title: A proteomics approach to cloning fenestrin from the nuclear exchange junction of tetrahymena.
abstract: 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.
authors.affiliation: Biology Department, St. Olaf College, Northfield, Minnesota 55057, USA. colee@stolaf.edu
authors.forename: Eric S
authors.initials: ES
authors.lastname: Cole
authors.forename: Paul C
authors.initials: PC
authors.lastname: Anderson
authors.forename: Ross B
authors.initials: RB
authors.lastname: Fulton
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Majerus
authors.forename: Megan G
authors.initials: MG
authors.lastname: Rooney
authors.forename: Johanna M
authors.initials: JM
authors.lastname: Savage
authors.forename: Douglas
authors.initials: D
authors.lastname: Chalker
authors.forename: Jerry
authors.initials: J
authors.lastname: Honts
authors.forename: Mary E
authors.initials: ME
authors.lastname: Welch
authors.forename: Amy L
authors.initials: AL
authors.lastname: Wentland
authors.forename: Erica
authors.initials: E
authors.lastname: Zweifel
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Beussman
mesh_terms: 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*
chemical_list: D008164:Luminescent Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins