Fig. 2. ATP13A1 mediates TM removal from the ER.

(A) Scheme to reconstitute TA protein dislocation from ER-derived rough microsomes (RMs). In vitro translated (IVT) radiolabeled TA protein is incubated with RMs from wildtype (WT), ATP13A1 knockout (KO), or KO Flp-In T-REx 293 cells stably re-expressing wildtype (WT) or D533A ATP13A1. RMs containing inserted TA protein (tot.) are re-isolated and incubated with an energy regenerating system and the cytosolic TM chaperone SGTA. TA proteins dislocated from RMs are separated by centrifugation (sup.). (B) SDS-PAGE and autoradiography of tot. and extracted (sup.) TA protein containing the OMP25 TM with the indicated RMs. Equal recovery of SGTA (Coomassie) and TA protein crosslinks to SGTA (x SGTA) are indicated. (C) As in B, after crosslinking with 250 μM BMH. (D) Immunofluorescence of endogenous TOM20 (purple; mitochondrial marker) and FLAG-tagged TA protein containing the OMP25 TM (FLAG-β-OMP25; green) stably expressed in (left) WT, ATP13A1 KO, or (right) KO Flp-In T-REx HeLa cells transfected with WT or D533A HA-tagged ATP13A1 (yellow arrowheads, transfected cells; red arrowheads, untransfected cells; also see fig. S5E). Scale bar, 10 μm. Panels below show high magnification views of boxed regions; intensity of FLAG-β-OMP25 is enhanced in ii, iv, and v. (E) Mean + s.e.m. and individual Manders’ colocalization coefficient (MCC) values of FLAG-β-OMP25 colocalization with TOM20. **** p<0.0001. (F) Volcano plot showing relation of the P-value and log₂ enrichment of proteins in ATP13A1 KO Flp-In T-REx HeLa cells re-expressing WT versus D533A FLAG-ATP13A1. Significantly enriched proteins are classified based on expected cellular localization and TM topology. mito., mitochondrial; N-term., N-terminal; SS, signal sequence; sol., soluble protein after SS cleavage. (G) Mean + s.e.m. and individual MCC of endogenous OMP25 colocalization with TOM20 in the indicated cells.