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 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.

A novel direct interaction of endoplasmic reticulum with microtubules.

The positioning and dynamics of organelles in eukaryotic cells critically depend on membrane-cytoskeleton interactions. Motor proteins play an important role in the directed movement of organelle membranes along microtubules, but the basic mechanism by which membranes stably interact with the microtubule cytoskeleton is largely unknown. Here we report that p63, an integral membrane protein of the reticular subdomain of the rough endoplasmic reticulum (ER), binds microtubules in vivo and in vitro. Overexpression of p63 in cell culture led to a striking rearrangement of the ER and to concomitant bundling of microtubules along the altered ER. Mutational analysis of the cytoplasmic domain of p63 revealed two determinants responsible for these changes: an ER rearrangement determinant near the N-terminus and a central microtubule-binding region. The two determinants function independently of one another as indicated by deletion experiments. A peptide corresponding to the cytoplasmic tail of p63 promoted microtubule polymerization in vitro. p63 is the first identified integral membrane protein that can link a membrane organelle directly to microtubules. By doing so, it may contribute to the positioning of the ER along microtubules.

The recycling of ERGIC-53 in the early secretory pathway. ERGIC-53 carries a cytosolic endoplasmic reticulum-exit determinant interacting with COPII.

Further investigation of the targeting of the intracellular membrane lectin endoplasmic reticulum (ER)-Golgi intermediate compartment-53 (ERGIC-53) by site-directed mutagenesis revealed that its lumenal and transmembrane domains together confer ER retention. In addition we show that the cytoplasmic domain is required for exit from the ER indicating that ERGIC-53 carries an ER-exit determinant. Two phenylalanines at the C terminus are essential for ER-exit. Thus, ERGIC-53 contains determinants for ER retention as well as anterograde transport which, in conjunction with a dilysine ER retrieval signal, control the continuous recycling of ERGIC-53 in the early secretory pathway. In vitro binding studies revealed a specific phenylalanine-dependent interaction between an ERGIC-53 cytosolic tail peptide and the COPII coat component Sec23p. These results suggest that the ER-exit of ERGIC-53 is mediated by direct interaction of its cytosolic tail with the Sec23p.Sec24p complex of COPII and that protein sorting at the level of the ER occurs by a mechanism similar to receptor-mediated endocytosis or Golgi to ER retrograde transport.