In vivo targeting of a sunflower oil body protein in yeast secretory (sec)mutants

A sunflower oleosin was expressed in yeast to study the in vivo insertion o f the protein into the endoplasmic reticulum (ER) and subsequent transfer t o lipid bodies. The oleosin cDNA was expressed in a range of yeast secretor y (sec) mutants to determine the precise targeting pathway of the oleosin t o the ER. Subcellular fractionation experiments indicated that the signal r ecognition particle (SRP) is required for oleosin targeting to the ER and h ence subsequent deposition on the lipid bodies in vivo. The expression of o leosin in a range of sec61 mutant alleles confirmed the role of the SEC61 t ranslocon in insertion of oleosin into the ER membrane, as well as indicati ng an unusual substrate/translocon interaction for one particular allele (s ec61-3). Mistargeting of the oleosin due to impaired SRP function resulted in enhanced proteolysis of the plant protein in the transformed yeast, as d etermined by pulse-chase analysis. These data therefore provide the first i n vivo evidence for the SRP-dependent targeting of the oleosin to the ER, a nd the subsequent requirement for a functional SEC61 translocon to mediate the correct insertion of the protein into the membrane.