Reconstitution of a Tail‐anchored Mitochondrial Membrane Protein

Abstract

Tail‐anchored (TA) proteins have a single transmembrane domain (TMD) located on the C‐terminus with the bulk of the protein facing the cytosol; in contrast with the conventional membrane protein that is translated directly into the membrane of interest. TA proteins are involved in organelle division, vesicle trafficking, and apoptosis. TA proteins lack an N‐terminal signal sequence and exhibit wide sequence variation in the TMD. Thus, TA proteins are likely inserted into the membrane post translationally. We asked if we can reconstitute Fis1, a mitochondrial outer membrane TA protein, in a detergent micelle to determine its structure. We were able to purify soluble recombinant Fis1 in the absence of detergent. Preliminary results suggest that in the absence of detergent, Fis1 exists in a soluble aggregate that can be disrupted by addition of detergent. This soluble aggregate is likely facilitated by the TMD of Fis1. We further show that in the detergent micelle Fis1 is oriented such that the cytoplasmic domain faces the solvent, reminiscent of the orientation on the mitochondrial surface. Using properly reconstituted Fis1 in detergent micelles, we collected CD, DLS, NMR, and EPR data with the goal of determining a high‐resolution structure of Fis1 in the membrane, which is an outstanding question in the mitochondrial fission field. More generally, this work has many implications for TA membrane protein reconstitution.Support or Funding InformationNIH R01‐GM067180This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.