Subdomain-specific localization of CLIMP-63 (p63) in the endoplasmic reticulum is mediated by its luminal alpha-helical segment

The microtubule-binding integral 63 kD cytoskeleton-linking membrane protei n (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is e xcluded 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 subdo main-specific localization, as visualized by confocal microscopy in living cells and by immunoelectron microscopy using ultrathin cryosections. Photob leaching/recovery analysis revealed that the luminal segment determines res tricted diffusion and immobility of the protein. The recombinant full-lengt h luminal segment of CLIMP-63 formed a-helical 91-nm long rod-like structur es as evident by circular dichroism spectroscopy and electron microscopy. I n the analytical ultracentrifuge, the luminal segment sedimented at 25.7 S, indicating large complexes. The complexes most likely arose by electrostat ic interactions of individual highly charged coiled coils. The findings ind icate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into cc-helical complexes that prevent nuclear envelope loc alization. Concentration of CLIMP-63 into patches may enhance microtubule b inding on the cytosolic side and contribute to ER morphology by the formati on of a protein scaffold in the lumen of the ER.