An approach to studying the localization and dynamics of eukaryotic translation factors in live yeast cells.
The discovery of Green Fluorescent Protein (GFP) and the development of technology that allows specific proteins to be tagged with GFP has fundamentally altered the types of question that can be asked using cell biological methods. It is now possible not only to study where a protein is within a cell, but also feasible to study the precise dynamics of protein movement within living cells. We have exploited these technical developments and applied them to the study of translation initiation factors in yeast, focusing particularly on the key regulated guanine nucleotide exchange step involving eIF2B and eIF2. This chapter summarizes current methodologies for the tagging and visualization of GFP-tagged proteins involved in translation initiation in live yeast cells.
Campbell
Susan G
SG
Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
Ashe
Mark P
MP
D000345:Affinity Labels / Q000378:metabolism; D001692:Biological Transport; D018929:Cell Culture Techniques; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014018:Tissue Distribution; D014170:Transformation, Genetic
D000345:Affinity Labels; D039642:Eukaryotic Initiation Factors; D011993:Recombinant Fusion Proteins; D012313:RNA