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

Abstract

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. 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 (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61β, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA 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, while 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), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins.

The topology, structure and PE interaction of LITAF underpin a Charcot-Marie-Tooth disease type 1C

“…In contrast to the assertion made by Lee et al [11] that LITAF domains are C-terminally inserted into membranes, our data show that the hydrophobic region does not traverse membranes, demonstrating that both the N-terminal and C-terminal halves of the LITAF domain reside on the cytosolic surface of membranes, in agreement with an earlier proposal made by Ponting et al [15] in 2002 and the recent publication by Qin et al [44]. The LITAF domain consists of five β-sheets, three N-terminal and two C-terminal to the predicted hydrophobic anchor region, and is stabilised by the coordination of a zinc atom by two pairs of evolutionarily-conserved cysteine residues.…”

Phenotypic spectrum of Charcot−Marie−Tooth disease due to LITAF/SIMPLE mutations: a study of 18 patients

“…LITAF/SIMPLE is a 161 amino acid protein associated with membranes of the endosomal compartment by a C‐terminal cysteine‐rich domain. The cysteine‐rich regions combine to form a zinc‐finger, necessary for stabilization into membranes . Our study confirms that CMT1C mutations are located in the C‐terminal part and, interestingly, extends the location of deleterious mutations to the extreme C‐terminal part of the protein (Arg160).…”

Novel EGR2 variant that associates with Charcot-Marie-Tooth disease when combined with lipopolysaccharide-induced TNF-α factor T49M polymorphism

“…8,16 Indeed, recent studies have shown that LITAF has a cysteine-rich region that coordinates zinc. 17,18 We explored whether LITAF could positively regulate the transcription of myelin genes. To this aim, HEK293 cells were cotransected with phPMP22enh-Luciferase and the plasmids encoding for the human LITAF sequence.…”