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Published online 11 June 2001. doi:10.1083/jcb.153.6.1287
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© The Rockefeller University Press, 0021-9525/2001/6/1287/ $5.00
The Journal of Cell Biology, Volume 153, Number 6, June 11, 2001 1287-1300

Original Article

Subdomain-specific Localization of CLIMP-63 (p63) in the Endoplasmic Reticulum Is Mediated by Its Luminal {alpha}-Helical Segment

Dieter R. Klopfensteina, Judith Klumpermanc, Ariel Lustigb, Richard A. Kammererb, Viola Oorschotc, and Hans-Peter Hauria
a Department of Pharmacology and Neurobiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland
b Department of Biophysical Chemistry, Biozentrum, University of Basel, CH-4056 Basel, Switzerland
c Department of Cell Biology, Institute of Biomembranes, Center for Biomedical Genetics, University Medical Center, 3584 CX Utrecht, Netherlands

Correspondence to: Hans-Peter Hauri, Department of Pharmacology and Neurobiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel:(41) 61-267-2222 Fax:(41) 61-267-2208 E-mail:hans-peter.hauri{at}unibas.ch.

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 {alpha}-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 {alpha}-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.

Key Words: coiled coils, CLIMP-63 (p63), endoplasmic reticulum, green fluorescent protein, microtubule-binding membrane protein


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