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In Vitro Studies of the Biosynthesis of Brain Tubulin on Membranes a
Author(s) -
GILBERT JEFFREY M.,
STROCCHI PAOLA
Publication year - 1986
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.1986.tb38386.x
Subject(s) - polysome , tubulin , biology , biochemistry , membrane protein , translation (biology) , cell fractionation , microtubule , membrane , microbiology and biotechnology , ribosome , rna , messenger rna , gene
Membrane elements in brain tissue contain relatively large amounts of alpha- and beta-tubulin (FIGURES 2 and 3). We have investigated the subcellular sites of tubulin biosynthesis in order to determine the origin of this membrane-associated tubulin. Free and membrane-bound polysomes from rat forebrain were separated by differential centrifugation, and the products of translation from these polysome populations were analyzed by 2DGE (FIGURES 4 and 6). Alpha- and beta-tubulin subunits were synthesized by the free polysome population (FIGURES 4 and 5A and B). The membrane-bound polysome fraction synthesized a protein with similar (but not identical) characteristics to alpha-tubulin (denoted as "MB" in FIGURE 6), including isoelectric point, molecular weight, peptide map, and copurification with microtubules after aggregation-disaggregation. Tubulin subunits synthesized in vitro by free polysomes could associate posttranslationally with a microsome fraction (FIGURE 7A). The association of the tubulin translation products with membranes was not disrupted by high salt; the associated tubulin, however, was susceptible to proteolytic digestion, with the exception of one of the beta-tubulin subunits (FIGURE 7B). There was an identical protease-resistant beta-tubulin subunit among the native proteins of the smooth microsome fractions. Our data is consistent with the conclusion that at least one beta subunit of membrane-associated tubulin is synthesized by free polysomes and becomes posttranslationally added to membrane structures. It is unlikely that a cotranslational mechanism is responsible, in which there is a signal-mediated insertion of a growing polypeptide chain to membrane. Our results, however, are consistent with a "membrane trigger" mechanism proposed by Wickner in which the membrane lipid bilayer triggers the folding of a polypeptide into a configuration that allows integral membrane insertion. The association of tubulin with membranes may also be secondary to the interaction of hydrophobic elements. The amino acid sequence of beta tubulin is known to contain several hydrophobic domains. Tubulin can be incorporated into phospholipid vesicles and various subcellular membrane elements. In our studies, in vitro synthesized tubulin from free polysome was found to be purified by hydrophobic affinity chromatography with ethane-sepharose (FIGURE 8). Thus, the hydrophobic characteristics of newly synthesized tubulin could be partially responsible for the posttranslational association of tubulin subunit with membranes. Native tubulin in a soluble fraction of CNS tissue was not purified by hydrophobic affinity chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)