Journal Article

An in vitro System to Examine the Effective Phospholipids and Structural Domain for Protein Targeting to Seed Oil Bodies

Jeff C.F. Chen and Jason T.C. Tzen

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 42, issue 11, pages 1245-1252
Published in print November 2001 | ISSN: 0032-0781
Published online November 2001 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pce160
An in vitro System to Examine the Effective Phospholipids and Structural Domain for Protein Targeting to Seed Oil Bodies

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An in vitro system was established to examine the targeting of proteins to maturing seed oil bodies. Oleosin, the most abundant structural protein, and caleosin, a newly identified minor constituent in seed oil bodies, were translated in a reticulocyte lysate system and simultaneously incubated with artificial oil emulsions composed of triacylglycerol and phospholipid. The results suggest that oil body proteins could spontaneously target to artificial oil emulsions in a co-translational mode. Incorporation of oleosin to artificial oil emulsions extensively protected a fragment of approximately 8 kDa from proteinase K digestion. In a competition experiment, in vitro translated caleosin and oleosin preferentially target to artificial oil emulsions instead of microsomal membranes. In oil emulsions with neutral phospholipids, relatively low protein targeting efficiency was observed. The targeting efficiency was substantially elevated when negatively charged phospholipids were supplemented to oil emulsions to mimic the native phospholipid composition of oil bodies. Mutated caleosin lacking various structural domains or subdomains was examined for its in vitro targeting efficiency. The results indicate that the subdomain comprising the proline knot motif is crucial for caleosin targeting to oil bodies. A model of direct targeting of oil-body proteins to maturing oil bodies is proposed.

Keywords: Key words: Caleosin — Negatively charged phospholipids — Oil body targeting — Oleosin — Proline knot.; Abbreviations: EGTA, ethylene-glycol-bis(β-aminoethyl ether)-tetraacetic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL(s), phospholipid(s); PS, phosphatidylserine; SRP; signal-recognition particle, TAG(s), triacylglycerol(s).

Journal Article.  5535 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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