FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins.
Nucleo-cytoplasmic shuttling is an important feature of proteins involved in nuclear export/import of RNAs, proteins, and also large ribonucleoprotein complexes such as ribosomes. The vast amount of proteomic data available shows that many of these processes are highly dynamic. Therefore, methods are needed to reliably assess whether a protein shuttles between nucleus and cytoplasm, and the kinetics with which it exchanges. Here we describe a combination of the classical heterokaryon assay with fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques, which allows an assessment of the kinetics of protein shuttling in the yeast Saccharomyces cerevisiae.
Wellcome Trust Centre for Cell Biology, University of Edinburgh, United Kingdom.
Katsiaryna
K
Belaya
David
D
Tollervey
Martin
M
Kos
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D002467:Cell Nucleus / Q000502:physiology*; D003593:Cytoplasm / Q000502:physiology*; D036681:Fluorescence Recovery After Photobleaching; D007700:Kinetics; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology / Q000235:genetics / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis*
D029701:Saccharomyces cerevisiae Proteins