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

Klopfenstein DR, Klumperman J, Lustig A, Kammerer RA, Oorschot V, Hauri HP - J. Cell Biol. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neurobiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

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 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.

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FRAP shows fast diffusion for luminal deletion mutants. Quantitative FRAP experiments with transiently transfected COS cells expressing CLIMP-63–GFP constructs. (A) The recovery profiles of five constructs are shown as the mean value of intensity in the region of interest as a function of time. (B) GFP-Δcytoplasmic (left) and GFP-Δcytoplasmic-Δ246–492 (right) show differences in diffusion comparing mobile, reticular ER (○) with immobile, clustered ER (•).
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Figure 6: FRAP shows fast diffusion for luminal deletion mutants. Quantitative FRAP experiments with transiently transfected COS cells expressing CLIMP-63–GFP constructs. (A) The recovery profiles of five constructs are shown as the mean value of intensity in the region of interest as a function of time. (B) GFP-Δcytoplasmic (left) and GFP-Δcytoplasmic-Δ246–492 (right) show differences in diffusion comparing mobile, reticular ER (○) with immobile, clustered ER (•).

Mentions: To determine whether the deletion mutants differ in protein mobility, we performed FRAP experiments in living cells. A strip of 2-μm width covering the reticular ER over the entire cell was bleached for 2 s and fluorescence recovery was quantified. Fig. 6 A shows that the recovery rate was faster for the luminal deletion construct than for GFP-Δcytoplasmic, whereas the differences among the deletion constructs were rather minimal (Table ). As a reference, we also included the Δlumen-GFP construct which showed high mobility and diffusion in the same range as the ER membrane enzyme cytochrome P450, Sec61β, and VSV-G protein (Table II; Cole et al. 1996; Szczesna-Skorupa et al. 1998; Rolls et al. 1999). The fraction of mobile protein was dependent on the presence of the luminal segment. The calculated diffusion coefficients ranging from 1.5–17.8 × 10−10cm2s−1 are shown in Table . The rate of fluorescence recovery in the nuclear envelope and in the reticular domain of the ER was comparable as judged from visual inspection. However, there was a considerable difference in mobility of a given construct if reticular and condensed ER areas were compared. Condensed ER regions of cells expressing the constructs Δcytoplasmic (Fig. 6 B, left) and ΔcytoplasmicΔ246–492 (right) displayed lower mobility than the corresponding reticular ER. These results suggest that molecular clustering renders the constructs nearly immobile and prevents them from mixing with the mobile reticular pool.


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

Klopfenstein DR, Klumperman J, Lustig A, Kammerer RA, Oorschot V, Hauri HP - J. Cell Biol. (2001)

FRAP shows fast diffusion for luminal deletion mutants. Quantitative FRAP experiments with transiently transfected COS cells expressing CLIMP-63–GFP constructs. (A) The recovery profiles of five constructs are shown as the mean value of intensity in the region of interest as a function of time. (B) GFP-Δcytoplasmic (left) and GFP-Δcytoplasmic-Δ246–492 (right) show differences in diffusion comparing mobile, reticular ER (○) with immobile, clustered ER (•).
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Related In: Results  -  Collection

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Figure 6: FRAP shows fast diffusion for luminal deletion mutants. Quantitative FRAP experiments with transiently transfected COS cells expressing CLIMP-63–GFP constructs. (A) The recovery profiles of five constructs are shown as the mean value of intensity in the region of interest as a function of time. (B) GFP-Δcytoplasmic (left) and GFP-Δcytoplasmic-Δ246–492 (right) show differences in diffusion comparing mobile, reticular ER (○) with immobile, clustered ER (•).
Mentions: To determine whether the deletion mutants differ in protein mobility, we performed FRAP experiments in living cells. A strip of 2-μm width covering the reticular ER over the entire cell was bleached for 2 s and fluorescence recovery was quantified. Fig. 6 A shows that the recovery rate was faster for the luminal deletion construct than for GFP-Δcytoplasmic, whereas the differences among the deletion constructs were rather minimal (Table ). As a reference, we also included the Δlumen-GFP construct which showed high mobility and diffusion in the same range as the ER membrane enzyme cytochrome P450, Sec61β, and VSV-G protein (Table II; Cole et al. 1996; Szczesna-Skorupa et al. 1998; Rolls et al. 1999). The fraction of mobile protein was dependent on the presence of the luminal segment. The calculated diffusion coefficients ranging from 1.5–17.8 × 10−10cm2s−1 are shown in Table . The rate of fluorescence recovery in the nuclear envelope and in the reticular domain of the ER was comparable as judged from visual inspection. However, there was a considerable difference in mobility of a given construct if reticular and condensed ER areas were compared. Condensed ER regions of cells expressing the constructs Δcytoplasmic (Fig. 6 B, left) and ΔcytoplasmicΔ246–492 (right) displayed lower mobility than the corresponding reticular ER. These results suggest that molecular clustering renders the constructs nearly immobile and prevents them from mixing with the mobile reticular pool.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Neurobiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

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 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.

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