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BIS 104 Lingappa b fall 08 001

BIS 104 Lingappa b fall 08 001 - expression cell-free...

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Unformatted text preview: expression cell-free translation Xenopus oocyle - system: microinjeotion ‘eirpression signal—gloom chimera globin signal-gloom plasmid; chimera . 12 6789.10 3 4 5 wow. + + + mombranssz— — protease: + - —- + + _+ _+_ detergent w r - H + __ __+ \I, . \'_\r ,«.—@@,;.. New Figure-A Signal globln expression and translooation in a cell-free translation system (with cartoon Interpreta- tion) and in Xenopus oocytes. This Full-length cDNA had been engineered into a site within the E. calf beta lactarnase gene. Beta lactamase, ' being a secretory protein of E. calf, has a signal sequence. Moreover, a convenient restriction endonuclease site (see p. 135) at'the start of the globin coding region made dele- tions of the upstream lactamase codons beyond the signal sequence technically quite easy. Thus, for the initial exper- iments we chose to use the beta lactamase signal se- quence. These DNA molecules were constructed behind an active promoter (see p. 539). This allowed us to make mRNA from the cloned DNA in a test tube and then translate this mRNA in a cell-free translation system. In the absence of added ER membranes, the full chi- mera was synthesized, which was larger than authentic giobin by the size of the signal sequence (Fig. A, lane 2, downward pointing arrowhead). When proteases were added to this translation product it was totally digested, as expected (lane 1). However it Eli-derived membrane vesicles were present during translation, a different pattern was observed A new product the size of authentic gl'obin was seen (lane 3, upward pointing arrowhead), in addition to some residual full-length chains. When these products were digested by added proteases, the authentic globin— sized product was protected from digestion while the re- sidual full-length chains were degraded (lane 4). Protection of authentic globin-sized chains from digestion by added proteases was lost if the vesicle biiayer was solubilized with a mild detergent, strongly suggesting that protection from proteases was due to translocation into the vesicle lumen (analogous to translocation into the lumen of the ER in viva) and not due to intrinsic protease resistance of the protein itself. Moreover, if membrane vesicles were added after Completion of protein synthesis. no authentic . globin-sized products were observad and the protein re- mained completely protease sensitive.z This demonstrated that translocation of globin occurred only while the signal sequence-bearing chain was being synthesized As a nec- essary control, globin without a signal sequence was shown not to be translocated even when membrane vesi- cles were present during. its synthesis.1 I To test whether these results applied to events occur- ring in viva, we used microinjection to introduce the mRNA For authentic globin or the signal-globin chimera into the cytoplasm of Xenapus oocytes. large living cells with an active secretory pathway. mRNA so introduced is translated by-ribosomes in the oocyte cytoplasm. Radiola- beled amino acids included in the medium which bathes the oocytes are taken up and incorporated into the newly synthesized proteins. Homogeniration of the oocytes con— verts the ER into vesicles, and analysis of localizationcan be carried out either by fractionation of the homogenate into cytosoI and vesicles or by protease digestion. as pre- ' viously described. Authentic-sized globin was synthesized from both mRN-As (lanes 6 and 8). However, globin syn- thesized from the chimeric mRNA was protected from added protease (lane 9) unless detergent was added (lane 10). while that synthesized from globin mRNA not an— coding a signal sequence was not protected from added proteases (lane 7). Moreover, fractionation demonstrated that authentic globin synthesized from globin mRNA was entirely in the cytosol while authentic-sized globin syn« thesized from the chimeric mRNA was entirely in the vesicles3 (not shown). These data suggested that, in the case of the chimera, authentic globin was generated asa result of cleavage of the signal sequence from the grow- ing chain as it translocated to the ER lumen. Thus, both in oitra and in viva, the beta. lactamase signal sequence was sufficient to direct chimpanzee alpha globin out of the cy- toplasm and into the secretory pathway.“3 An. ironic twist to this story was that, some time later, an insect globin gene was cloned and'found to have a remarkable structure. Globin‘ is found in the cytoplasm of red blood cells in most animal species. However insects lack red blood cells. Instead, they secrete their globin into a fluid, called hemolymph. which bathes the cells. Thus in— sect globin. whose mature sequence is quite conserved with other animal globins, is naturally a secreted rather than a cytoplasmic protein. When the mRNA for insect globin was examined, it was found to encode a signal se- quence at its amino terminus. Unbelmownst to us. we had been “scooped" by evolution 800 million years ago (when insects branched off from the lineage leading to verte- brates) on the neat idea of redirecting cytoplasmic globin- into the secretory pathway! Subsequent work over the years has shown that these findings are true for a wide range of signal and pas— senger sequences, although passenger sequences are not Mechanisms Targeting Proteins to the ER 841 ...
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