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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. These mutations map to a highly conserved C-terminal region, termed the LI...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Portland Press Ltd.
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095921/ https://www.ncbi.nlm.nih.gov/pubmed/27582497 http://dx.doi.org/10.1042/BCJ20160657 |
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author | Qin, Wenxia Wunderley, Lydia Barrett, Anne L. High, Stephen Woodman, Philip G. |
author_facet | Qin, Wenxia Wunderley, Lydia Barrett, Anne L. High, Stephen Woodman, Philip G. |
author_sort | Qin, Wenxia |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5095921 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Portland Press Ltd. |
record_format | MEDLINE/PubMed |
spelling | pubmed-50959212016-11-08 The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein Qin, Wenxia Wunderley, Lydia Barrett, Anne L. High, Stephen Woodman, Philip G. Biochem J Research Articles 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. Portland Press Ltd. 2016-11-01 2016-10-27 /pmc/articles/PMC5095921/ /pubmed/27582497 http://dx.doi.org/10.1042/BCJ20160657 Text en © 2016 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0) . |
spellingShingle | Research Articles Qin, Wenxia Wunderley, Lydia Barrett, Anne L. High, Stephen Woodman, Philip G. The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein |
title | The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein |
title_full | The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein |
title_fullStr | The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein |
title_full_unstemmed | The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein |
title_short | The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein |
title_sort | charcot marie tooth disease protein litaf is a zinc-binding monotopic membrane protein |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095921/ https://www.ncbi.nlm.nih.gov/pubmed/27582497 http://dx.doi.org/10.1042/BCJ20160657 |
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