The zinc metalloprotease ZMPSTE24 binds a distinct topological isoform of the tail-anchored protein IFITM3

The biogenesis of integral membrane proteins is complex, as revealed by an ever-growing number of cellular components shown to be dedicated to the insertion, folding, surveillance, rectification, or quality control of specific client membrane proteins. The zinc metalloprotease ZMPSTE24 and its yeast homolog Ste24 have well-established roles in the proteolytic maturation of the nuclear scaffold protein lamin A and yeast a-factor, respectively. Additionally, Ste24 has been implicated through yeast genetic screens in a variety of membrane processes, including ER- associated degradation (ERAD), Sec61 translocon “unclogging,” the unfolded protein response (UPR), and potentially as a membrane protein topology determinant. Recently, an interaction was demonstrated between ZMPSTE24 and the antiviral interferon induced transmembrane protein IFITM3, although the functional significance of this interaction is not well-understood. IFITM3 is a tail-anchored protein with a cytoplasmic N-terminus, a single transmembrane span, and a lumenal/exocellular C-terminus. Here, we show that a catalytic-dead version of ZMPSTE24, ZMPSTE24E336A, exhibits enhanced binding to IFITM3, and this bound species of IFITM3 is hypo-palmitoylated. Using a split fluorescence topology reporter, we demonstrate that ZMPSTE24E336A “traps” and stabilizes a subpopulation of IFITM3 molecules with an atypical membrane topology, whose C-terminus is cytosolic instead of lumenal. Such inverted forms of IFITM3 are also detected in the presence of ERAD inhibitors when ZMPSTE24E336A is absent. We hypothesize the ZMPSTE24E336A trap mutant reveals a normally transient isoform of IFITM3 whose transmembrane span is inverted and that ZMPSTE24 is involved in the quality control of IFITM3 topology, either inverting, correcting or assisting in removal of aberrant IFITM3 molecules. ### Competing Interest Statement The authors have declared no competing interest. NIH Common Fund, https://ror.org/001d55x84, R35GM127073