Subdomain-Specific Localization of Climp-63 (P63) in the Endoplasmic Reticulum Is Mediated by Its Luminal α-Helical Segment

Abstract

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.

Interaction of Microtubule-associated Protein-2 and p63

“…Here we report a novel association of MAP2 with RER membranes using subcellular fractionation, electron microscopy immunocytochemistry, and electron microscopy in an in vitro reconstitution assay. Moreover, we showed that this association involves the interaction of the MAP2 projection domain with a 63-kDa nonglycosylated type II integral RER membrane protein, termed p63, that was found to mediate the interaction between RER membranes and microtubules in a previous study (37)(38)(39). Taken together, our results suggest that the interaction between MAP2 and p63 might contribute to the preferential distribution of RER membranes in the dendritic processes.…”

“…More specifically, Hook3 links the Golgi membranes to microtubules (36). Furthermore, p63 was shown to be involved in the interaction of ER with microtubules in a non-neuronal cell line (37)(38)(39). Because p63 is an integral membrane protein, it was termed a CLIMP (cytoskeleton-linking membrane protein).…”

Functional Regulation of Tissue Plasminogen Activator on the Surface of Vascular Smooth Muscle Cells by the Type-II Transmembrane Protein p63 (CKAP4)

“…The three-dimensional structure of p63 is not known, but the C-terminal extra-cytoplasmic part of the protein (residues 132-602) has been suggested to have a largely ␣-helical structure based on its CD spectrum (39). This is consistent with sequence analysis (41), which suggests that up to 30% of this domain will form ␣-helical coiled-coils due to repeating heptad motifs.…”

“…Fibrin contains multiple binding sites for tPA, at least one of which (AA 148 -160) is contained within a coiledcoil region of the protein (43), and the binding sites for tPA in the other two proteins are retained in fragments containing their coiled-coil regions. The isolated extra-cytoplasmic domain of p63 expressed in Escherichia coli has been shown to form insoluble oligomers and rod-like multimeric structures (39). The p63 that we detected on the surface of VSMC appeared to be highly clustered, which may similarly be due to oligomerization.…”

“…Previous studies of trafficking and localization have been performed in cells overexpressing p63, which leads to rearrangements of both the ER and microtubules mediated by the cytoplasmic domain of p63 (38). At endogenous expression levels p63 may behave differently and, possibly, in a cell type-specific manner (39). Without the close association with microtubules as a consequence of overexpression, a proportion of p63 may escape its sub-domain-specific localization and be free to reach the plasma membrane.…”