APEX2 proximity labeling of PI(3)P dependent ER and early endosome membrane contact tether proteins

To understand the nature of ER and early endosome membrane contact site, we capitalized the MTM1 knock out (KO) HeLa cells that do not dissociate the ER and early endosome membrane contact upon nutrient starvation. By using the Doxycyclin inducible APEX2 fused Rab5A (early endosome marker) expressing stable cell lines of wildtype (WT) or KO cells, early endsome proximity proteins were acutely biotinyated during starvation. Subsequent biochemical fractionation were conducted to enrich ER membrane and biotinylated proteins were furhter enriched for Mass spectrometry analysis.