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.
Highlights
Cells communicate with their environment by the internalisation of lipids and membrane proteins from their surface
Quantitation showed that
We identify LITAF and cell death involved p53 target 1 (CDIP1) as belonging to a novel, conserved class of monotopic integral membrane proteins that localise to the plasma membrane and endocytic compartments
Summary
Cells communicate with their environment by the internalisation of lipids and membrane proteins from their surface. Mammalian cells express a related protein, CDIP1 (cell death involved p53 target 1), like LITAF possessing conserved PT/SAP and PPXY motifs and the C-terminal ‘SIMPLE-like’ domain (Figure 1A) [28]. We show that LITAF defines a novel class of monotopic integral membrane proteins, in which the hydrophobic region is stably inserted into the membrane by virtue of a zinc-binding domain formed by the neighbouring cysteine-rich regions. This organisation is likely a general feature of LITAF domain proteins, since the same membrane topology is found in CDIP1
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