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Subdomain-Specific Localization of Climp-63 (P63) in the Endoplasmic Reticulum Is Mediated by Its Luminal α-Helical Segment
The microtubule-binding integral 63 kD cytoskeleton-linking membrane protein (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is excluded from the nuclear envelope. We studied the mechanism underlying this ER subdomain–specific localization by mutagenesis and structural analysis....
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
The Rockefeller University Press
2001
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2192027/ https://www.ncbi.nlm.nih.gov/pubmed/11402071 |
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author | Klopfenstein, Dieter R. Klumperman, Judith Lustig, Ariel Kammerer, Richard A. Oorschot, Viola Hauri, Hans-Peter |
author_facet | Klopfenstein, Dieter R. Klumperman, Judith Lustig, Ariel Kammerer, Richard A. Oorschot, Viola Hauri, Hans-Peter |
author_sort | Klopfenstein, Dieter R. |
collection | PubMed |
description | The microtubule-binding integral 63 kD cytoskeleton-linking membrane protein (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is excluded from the nuclear envelope. We studied the mechanism underlying this ER subdomain–specific localization by mutagenesis and structural analysis. Deleting the luminal but not cytosolic segment of CLIMP-63 abrogated subdomain-specific localization, as visualized by confocal microscopy in living cells and by immunoelectron microscopy using ultrathin cryosections. Photobleaching/recovery analysis revealed that the luminal segment determines restricted diffusion and immobility of the protein. The recombinant full-length luminal segment of CLIMP-63 formed α-helical 91-nm long rod-like structures as evident by circular dichroism spectroscopy and electron microscopy. In the analytical ultracentrifuge, the luminal segment sedimented at 25.7 S, indicating large complexes. The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils. The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into α-helical complexes that prevent nuclear envelope localization. Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER. |
format | Text |
id | pubmed-2192027 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2001 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-21920272008-05-01 Subdomain-Specific Localization of Climp-63 (P63) in the Endoplasmic Reticulum Is Mediated by Its Luminal α-Helical Segment Klopfenstein, Dieter R. Klumperman, Judith Lustig, Ariel Kammerer, Richard A. Oorschot, Viola Hauri, Hans-Peter J Cell Biol Original Article The microtubule-binding integral 63 kD cytoskeleton-linking membrane protein (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is excluded from the nuclear envelope. We studied the mechanism underlying this ER subdomain–specific localization by mutagenesis and structural analysis. Deleting the luminal but not cytosolic segment of CLIMP-63 abrogated subdomain-specific localization, as visualized by confocal microscopy in living cells and by immunoelectron microscopy using ultrathin cryosections. Photobleaching/recovery analysis revealed that the luminal segment determines restricted diffusion and immobility of the protein. The recombinant full-length luminal segment of CLIMP-63 formed α-helical 91-nm long rod-like structures as evident by circular dichroism spectroscopy and electron microscopy. In the analytical ultracentrifuge, the luminal segment sedimented at 25.7 S, indicating large complexes. The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils. The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into α-helical complexes that prevent nuclear envelope localization. Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER. The Rockefeller University Press 2001-06-11 /pmc/articles/PMC2192027/ /pubmed/11402071 Text en © 2001 The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Original Article Klopfenstein, Dieter R. Klumperman, Judith Lustig, Ariel Kammerer, Richard A. Oorschot, Viola Hauri, Hans-Peter Subdomain-Specific Localization of Climp-63 (P63) in the Endoplasmic Reticulum Is Mediated by Its Luminal α-Helical Segment |
title | Subdomain-Specific Localization of Climp-63 (P63) in the Endoplasmic Reticulum Is Mediated by Its Luminal α-Helical Segment |
title_full | Subdomain-Specific Localization of Climp-63 (P63) in the Endoplasmic Reticulum Is Mediated by Its Luminal α-Helical Segment |
title_fullStr | Subdomain-Specific Localization of Climp-63 (P63) in the Endoplasmic Reticulum Is Mediated by Its Luminal α-Helical Segment |
title_full_unstemmed | Subdomain-Specific Localization of Climp-63 (P63) in the Endoplasmic Reticulum Is Mediated by Its Luminal α-Helical Segment |
title_short | Subdomain-Specific Localization of Climp-63 (P63) in the Endoplasmic Reticulum Is Mediated by Its Luminal α-Helical Segment |
title_sort | subdomain-specific localization of climp-63 (p63) in the endoplasmic reticulum is mediated by its luminal α-helical segment |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2192027/ https://www.ncbi.nlm.nih.gov/pubmed/11402071 |
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