The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein

LITAF (LPS-Induced TNF-Activating Factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal dominant Charcot Marie Tooth disease type 1C (CMT1C). These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here we have investigated whether LITAF is a tail-anchored membrane spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared to Sec61 b , a bona fide tail-anchored protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal or C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of a LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a tail-anchored protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, whilst mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc-finger. The related human protein, CDIP1 (Cell Death Involved p53 Target 1), displayed identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins.