title: 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.
authors: Faculty of Biology Medicine and Health, University of Manchester, Manchester M13 9PT, U.K.
authors: Wenxia
authors: W
authors: Qin
authors: Faculty of Biology Medicine and Health, University of Manchester, Manchester M13 9PT, U.K.
authors: Lydia
authors: L
authors: Wunderley
authors: Faculty of Biology Medicine and Health, University of Manchester, Manchester M13 9PT, U.K.
authors: Anne L
authors: AL
authors: Barrett
authors: Faculty of Biology Medicine and Health, University of Manchester, Manchester M13 9PT, U.K.
authors: Stephen
authors: S
authors: High
authors: Faculty of Biology Medicine and Health, University of Manchester, Manchester M13 9PT, U.K.
authors: Philip G
authors: PG
authors: Woodman
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D002607:Charcot-Marie-Tooth Disease / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D021381:Protein Transport; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C522864:CDIP1 protein, human; D002352:Carrier Proteins; C118738:LITAF protein, human; D008565:Membrane Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; C017800:zinc-binding protein
keywords: CDIP1; ESCRT; endosome; membrane integration