Secretion trap tagging of secreted and membrane-spanning proteins using Arabidopsis gene traps.
Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a beta-glucuronidase reporter enzyme that is inhibited by N-linked glycosylation specific to the secretory pathway. Treatment of seedlings with tunicamycin inhibits glycosylation, resulting in increased activity of secreted beta-glucuronidase fusions that result from gene trap integration downstream of exons encoding signal peptides. In the 2,059 gene trap lines that we screened, 32 secretion trap expression patterns were identified in a wide variety of tissues including embryos, meristems, and the developing vasculature. Genes disrupted by the secretion traps encode putative extracellular signaling proteins, membrane transport proteins, and novel secreted proteins of unknown function missed by conventional mutagenesis and gene prediction. Secretion traps provide a unique reagent for gene expression studies and can guide the genetic combination of loss of function alleles in related genes.
Groover
Andrew T
AT
Cold Spring Harbor Laboratory, Box 100, New York 11724, USA.
Fontana
Joseph R
JR
Arroyo
Juana M
JM
Yordan
Cristina
C
McCombie
W Richard
WR
Martienssen
Robert A
RA
D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics*; D002462:Cell Membrane / Q000235:genetics; D017343:Genes, Plant; D016254:Mutagenesis, Insertional; D018517:Plant Roots / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics
D029681:Arabidopsis Proteins