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In vivo targeting of a sunflower oil body protein in yeast secretory ( sec ) mutants
Author(s) -
Beaudoin Frédéric,
Wilkinson Barrie M.,
Stirling Colin J.,
Napier Johnathan A.
Publication year - 2000
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2000.00769.x
Subject(s) - mutant , yeast , in vivo , sunflower oil , sunflower , chemistry , microbiology and biotechnology , biology , food science , biochemistry , genetics , gene , agronomy
Summary A sunflower oleosin was expressed in yeast to study the in vivo insertion of the protein into the endoplasmic reticulum (ER) and subsequent transfer to lipid bodies. The oleosin cDNA was expressed in a range of yeast secretory ( sec ) mutants to determine the precise targeting pathway of the oleosin to the ER. Subcellular fractionation experiments indicated that the signal recognition particle (SRP) is required for oleosin targeting to the ER and hence subsequent deposition on the lipid bodies in vivo . The expression of oleosin in a range of sec61 mutant alleles confirmed the role of the SEC61 translocon in insertion of oleosin into the ER membrane, as well as indicating an unusual substrate/translocon interaction for one particular allele ( sec61‐3 ). Mistargeting of the oleosin due to impaired SRP function resulted in enhanced proteolysis of the plant protein in the transformed yeast, as determined by pulse–chase analysis. These data therefore provide the first in vivo evidence for the SRP‐dependent targeting of the oleosin to the ER, and the subsequent requirement for a functional SEC61 translocon to mediate the correct insertion of the protein into the membrane.