Figure 6: The rate limiting step in Msp1 activity is likely removal of the substrate from the lipid bilayer.

A) Diagram of Sumo-Sec22 and DHFR-Sec22 model substrates. Note that the only difference is in the cytosolic domain.

B) Protease protection assay for the DHFR-Sec22 substrate.

C) Standard curves for reconstituted substrates show significantly lower levels of luminescence for DHFR-Sec22 than Sumo-Sec22 despite identical reconstitution conditions. This indicates that DHFR-Sec22 reconstitution is inefficient. Reconstitutions and standard curves were performed in parallel for both substrates.

D) Extraction assay for DHFR-Sec22 model substrate shows ATP dependent extraction that is stimulated upon addition of methotrexate.

E) Extraction assay for Sumo-Sec22 model substrate shows a 44% stimulation of extraction activity upon addition of methotrexate.

F) Extraction of DHFR-Sec22 corrected for stimulating effects of methotrexate. Same data as in D, but extraction activity of the +ATP, +MTX sample was reduced by 44%. After this correction factor, the ATP dependent extraction is comparable to the DMSO negative control, indicating that methotrexate does not inhibit extraction of DHFR-Sec22.

G) Diagram of the lateral diffusion model. Mislocalized ER-TA proteins have a hydrophobic mismatch with the OMM, leading to interaction between the solvent exposed TMD and hydrophobic residues in the seam of the Msp1 hexamer. The substrate enters the central pore of Msp1 by laterally diffusing through the seam, potentially bypassing the need for unfolding of cytosolic domains⁴⁵.