Plastid LPAT1 is an integral inner envelope membrane protein with the acyltransferase domain located in the stroma.

Publisher: Springer Country of Publication: Germany NLM ID: 9880970 Publication Model: Electronic Cited Medium: Internet ISSN: 1432-203X (Electronic) Linking ISSN: 07217714 NLM ISO Abbreviation: Plant Cell Rep Subsets: MEDLINE

Original Publication: Berlin ; New York : Springer, 1981-

Arabidopsis*/genetics ; Arabidopsis*/metabolism ; Arabidopsis*/enzymology ; Acyltransferases*/metabolism ; Acyltransferases*/genetics; Protein Domains ; Plastids/metabolism ; Arabidopsis Proteins/metabolism ; Arabidopsis Proteins/genetics ; Plants, Genetically Modified ; Chloroplasts/metabolism ; Membrane Proteins/metabolism ; Membrane Proteins/genetics ; Nicotiana/genetics ; Nicotiana/metabolism

Key Message: The N-terminal transmembrane domain of LPAT1 crosses the inner membrane placing the N terminus in the intermembrane space and the C-terminal enzymatic domain in the stroma. Galactolipids mono- and di-galactosyl diacylglycerol are the major and vital lipids of photosynthetic membranes. They are synthesized by five enzymes hosted at different sub-chloroplast locations. However, localization and topology of the second-acting enzyme, lysophosphatidic acid acyltransferase 1 (LPAT1), which acylates the sn-2 position of glycerol-3-phosphate (G3P) to produce phosphatidic acid (PA), remain unclear. It is not known whether LPAT1 is located at the outer or the inner envelope membrane and whether its enzymatic domain faces the cytosol, the intermembrane space, or the stroma. Even the size of mature LPAT1 in chloroplasts is not known. More information is essential for understanding the pathways of metabolite flow and for future engineering endeavors to modify glycerolipid biosynthesis. We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. We show that LPAT1 traverses the inner membrane via an N-terminal transmembrane domain, with its N terminus protruding into the intermembrane space and the C-terminal enzymatic domain residing in the stroma, hence displaying a different membrane topology from its bacterial homolog, PlsC.
(© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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112-2923-B-001-001-MY2 National Science and Technology Council; 113-2311-B-002-007 National Science and Technology Council; JPMJGX23B0 GteX Program Japan

Keywords: Chloroplast; Galactolipid; Inner envelope membrane; Lysophosphatidic acid acyltransferase; Phosphatidic acid; Topology

EC 2.3.- (Acyltransferases)
0 (Arabidopsis Proteins)
EC 2.3.1.52 (2-acylglycerophosphate acyltransferase)
0 (Membrane Proteins)

Date Created: 20241009 Date Completed: 20241009 Latest Revision: 20241113

20241114

10.1007/s00299-024-03347-z

39382709