Counting cytokinesis proteins globally and locally in fission yeast.
We used fluorescence microscopy to measure global and local concentrations of 28 cytoskeletal and signaling proteins fused to yellow fluorescent protein (YFP) in the fission yeast Schizosaccharomyces pombe. Native promoters controlled the expression of these functional YFP fusion proteins. Fluorescence measured by microscopy or flow cytometry was directly proportional to protein concentration measured by quantitative immunoblotting. Global cytoplasmic concentrations ranged from 0.04 (formin Cdc12p) to 63 micromolar (actin). Proteins concentrated up to 100 times in contractile rings and 7500 times in spindle pole bodies at certain times in the cell cycle. This approach can be used to measure the global and local concentrations of any fusion protein.
Wu
Jian-Qiu
JQ
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.
Pollard
Thomas D
TD
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000235:genetics; D048749:Cytokinesis; D036341:Intercellular Signaling Peptides and Proteins / Q000032:analysis* / Q000235:genetics; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012568:Schizosaccharomyces / Q000737:chemistry*; D029702:Schizosaccharomyces pombe Proteins / Q000032:analysis* / Q000235:genetics
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; C054562:yellow fluorescent protein, Bacteria