Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast.
We have developed a method for monitoring autophagy using Rosella, a biosensor comprised of a fast-maturing pH-stable red fluorescent protein fused to a pH-sensitive green fluorescent protein variant. Its mode of action relies upon differences in pH between different cellular compartments and the vacuole. Here we demonstrate its utility in yeast (Saccharomyces cerevisiae) by expression in the cytosol, and targeting to mitochondria or to the nucleus. When cells were cultured in nitrogen depleted medium, uptake of the compartment labelled with the biosensor (i.e., cytosol, mitochondria, or nucleus) into the vacuole was observed. We showed that this vacuolar uptake was, for cytosol and mitochondria, an ATG8-dependent process while the uptake of the nucleus was significantly reduced in the absence of Atg8p and can be said to be partially ATG8-dependent. We further demonstrated the value of the biosensor as a reporter of autophagy by employing fluorescence-activated cell sorting of discrete populations of cells undergoing autophagy.
Rosado
Carlos J
CJ
Department of Biochemistry & Molecular Biology, Monash University, Clayton Campus, Victoria, Australia.
Mijaljica
Dalibor
D
Hatzinisiriou
Irene
I
Prescott
Mark
M
Devenish
Rodney J
RJ
D001343:Autophagy / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D015374:Biosensing Techniques; D003600:Cytosol / Q000378:metabolism*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism; D015388:Organelles / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012107:Research Design; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000502:physiology*; D014617:Vacuoles / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein