Oleosin of Plant Seed Oil Bodies Is Correctly Targeted to the Lipid Bodies in Transformed Yeast

Julie T.L Ting,Ronald A Balsamo,Chandra Ratnayake,Anthony H.C Huang

doi:10.1074/jbc.272.6.3699

Julie T.L Ting, Ronald A Balsamo + Show 2 more

Open Access

https://doi.org/10.1074/jbc.272.6.3699

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Abstract

Yeast (Saccharomyces cerevisiae) has been used extensively as a heterologous eukaryotic system to study the intracellular targeting of proteins to different organelles. The lipid bodies in yeast have not been previously subjected to such studies. These organelles are functionally equivalent to the subcellular storage oil bodies in plant seeds. A plant oil body has a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins. We tested whether plant oleosin could be correctly targeted to the lipid bodies in transformed yeast. The coding region of a maize (Zea mays L.) oleosin gene was incorporated into yeast high copy and low copy number plasmids in which its expression was under the control of GAL1 promoter. Yeast strains transformed with these plasmids produced oleosin when grown in a medium containing galactose but not glucose. The oleosin produced in yeast had a molecular mass slightly higher than that of the native protein in maize. Oleosin accumulated concomitantly with the storage lipids during growth of the transformed yeast, and it was not secreted. Subcellular fractionation of the cell extracts obtained by two different cell breakage procedures revealed that the oleosin was largely restricted to the lipid bodies. Oleosin apparently did not affect the lipid contents and composition of the transformed yeast lipid bodies but replaced some of the native proteins associated with the organelles. Immunocytochemistry of the transformed yeast cells showed that the oleosin was present mostly on the periphery of the lipid bodies. Oleosin isolated from maize or transformed yeast strain, alone or in the presence of phospholipids or SDS, did not bind to the yeast lipid bodies in vitro. We conclude that plant oleosin is correctly targeted to the lipid bodies in transformed yeast and that yeast may be used as a heterologous system to dissect the intracellular targeting signals in the oleosin.

Highlights

Diverse organisms store lipids in subcellular particles as food reserves that will be mobilized during a forthcoming period of active metabolism
We show that plant seed oleosin is correctly targeted to the lipid bodies in yeast transformed with the plant oleosin gene
In a transformed yeast strain that contains a high amount of oleosin, we have demonstrated by biochemical means that the oleosin is present mostly in the lipid bodies

Summary

Plant Oleosin Targeted to Yeast Lipid Bodies

Polyribosomes bound to the ER, no cleavable N-terminal signal sequence in the protein has been identified [1]. Oleosin does not enter the lumen of the ER, otherwise budding would occur at the luminal side of the ER and the newly formed oil body would enter the intracellular secretary pathway. Oleosin has a conserved central hydrophobic domain that could act as the targeting signal for the hydrophobic matrix of the oil bodies This targeting signal would be equivalent to the internal hydrophobic targeting sequences in many membrane proteins of diverse organisms [15, 16]. The lipid bodies, of about 0.2– 0.5 ␮m in diameter, contain about 50% TAGs and 50% steroid esters, which are accumulated as food reserves during the log phase and the late stage of growth [5, 6]. We show that plant seed oleosin is correctly targeted to the lipid bodies in yeast transformed with the plant oleosin gene

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DISCUSSION