Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) are conserved constituents of membrane contact sites that involve ER membranes. VAPs are ER-integral proteins that interact with oxysterol-binding protein and its homologs, which bind to specific lipids in target membranes. VAP27 proteins are the VAPs in plants, and the Arabidopsis genome encodes ten homologues. VAP27-1 binds to OPR2A at contact sites between the ER membrane and either plasma membrane or autophagosome membranes. Whether VAP27 proteins and OPR2A are also involved in formation of contact sites between the ER and chloroplast membranes was previously unknown.
The researchers find that VAP27-1 and VAP27-3 accumulate in ER membranes that are in physical proximity to chloroplasts. The half time of fluorescence recovery after photobleaching is longer in these regions close to chloroplasts, suggesting reduced protein movement there compared to in ER tubules. Fluorescence complementation could also be shown between VAP27-1 or VAP27-3 and the outer chloroplast envelope protein 7 (OEP7), showing physical proximity. VAP27-1 and VAP27-3 interact with OPR2A, and in vitro translated OPR2A as well as VAP27-1 and VAP27-3 lacking transmembrane domains could be co-isolated with intact chloroplasts and are sensitive to trypsin digestion, which is characteristic for proteins associated with the outer envelope membrane. OPR2A and the two VAP27 paralogues can interact with monogalactosyldiacylglycerol, a typical lipid of chloroplast membranes. Interestingly, loss of VAP27 and OPR2A has only minor effects on total acyl composition but leads to accumulation of β-sitosterol and campesterol in chloroplast membranes, suggesting that these proteins may be involved in regulation of sterol homeostasis in chloroplasts.
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