A proteomics approach to cloning fenestrin from the nuclear exchange junction of tetrahymena.
We set out to find the "fenestrin" gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila. First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS-PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP-localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting.
Cole
Eric S
ES
Biology Department, St. Olaf College, Northfield, Minnesota 55057, USA. colee@stolaf.edu
Anderson
Paul C
PC
Fulton
Ross B
RB
Majerus
Matthew E
ME
Rooney
Megan G
MG
Savage
Johanna M
JM
Chalker
Douglas
D
Honts
Jerry
J
Welch
Mary E
ME
Wentland
Amy L
AL
Zweifel
Erica
E
Beussman
Douglas J
DJ
D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D018503:Genome, Protozoan; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D040901:Proteomics / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013768:Tetrahymena / Q000235:genetics / Q000378:metabolism*
D008164:Luminescent Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins