as it was deemed unrelated to local protein synthesis (Black and Lasek, 1977;Ingoglia et al, 1983;Jirikowski et al, 1990;Mohr and Richter, 1992;Wensley et al, 1995) since the notion that axons do not synthesize proteins prevailed.…”

“…Notwithstanding, the role of axoplasmic RNAs has been controversial as it was deemed unrelated to local protein synthesis (Black and Lasek, 1977;Ingoglia et al, 1983;Jirikowski et al, 1990;Mohr and Richter, 1992;Wensley et al, 1995) since the notion that axons do not synthesize proteins prevailed. If we track this deeply rooted notion back to its roots, we fi nd that electron microscopists did not include ribosomes in the inventory of the axoplasm, and that cell biologists considered axons to be ribosome-free, based on this one and only datum.…”