STARD3 or STARD3NL and VAP form a novel molecular tether between late endosomes and the ER.
Description of experimental evidence
The protein was validated by immunoprecipitation, electron microscopy, immunofluorescence, in situ proximity ligation assay, mSDS-PAGE and western blot analysis in HeLa cells.
Corroborating this, in vitro reconstitution assays indicated that STARD3 and its ER-anchored partner, Vesicle-associated membrane protein-associated protein (VAP), assemble into a machine that allows a highly efficient transport of cholesterol within membrane contacts.
Description of experimental evidence
The protein was validated by immunofluorescence, colocalization analysis, filipin staining, GFP-D4, filipin co-staining, flotation experiments, electron microscopy and stereology in Hela cells, and the complex allows a highly efficient transport of cholesterol within membrane contacts.
Consequently, MOSPD2 and these organelle‐bound proteins mediate the formation of contact sites between the ER and endosomes, mitochondria, or Golgi.; ll these proteins, by binding VAP proteins, are known to build contact sites between the ER and endosomes (STARD3, STARD3NL, ORP1L), mitochondria (PTPIP51), and Golgi (STARD11).
Description of experimental evidence
The protein was validated by immunofluorescence, colocalization analysis, SDS–PAGE, western blot, coomassie blue staining, pull‐down assays, GFP‐Trap, mass spectrometry analysis, electron microscopy and immunoprecipitation in HeLa cells and HEK293T cells.
The endoplasmic reticulum possesses three major receptors, VAP‐A, VAP‐B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts; a novel type of FFATs that we named Phospho‐FFATs (illustrated here with STARD3), which strictly depend on phosphorylation to be active. Thus, phosphorylation acts as a switch mechanism to turn on the interaction between VAPs/MOSPD2 and their partners possessing a Phospho‐FFAT, and thus membrane contact site formation.
Description of experimental evidence
The protein was validated by pull‐down assays, immunoprecipitation,SDS–PAGE, western blot, CIP treatment, coomassie blue staining, mass spectrometry analysis, immunofluorescence and colocalization analysis in HeLa cells.
