This proposal combined recombinant DNA technology with the use of reconstituted cell-free systems towards the analysis of key problems in protein topogenesis (the processes by which newly synthesized proteins are transported to their correct subcellular locations). Using recombinant expression plasmids containing restriction endonuclease directed deletions in genes for eukaryotic secretory proteins as a source of mRNA we shall use a transcription linked, translation coupled translocation system to define rigorously several currently debated concepts involving the location and structure of signal sequences. By constructing expression plasmids of non-secretory proteins into which sections of signal sequence encoding DNA have been inserted (either intact or altered by site directed mutagenesis) we will determine the functional domains within wignal sequences and try to elucidate the mechanisms by which they function. If these experiments uphold our working hypothesis that discrete polypeptide sequences are primary determinants of protein localization, we shall initiate the construction of a 'topogeneic library', containing DNA segments whose insertion into the coding region of the gene for any protein will alter that protein's topogenic fate on expression in vivo or in vitro. Through this work, a more precise molecular understanding of topogenic sequences will emerge: 1) the functional domains within topogenic sequences will be identified; 2) the rules by which gene recombination may confer new locations (topogenic fates) to proteins will be determined; 3) a variety of currently debated issues in the field of protein topogenesis will be resolved. These studies would be an invaluable adjunct to future attempts to selectively (re)direct any particular newly synthesized protein to any chosen intracellular compartment by reprogramming its complement of topogenic sequences.
Showing the most recent 10 out of 12 publications
